Emerging Technologies for Food Processing 9780126767575, 0126767572

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About the Editor

Born in Southern China, Professor Da-Wen Sun is an internationally recognized figure for his leadership in food engineering research and education. His main research activities include cooling, drying and refrigeration processes and systems, quality and safety of food products, bioprocess simulation and optimization and computer vision technology. In particular, his innovative work on vacuum cooling of cooked meats, pizza quality inspection by computer vision and edible films for shelf-life extension of fruit and vegetables has been widely reported in national and international media. Results of his work have been published in over 150 peer reviewed journal papers and more than 200 conference papers. Dr Sun received First Class Honours BSc and MSc degrees in Mechanical Engineering and a PhD degree in Chemical Engineering in China before working in various universities in Europe. Dr Sun became the first Chinese to be permanently employed in an Irish University when he was appointed College Lecturer at National University of Ireland, Dublin (University College Dublin) in 1995 and was then promoted to Senior Lecturer. Dr Sun is now a professor and director of the Food Refrigeration and Computerised Food Technology Research Group at Department of Biosystems Engineering, University College Dublin. As a leading educator in food engineering, Professor Sun has significantly contributed to the field of food engineering. He has trained many PhD students, who have made their own contributions to the industry and academia. Professor Sun has also given lectures on advances in food engineering on a regular basis to academic institutions internationally and delivered keynote speeches at international conferences. As a recognized authority in food engineering, he has been conferred adjunct/visiting/consulting professorships from ten top universities in China including Shanghai Jiao Tong University, Zhejiang University, Harbin Institute of Technology, China Agriculture University, South China University of Technology, Southern Yangtze University, etc. In recognition of his significant contribution to food engineering worldwide, the

xiv About the Editor

International Commission of Agricultural Engineering (CIGR) awarded him the CIGR Merit Award in 2000 and the Institution of Mechanical Engineers (IMechE) based in the UK awarded him ‘Food Engineer of the Year 2004’. Professor Sun is a Fellow of the Institution of Agricultural Engineers. He has also received numerous awards for teaching and research excellence, including the President’s Research Award of University College Dublin twice. He is Chair of CIGR Section VI on Postharvest Technology and Process Engineering, Guest Editor of Journal of Food Engineering and Computer and Electronics in Agriculture and editorial board member for Journal of Food Process Engineering. He is also a Chartered Engineer registered in the UK Engineering Council.

Contributors

Ana Allende (Ch. 26), Technical University of Cartagena, Department of Food Engineering, Postharvest and Refrigeration Group, Cartagena, Murcia, Spain Francisco Artés (Ch. 26), Technical University of Cartagena, Department of Food Engineering, Postharvest and Refrigeration Group, Cartagena, Murcia, Spain B Ravindra Babu (Ch. 10), Department of Food Engineering, Central Food Technological Research Institute, Mysore, India V M Balasubramaniam (Ch. 2), The Ohio State University, Department of Food Science and Technology, Columbus, Ohio, USA Domenico Cacace (Ch. 11), Stazione Sperimentale per l’Industria delle Conserve Alimentari (Experimental Station for the Food Preserving Industry), Angri (SA), Italy Siaw Kiang Chou (Ch. 20), Department of Mechanical Engineering, National University of Singapore, Singapore Kian Jon Chua (Ch. 20), Department of Mechanical Engineering, National University of Singapore, Singapore Timothy D Durance (Ch. 19), University of British Columbia, Food, Nutrition and Health, Vancouver, Canada Marianne Dyrby (Ch. 21), The Royal Veterinary and Agricultural University, Quality and Technology, Frederiksberg, Denmark Pedro Elez-Martínez (Chs 7 and 8), University of Lleida, Department of Food Technology, UTPV-CeRTA, Lleida, Spain Søren B Engelsen (Ch. 21), The Royal Veterinary and Agricultural University, Quality and Technology, Frederiksberg, Denmark David J Geveke (Ch. 12), US Department of Agriculture, Eastern Regional Research Center, Wyndmoor, PA, USA Adeline Goullieux (Ch. 18), Université de Picardie Jules Verne, Laboratoire des Technologies Innovantes, IUT-GB, Amiens, France Mansel W Griffiths (Ch. 5), University of Guelph, Ontario, Canada Magnus Gudmundsson (Ch. 6), Matra, Technological Institute of Iceland, Keldnaholt, Reykjavik, Iceland Hannes Hafsteinsson (Ch. 6), Matra, Technological Institute of Iceland, Keldnaholt, Reykjavik, Iceland

xvi Contributors

Flemming Hansen (Ch. 15), Danish Meat Research Institute, Roskilde, Denmark Volker Heinz (Ch. 4), Berlin University of Technology, Department of Food Biotechnology and Food Process Engineering, Berlin, Germany Eamonn Hogan (Ch. 1), Food Refrigeration and Computerised Food Technology Group, National University of Ireland, Dublin, Ireland C James (Ch. 27), University of Bristol, Food Refrigeration and Process Engineering Research Centre, Langford, North Somerset, UK S J James (Ch. 27), University of Bristol, Food Refrigeration and Process Engineering Research Centre, Langford, North Somerset, UK Zongchao Jia (Ch. 25), Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada Alan L Kelly (Ch. 1), Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland Dietrich Knorr (Ch. 4), Berlin University of Technology, Department of Food Biotechnology and Food Process Engineering, Berlin, Germany Monique Lacroix (Ch. 14), INRS-Institut Armand-Frappier, Canadian Irradiation Center, Université du Québec, Laval City, Québec, Canada Robert W Lencki (Ch. 28), University of Guelph, Department of Food Science, Guelph, Ontario, Canada Pascual Lopez-Buesa (Ch. 13), Universidad de Zaragoza, Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Zaragoza, Spain Olga Martín-Belloso (Chs 7 and 8), University of Lleida, Department of Food Technology, UTPV-CeRTA, Lleida, Spain Timothy J Mason (Ch. 13), Coventry University, Sonochemistry Centre, School of Science and the Environment, Coventry, UK Gauri S Mittal (Ch. 5), University of Guelph, Ontario, Canada Montserrat Mor-Mur (Ch. 3), Universitat Autònoma de Barcelona, Centre Especial de Recerca Planta de Tecnologia dels Aliments (CeRTA, XIT), Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Barcelona, Spain Naveen Nagaraj (Ch. 10), Department of Food Engineering, Central Food Technological Research Institute, Mysore, India K Niranjan (Chs 9 and 10), School of Food Biosciences, The University of Reading, Reading, Berkshire, UK Valerie Orsat (Ch. 17), Bioresource Engineering Department, McGill University, Ste-Anne de Bellevue, Québec, Canada Laura Otero (Ch. 24), Instituto del Frío (CSIC), Department of Engineering, Madrid, Spain Jean-Pierre Pain (Ch. 18), Université Montpellier II, Département Agro-Ressources et Procédés Biologiques UMR1028, Montpellier, France Luigi Palmieri (Ch. 11), Stazione Sperimentale per l’Industria delle Conserve Alimentari (Experimental Station for the Food Preserving Industry), Angri (SA), Italy Srilatha Pandrangi (Ch. 2), The Ohio State University, Department of Food Science and Technology, Columbus, Ohio, USA

Contributors xvii

Ganapathi Patil (Ch. 10), Department of Food Engineering, Central Food Technological Research Institute, Mysore, India G S Vijaya Raghavan (Ch. 17), Bioresource Engineering Department, McGill University, Ste-Anne de Bellevue, Québec, Canada K S M S Raghavarao (Chs 9 and 10), Department of Food Engineering, Central Food Technological Research Institute, Mysore, India N K Rastogi (Ch. 9), Department of Food Engineering, Central Food Technological Research Institute, Mysore, India Enrique Riera (Ch. 13), Instituto de Acústica, CSIC, Ultrasonics Department, Madrid, Spain Serpil Sahin (Ch. 16), Middle East Technical University, Food Engineering Department, Ankara, Turkey Pedro D Sanz (Ch. 24), Instituto del Frío (CSIC), Department of Engineering, Madrid, Spain Christine H Scaman (Ch. 19), University of British Columbia, Food, Nutrition and Health, Vancouver, Canada Jakob Søltoft-Jensen (Ch. 15), Danish Meat Research Institute, Roskilde, Denmark Gülüm Sumnu (Ch. 16), Middle East Technical University, Food Engineering Department, Ankara, Turkey Da-Wen Sun (Chs 1, 22 and 23), Food Refrigeration and Computerised Food Technology Group, National University of Ireland, Dublin, Ireland Stefan Toepfl (Ch. 4), Berlin University of Technology, Department of Food Biotechnology and Food Process Engineering, Berlin, Germany Antonio Vercet (Ch. 13), Universidad de Zaragoza, Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Zaragoza, Spain Nanna Viereck (Ch. 21), The Royal Veterinary and Agricultural University, Quality and Technology, Frederiksberg, Denmark Brent Wathen (Ch. 25), Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada Josep Yuste (Ch. 3), Universitat Autònoma de Barcelona, Centre Especial de Recerca Planta de Tecnologia dels Aliments (CeRTA, XIT), Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Barcelona, Spain Liyun Zheng (Chs 22 and 23), Food Refrigeration and Computerised Food Technology Group, National University of Ireland, Dublin, Ireland

Preface

Emerging Technologies for Food Processing presents a comprehensive review of innovations in food processing, stresses topics vital to the food industry today and pinpoints the trends in future research and development. This volume contains 28 chapters and is divided into six parts. The first three parts cover latest advances in non-thermal processing including high pressure, pulsed electric fields, radiofrequency, high intensity pulsed light, ultrasound, irradiation and new hurdle technology, with emphasis particularly on high pressure and pulsed electric fields as extensive research has been carried out in these two areas in recent years. The fourth part provides alternative technologies and strategies for thermal processing; topics discussed include recent developments in microwave, ohmic, dielectric heating, dehydration using combined microwave vacuum techniques, new hybrid drying technologies and nuclear magnetic resonance technology. The latest developments in food refrigeration are discussed in part five, addressing innovative applications of vacuum cooling for rapid cooling of foods, acceleration of freezing process using high pressure and ultrasound and freezing with antifreeze protein and ice nucleation. The book concludes with current topics in minimal processing of vegetables, fruits, juices and cook-chill ready meals and modified atmosphere packaging for minimally processed foods. In this volume, each chapter is written by an international expert (or experts), presenting thorough research results and critical reviews of one aspect of the relevant issues and including a comprehensive list of recently published literature. It should, therefore, provide valuable sources of information for further research and developments for the food processing industry. This volume is written for food engineers and technologists in the food industry. It will also serve as an essential and comprehensive reference source to undergraduate and postgraduate students and researchers in universities and research institutions.

High Pressure Processing of Foods: An Overview Eamonn Hogan1, Alan L Kelly 2 and Da-Wen Sun1 1

Food Refrigeration and Computerised Food Technology Group, National University of Ireland, Dublin, Ireland 2 Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland

The quality and safety of food products are the two factors that most influence the choices made by today’s increasingly demanding consumers. Conventional food sterilization and preservation methods often result in a number of undesired changes in foods, such as loss of smell, colour, flavour, texture and nutritional value – in short, a reduction in the apparent freshness and quality of the final product. High-pressure (HP) processing, also sometimes known as high hydrostatic pressure (HHP), or ultra high pressure (UHP) processing, is a relatively new non-thermal food processing method that subjects liquid or solid foods, with or without packaging, to pressures between 50 and 1000 MPa. Extensive investigations have revealed the potential benefits of high pressure processing as an alternative to heat treatments. These benefits are apparent in various areas of food processing, such as the inactivation of microorganisms and enzymes, denaturation and alteration of the functionality of proteins and structural changes to food materials.

1 Introduction Food processing involves the transformation of raw animal or plant materials into consumer-ready products, with the objective of stabilizing food products by preventing or reducing negative changes in quality. Without these processes, we would neither be able to store food from time of plenty to time of need nor to transport food over long distances (Lund, 2003). To consumers, the most important attributes of a food product are its sensory characteristics (e.g. texture, flavour, aroma, shape and colour). These determine an individual’s preference for specific products and minor differences between brands of similar products can have a substantial influence on acceptability. A goal of food manufacturers is to develop and employ processing technologies that retain or create desirable sensory qualities or reduce undesirable changes in food due to processing. Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

1

4 High Pressure Processing of Foods: An Overview

Physical (e.g. heating, freezing, dehydration, and packaging) and chemical (e.g. reduction of pH or use of preservatives) preservation methods continue to be used extensively and technological advances to improve the efficiency and effectiveness of these processes are being made at a rapid rate. The basis of these traditional methods involves reducing microbial growth and metabolism to prevent undesirable chemical changes in food. Probably the most common method of food preservation used today is thermal treatment (e.g. pasteurization, sterilization). Although heating food effectively reduces levels of microorganisms such as bacteria, such processing can alter the natural taste and flavour of food and destroy vitamins. Therefore, alternative or novel food processing technologies are being explored and implemented to provide safe, fresher-tasting, nutritive foods without the use of heat or chemical preservatives. Innovative non-thermal processes for preservation of food have attracted the attention of many food manufacturers. In the search for new processing methods, particularly for certain products, the application of high-pressure (HP) processing has shown considerable potential as an alternative technology to heat treatments, in terms of assuring safety and quality attributes in minimally-processed food products (Palou et al., 2002). Consumer demand for minimally-processed food products has presented particular challenges to food processors. Most retailers are reporting up to 30 per cent growth in fresh, chilled and healthy food sales. These products all face the same problem: how to keep the food fresh and healthy with high retention of vitamin and nutrient levels, while offering a reasonable shelf-life and convenience and assuring food safety. HP technology potentially answers many, if not all, of these challenges. Unlike heat treatment, HP treatment does not reduce the quality of foods and pressure is evenly and instantaneously transmitted throughout the sample, which allows products without over-treated parts to be obtained. HP processing can thus facilitate the production of foods that have the quality of fresh foods but the convenience and profitability associated with shelf-life extension (McClements et al., 2001).

2 Principles of high pressure processing Currently, a great deal of research is being directed towards understanding the effects of high pressure (HP) on food and food ingredients. Some key findings in this regard will be summarized in this chapter.

2.1 Background Studies of the effects of high pressures on foods date back over a century. In 1899, Bert Hite of the Agriculture Research Station in Morganstown, West Virginia, USA, designed and constructed a high-pressure unit to pasteurize milk and other food products (Hite, 1899). Hite constructed a machine that could reach pressures in excess of about 6800 atmospheres (approximately 700 MPa) and he and his co-workers examined the potential use of HP processing for a wide range of foods and beverages, including the pressure inactivation of viruses. The level of sophistication that was accomplished is remarkable, given the technological disadvantages of that time period regarding

Principles of high pressure processing 5

Figure 1.1

Modern 35 l high pressure food processing unit.

processing systems and packaging materials (Hoover, 1993). In 1899, Hite reported that treatment at pressures of 450 MPa or greater could improve the keeping quality of milk (Hite, 1899). In 1914 he showed that yeasts and lactic acid bacteria associated with sweet, ripe fruit were more susceptible to pressure than other organisms, especially spore-forming bacteria associated with vegetables (Patterson et al., 1995). Compared to today’s HP processing equipment, the prototype system utilized in the 1890s by Hite is very primitive. Today, with advances in computational stress analysis and new materials, high capacity pressure systems can be manufactured to allow reliable HP treatment of food products at even higher pressures (Hoover, 1993). Figure 1.1 shows an example of a modern high-pressure processing unit. Although the potential for HP processing of foods has thus been known since the late nineteenth century, its application and potential have only recently been widely recognized. While this potential was largely ignored through most of the last century, basic work on the effects of hydrostatic pressures on biological systems steadily developed. In recent years, the use of HP as a food preservation technique has gained momentum throughout the world as an alternative to traditional heat-based methods, for the reasons cited earlier. Much of the research regarding the use of HP for food preservation has concerned inactivation of microorganisms; the pressure stability of food enzymes is now also beginning to attract increasing attention (Ashie and Simpson, 1996; Krebbers et al., 2003).

2.2 Description of the process In a HP process, the food product to be treated is placed in a pressure vessel capable of sustaining the required pressure; the product is submerged in a liquid, which acts as the

6 High Pressure Processing of Foods: An Overview

pressure-transmitting medium. Water may be used as the pressure-transmitting medium, but media containing castor oil, silicone oil, sodium benzoate, ethanol or glycol are also used. The ability of the pressure-transmitting fluid to protect the inner vessel surface from corrosion, the specific HP system being used, the process temperature range and the viscosity of the fluid under pressure are some of the factors involved in selecting the medium. Industrial HP treatment is currently a batch or semi-continuous process. The selection of equipment depends on the kind of food product to be processed. Solid food products or foods with large solid particles can only be treated in a batch mode. Liquids, slurries and other pumpable products have the additional option of semi-continuous production (Ting and Marshall, 2002). Currently, most HP machines in industrial use for food processing are batch systems, whereby the product is placed in a highpressure chamber and the vessel is closed, filled with pressure-transmitting medium and pressurized either by pumping medium into the vessel or by reducing the volume of the pressure chamber, for example by using a piston. If water is used as the pressurizing medium, its compressibility must be accounted for; water is compressed by up to 15 per cent of volume at pressures above 600 MPa. Once the desired pressure is reached, the pump or piston is stopped, the valves are closed and the pressure is maintained without further energy input. After the required hold time has elapsed, the system is depressurized, the vessel opened and the product unloaded. The system is then reloaded with product, either by operators or machines, depending on the degree of automation possible (Ting and Marshall, 2002). The total time for pressurization, holding and depressurization is referred to as the ‘cycle time’. The cycle time and the loading factor (i.e. the percentage of the vessel volume actually used for holding packaged product, primarily a factor of package shape) determines the throughput of the system. In a commercial situation, with this sort of batch process, a short holding time under pressure is desirable in order to maximize throughput of product. If the product is pumpable, it may be advantageous to pump it into and out of the processing vessel through special high-pressure transfer valves and isolators. To package the product after treatment, additional systems, such as an aseptic filling station, are then required (Ting and Marshall, 2002). Current semi-continuous systems for treating liquids use a pressure vessel with a free piston to compress liquid foods. A low-pressure food pump is used to fill the pressure vessel and, as the vessel is filled, the free piston is displaced. When filled, the inlet port is closed and high-pressure process water is introduced behind the free piston to compress the liquid food. After an appropriate holding time, releasing the pressure on the high-pressure process water decompresses the system. The treated liquid is discharged from the pressure vessel to a sterile hold tank through a discharge port. A low-pressure water pump is used to move the free piston towards the discharge port. The treated liquid food can be filled aseptically into pre-sterilized containers. A semi-continuous system with a processing capacity of 600 l/h of liquid food and a maximum operating pressure of 400 MPa is used commercially to process grapefruit juice in Japan. Multiple units can be sequenced so that, while one unit is being filled, others are in various stages of operation (Palou et al., 2002). For any HP system, the working pressure is a very important parameter, not only because the initial price of the equipment increases significantly with its maximum

Principles of high pressure processing 7

5000 kg

5000 kg

1 cm2 (⫽1000 MPa) Figure 1.2

A 10 000 kg weight on 1 cm2 equals a pressure of 1000 MPa (Source: www.relayresearch.ie).

working pressure, but also because a decrease in working pressure can reduce significantly the number of failures, increasing the working life of the equipment (Otero et al., 2000). Pressures anywhere between 50 and 1000 MPa are commonly used. To put that into perspective, two 5000 kg elephants balancing on a 1 cm2 area will produce a pressure force of 1000 MPa, as illustrated in Figure 1.2. Keeping the sample under pressure for extended periods of time does not require any additional energy (Cheftel and Culioli, 1997). The work of compression during HP treatment will increase the temperature of foods through adiabatic heating, by approximately 3°C per 100 MPa, depending on the composition of the food (Balasubramanian and Balasubramaniam, 2003). For example, if the food contains a significant amount of fat, such as butter or cream, the temperature rise can be larger. Foods cool down to their original temperature on decompression if no heat is lost to, or gained through, the walls of the pressure vessel during the hold time at pressure.

2.3 Process principles There are two general scientific principles of direct relevance to the use of high pressures in food processing. The first is Le Chatelier’s Principle, which applies to all physical processes and states that, when a system at equilibrium is disturbed the system responds in a way that tends to minimize the disturbance (Pauling, 1964). This means that HP stimulates reactions that result in a decrease in volume but opposes reactions that involve an increase in volume. Any phenomenon (e.g. phase transition, change in molecular configuration, chemical reaction) that is accompanied by a decrease in volume will be enhanced by pressure. Secondly, the Isostatic Rule states that pressure is instantaneously and uniformly transmitted throughout a sample under pressure, whether the sample is in direct contact

8 High Pressure Processing of Foods: An Overview

with the pressure medium or hermetically sealed in a flexible package that transmits pressure (Olsson, 1995). Pressure is transmitted in a uniform (isostatic) and quasiinstantaneous manner throughout the sample; the time necessary for pressure processing is therefore independent of sample size, in contrast to thermal processing.

2.4 Packaging requirements In batch-wise HP processing systems, the product is generally treated in its final primary package; commonly, the food and its package are treated together and so the entire pack remains a ‘secure unit’ until the consumer opens it. When considering new technologies involving the treatment of packaging materials, it is very important to study the safety of the material, the possible formation of compounds that influence the odour and taste of the food and the effects of pressure on mechanical and physical properties of the packaging material, e.g. strength and barrier properties. HP processing requires airtight packages that can withstand a change in volume corresponding to the compressibility of the product (Hugas et al., 2002), as foods decrease in volume as a function of the pressure applied, while an equal expansion occurs on decompression. For this reason, the packaging used for HP treated foods must be able to accommodate up to a 15 per cent reduction in volume and return to its original volume without loss of seal integrity or barrier properties. Therefore, selection of packaging materials is very important. Plastic films are generally accepted for HP processing, although they are frequently not suitable for high temperature processing. On the other hand, metal cans and glassware are generally not suitable for HP treatment. Packaging materials which are oxygen-impermeable and opaque to light may be developed especially for keeping fresh colour and flavour of certain HP-treated foods (Hayashi, 1995). In production, the use of flexible pouches can achieve high packing ratios; the use of semi-rigid trays is also possible; vacuum-packed products are ideally suited for HP. Since the size and shape of the product will have major effects on the stacking effectiveness of the product carrier, they must be optimized for the most cost-effective process; it is obviously uneconomical to treat empty space. Glass bottles or gable cartons can be used for HP-processed foods if filling is performed after exposure to pressure. This further allows innovative package shapes and printing graphics (Ting and Marshall, 2002). A critical control point in the food industry is the handling of certain products after processing; improper handling can lead to products becoming re-contaminated before packaging. Not even with strict hygienic measures is the risk of microbial recontamination, mainly through personnel and equipment, completely eliminated, unless completely aseptic packaging is used. Thus, subsequent processing is frequently necessary to guarantee the safety and shelf-life of the product. At present, heating after vacuum packaging is the most common process carried out for this purpose; however, this requires the use of expensive heat-resistant packages. This heat treatment step could in principle be replaced by HP treatment (Yuste et al., 2000); this would be especially suitable for foods with attributes and properties that are heat-sensitive.

Use of high pressure to improve food safety and stability 9

Table 1.1 Some commercially-available HP-processed food products (adapted from Leadley et al., 2003) Product

Manufacturer

Country

Jams, fruit sauces, yoghurt and jelly Mandarin juice Tropical fruits Beef Guacamole, salsa dips, ready meals and fruit juices Hummus Fruit and vegetable juices Ham Processed poultry products Oysters Oysters Oysters Orange juice Fruit juices Apple juice Sliced ham and tapas Fruit juices and smoothies

Meida-Ya Wakayama Food Industries Nishin Oil Mills Fuji Ciku Mutterham Avomex

Japan Japan Japan Japan USA

Hannah International Odwalla Hormel Foods Purdue Farms Motivatit Seafoods Goose Point Oysters Joey Oysters Ultifruit Pampryl Frubaca Espuña Orchard House

USA USA USA USA USA USA USA France France Portugal Spain UK

2.5 Current commercial status of high pressure processing HP processing can be applied to a wide range of different foods, including meat-based products (cooked and dry ham, etc.), fish, pre-cooked dishes and fruit, vegetables and juices. The main applications today are in the production of jams, fruit juice, soups, oysters and, more recently, processed meats such as hams. Table 1.1 lists commercial food products available which are processed by high pressure. The product range is increasing and spreading from its origins in Japan, followed by the USA and now Europe.

3 Use of high pressure to improve food safety and stability The effectiveness of any food preservation technique is primarily evaluated on the basis of its ability to eradicate pathogenic microorganisms present and so to enhance the product’s safety; a secondary objective is inactivation of spoilage microorganisms to improve the shelf-life of the food (McClements et al., 2001). Growth of microorganisms in foods can cause spoilage by producing unacceptable changes in taste, odour, appearance and texture. Microorganisms are a heterogeneous group of organisms, different members of which are capable of growth at temperatures from well below freezing (extreme psychrophiles) to temperatures above 100°C (extreme thermophiles). However, each species has a particular temperature range in which it can grow best; this range is determined

10 High Pressure Processing of Foods: An Overview

largely by the influence of temperature on cell membranes and enzymes, and growth is restricted to those temperatures at which cellular enzymes and membranes can function. As with heat, large differences in pressure resistance can be apparent among various strains of the same species. HP treatment is also known to cause sublethal injury to microbes, which is a particularly important consideration for any preservation method. Given favourable conditions, such as prolonged storage in a suitable substrate, sublethally injured cells may be able to recover (McClements et al., 2001). On the other hand, cell death is associated with irreversible damage to cell components essential for cell growth and reproduction.

3.1 Effect of high pressure on microorganisms Microbial inactivation by HP has been extensively studied and has been concluded to be the result of a combination of factors. The primary site for pressure-induced microbial inactivation is the cell membrane (e.g. modifications in permeability and ion exchange) (McClements et al., 2001). Microorganisms are resistant to selective chemical inhibitors due to their ability to exclude such agents from the cell, mainly by the action of the cell membrane; however, if the membrane becomes damaged, this tolerance is lost. In addition, HP causes changes in cell morphology and biochemical reactions, protein denaturation and inhibition of genetic mechanisms. Other mechanisms of action which may be responsible for microbial inactivation include the denaturation of key enzymes and the disruption of ribosomes (Linton and Patterson, 2000). Different microorganisms react to high pressure treatment with different degrees of resistance. Table 1.2 summarizes the pressure required to achieve a 5-log inactivation ratio for various microorganisms, for a treatment duration of 15 minutes.

3.1.1 Bacteria

Bacteria are relatively simple, single-celled organisms and are among the smallest freeliving organisms known. The main bacteria that cause food poisoning are Campylobacter

Table 1.2 Pressures required to achieve a 5-log cycle inactivation ratio for certain microorganisms, for a 15-minute treatment (Patterson et al., 1995) Microorganism

Pressure (MPa)

Yersinia enterocolitica Salmonella typhimurium Listeria monocytogenes Salmonella enteritidis Escherichia coli O157:H7 Staphylococcus aureus

275 350 375 450 680 700

Use of high pressure to improve food safety and stability 11

spp., Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Vibrio spp. Among these, Listeria monocytogenes and Staphylococcus aureus are probably the two most intensively studied species in terms of use of HP processing. Listeria monocytogenes is a Gram-positive rod that is an important pathogen in acidified and other foods, such as dairy products and ready-to-eat meats. As a foodborne bacterium L. monocytogenes requires particular care for processing and storage because it is moderately heat resistant and can grow anaerobically under refrigeration. Staphyloccocus aureus appears to have a high resistance to pressure (Erkmen and Karatas, 1997). E. coli O157:H7 also has a high barotolerance and is considered to be an important pathogen that can cause serious illness (Linton et al., 2001). Outbreaks of food poisoning due to E. coli O157:H7 have been associated with a range of foods, including ground beef (Doyle, 1991), and raw and skimmed milk (Garcia-Graells et al., 1999; Linton et al., 2001); it has also been isolated from pork, lamb and poultry (Doyle, 1991; Patterson and Kilpatrick, 1998). In addition, high-acid foods, such as apple cider (Besser et al., 1993), mayonnaise (Weagent et al., 1994) and yoghurt (Morgan et al., 1993) have also been implicated in E. coli outbreaks. Microbiological safety can be assured in many, if not all, of the products mentioned here if they are processed using HP. Treating food samples using HP can destroy both pathogenic and spoilage microorganisms; however, there is a large variation in the pressure resistance of different bacterial strains and the nature of the medium can even effect the response of microorganisms to pressure. Table 1.3 compares the different pressures required to inactivate two different bacteria at various temperatures, in milk and poultry meat. The stage of growth of the bacteria is also important in determining pressure resistance, with cells in the stationary phase being more resistant than those in the exponential phase (McClements et al., 2001). Also, Gram-positive and Gram-negative bacteria differ significantly in terms of the chemical structure of their cell walls. The cell walls of Gram-negative bacteria are significantly weaker and Gram-negative bacteria consequently tend to be more pressure-sensitive than Gram-positive bacteria (Patterson and Kilpatrick, 1998).

Table 1.3 Predicted treatment pressure required at various temperatures for a 5-log10 inactivation of Escherichia coli and Staphylococcus aureus in poultry meat and milk, for a treatment time of 15 minutes (Patterson and Kilpatrick, 1998) Temperature (°C) during pressure treatment

Estimated pressure (MPa) Escherichia coli

10 20 30 40 50 60

Staphylococcus aureus

Poultry meat

UHT milk

Poultry meat

UHT milk

850 779 681 544 371 125

1014 966 840 638 392 133

647 694 735 701 524 177

749 625 602 583 478 196

12 High Pressure Processing of Foods: An Overview

Further work is required to understand more fully the factors that can affect the response of microorganisms, including pathogens, to pressure so that treatments can be optimized and microbiological safety can be assured. 3.1.2 Bacterial spores

The elimination of bacterial endospores from food probably represents the greatest food processing and food-safety challenges to the industry. It is well established that spores are the most pressure-resistant life forms known; in general, only very high pressures (⬎800 MPa) can kill bacterial spores around ambient temperatures. Alternatively, other processing methods, applied in combination with HP, can be effective for elimination of bacterial spores, by achieving a synergistic or hurdle effect. In particular, HP treatment at elevated temperatures (e.g. HP treatment at up to 90°C) is very effective in the elimination of bacterial spores in foods. Pressureinduced inactivation of bacterial spores is also markedly enhanced at temperatures of 50–70°C and perhaps also at or below 0°C. The most heat-resistant pathogenic bacterium is Clostridium botulinum and spores of C. botulinum are also among the most pressure-resistant microorganisms known. Among other spore-forming bacteria of concern, Bacillus cereus has been widely studied because of its anaerobic nature and very low rate of lethality. Bacillus cereus is a spore-forming food-borne pathogen, which is ubiquitous in nature and hence occurs frequently in a wide range of food raw materials. It is recognized as a leading cause of bacterial food poisoning, with a variety of proteinaceous and starchy foods being implicated (Van Opstal et al., 2004). An alternative to using treatments combining heat and pressure for enhanced killing of bacterial spores is first to cause bacterial spore germination and then use HP to kill the much more pressure-sensitive vegetative cells. Germination is the process by which a dormant spore changes into a vegetative cell. Interestingly, bacterial spores can be stimulated to germinate by treatment at relatively low pressures, e.g. 50–300 MPa; germinated spores can then be killed by relatively mild heat treatments or higher pressure treatments (Smelt, 1998). Process temperatures in the range of 80–110°C in conjunction with pressures of about 600 MPa have been used to inactivate spore-forming bacteria such as B. cereus (Van Opstal et al., 2004). Cycling treatments, where spores are exposed to alternating low and high pressures, or alternating cycles of pressurization and depressurization, are also of interest for sterilization processes. A matrix of conditions for inactivation of spores of Bacillus and Clostridia was presented by Meyer et al. (2000). The mode of action of HP on bacterial spores is still largely a matter of speculation. 3.1.3 Fungi

Fungi can be divided into two groups based on their vegetative structures: unicellular fungi (yeasts) and those producing hyphae (moulds, mushrooms, etc.). Vegetative bacterial cells, yeasts and moulds are, in general, more susceptible to pressure than bacterial spores; thus, they can be inactivated using relatively low pressures. Yeasts are simply single-celled fungi that reproduce by budding or fission. The group includes members of the ascomycetes and imperfect fungi. Yeasts are an important

Use of high pressure to improve food safety and stability 13

group of spoilage microorganisms, but are generally not food pathogens, although toxic mould growth may be a safety concern in foods. Treatment at pressures less than 400 MPa for a few minutes is sufficient to inactivate most yeasts. Smelt (1998) reported that, at about 100 MPa, the nuclear membrane of yeasts was affected and that at more than 400–600 MPa further alteration occurred in the mitochondria and the cytoplasm. Moulds are mycelial fungi and many of these organisms are important industrially, e.g. in food spoilage, food fermentations and biodegradation processes. Pressures between 300 and 600 MPa can inactivate most moulds (Smelt, 1998). O’Reilly et al. (2000) demonstrated that HP was effective for inactivation of Penicillium roqueforti spores in cheese systems. 3.1.4 Viruses

Viruses are very different from other groups of microorganisms in terms of their structure and the way in which they function; there is also considerable diversity within the virus family. With the exception of nucleic acid, viruses do not have the structures that one normally associates with living cells; they simply consist of a protein coat, called a capsid, made up of a number of protein subunits (capsomeres) that enclose a central core of nucleic acid. Viruses may also contain a small number of enzymes required for the infection of host cells. Among viruses there is a high degree of structural diversity and this is reflected in a wide range of pressure resistances (Smelt, 1998). The most common human enteric viruses are Norwalk-like viruses (SRSVs), hepatitis A, rotavirus and human astrovirus. Complete inactivation of suspensions of feline calicivirus (a Norwalk-like virus surrogate), adenovirus, and adenovirus and hepatitis A can be achieved by treatment at 275 MPa for 5 minutes (Kingsley et al., 2002), 400 MPa for 15 minutes (Wilkinson et al., 2001) and at 450 MPa for 5 minutes (Kingsley et al., 2002), respectively. In contrast, several studies have demonstrated the remarkable baroresistance of poliovirus (Nakagami et al., 1992; Oliveira et al., 1999; Wilkinson et al., 2001; Kingsley et al., 2002). Foot and mouth disease virus was reduced by 102.9 plaque-forming units (PFU) by treatment at 220 MPa for 1 h (Kingsley et al., 2002). The mode of inactivation of viruses by high pressure has not been fully elucidated, although the viral envelope, when present, appears to be one target for HP inactivation. Treatment at pressures above 300 MPa damages the envelopes of human immunodeficiency virus (HIV) and cytomegalovirus (CMV), preventing the binding of virus particles to cells (Nakagami et al., 1992). Pressure can also cause the dissociation of virus particles; depending on the virus and the treatment conditions, pressure-induced dissociation may be fully reversible or irreversible (Da Poian et al., 1994). High pressure can also induce minor changes in viral structures without disassembling the whole particle (Gaspar et al., 2002). The formation of non-infectious particles after HP treatment has been observed for many viruses, including rotavirus (Pontes et al., 2001), HIV (Nakagami et al., 1996), lambda phage (Bradley et al., 2000) and picornaviruses (Oliveira et al., 1999). 3.1.5 Prions

Prions are associated with certain neurological disorders, including bovine spongiform encephalopathy (BSE) in cattle and Creutzfeld-Jakob disease (CJD) in humans.

14 High Pressure Processing of Foods: An Overview

In general, prions are even more difficult to destroy than bacterial spores, with certain prions surviving autoclaving at 134°C (Taylor, 1999). Recently, it was reported that high pressure treatment of prion-contaminated meat at 690–1200 MPa and 121–137°C reduced the infectivity of the prions therein (Brown et al., 2003). Such treatments may have the advantage of ensuring safety of samples without the excessive damage that may be associated with autoclaving alone.

3.2 Factors influencing microbial sensitivity to high pressure As stated already, the pressure resistance of microorganisms varies considerably, depending on factors such as species, strain, stage of growth and food composition. Factors that can affect the response of microorganisms, including pathogens, to pressure must be considered so that treatments can be optimized and microbiological safety can be assured. 3.2.1 pH

The likelihood that a particular organism will grow in a particular food depends on the interaction between all the factors that influence the growth of microorganisms, including competition between species. The pH of the food is one of the main factors affecting the growth and survival of microorganisms; all microorganisms have a pH range in which they can grow and an optimum pH at which they grow best. The pH of a food, if not optimal for a particular species, can thus not only enhance inactivation during treatment but also inhibit outgrowth of sublethally injured cells. Bacterial spores are generally most resistant to the direct effects of pressure treatment at neutral pH (Smelt, 1998). Extents of pressure-induced inactivation will generally be enhanced and recovery of sublethally injured cells inhibited, for most species, at acidic pH values. For example, the pressure resistance of E. coli O157:H7 in orange juice is dependent on the pH of the juice, the degree of inactivation increasing as pH decreases; survival of E. coli O157:H7 in orange juice during storage is also dependent on pH (Linton and Patterson, 2000). Compression of foods during HP treatment may shift the pH of the food as a function of applied pressure; the direction of pH shift and its magnitude must be determined for each food treatment process. 3.2.2 Water activity (aw) Water in the liquid state is essential for the existence of all living organisms. The amount of water available for microbial growth is generally expressed in terms of the water activity (aw) of the system. Lowering the water activity of a food can significantly influence the growth of food spoilage or food-poisoning organisms that may be present in the raw materials or introduced during processing; this is the principle of the very old method of food preservation by drying. Reducing the aw appears to protect microbes against inactivation by HPP; however, on the other hand, recovery of sublethally injured cells can be inhibited by low aw (Smelt, 1998). Consequently, the net effect of water activity on microbial inactivation by HP treatment may be difficult to predict.

Use of high pressure to improve food safety and stability 15

3.2.3 Temperature, pressure and holding time

Increasing treatment pressure, holding time, or temperature will generally increase the number of microorganisms inactivated (with bacterial spores being the important exception). While many HP processes are performed at ambient temperature, increasing or, to a lesser extent, decreasing temperature has been found to increase the inactivation rate of microorganisms during HP treatment (Kalchayanand et al., 1998). Temperatures above 45–50°C increase the rate of inactivation of food pathogens and spoilage microbes (Palou et al., 2002). The use of high temperatures for food processing, the advantages of which for inactivation of spores have been discussed earlier, is complicated by the fact that the large steel cylinders in which the food is held during treatment are very slow to change in temperature and that the food itself can undergo a significant increase in temperature during processing due to adiabatic heating. Temperature increases due to adiabatic compressions can be 3°C or more per 100 MPa and, while these increases in temperature are generally transient, in some processes use of sample insulation may retain this heat and add to the thermal dimension of the processing conditions (Ting et al., 2002). The choice of processing temperature will also influence the selection of suitable pressure-transmitting media. There is a minimum critical pressure below which microbial inactivation by HP will not take place regardless of process time. Important processing parameters to be considered in HPP are the come-up times (period necessary to reach treatment pressure) and pressure-release times. Obviously, long come-up times will add appreciably to the total process time and affect the product throughput, but these periods will also affect inactivation kinetics of microorganisms. Therefore, consistency and control of these times are important in HP process development.

3.3 High pressure regulations Today, in most countries, food safety is tightly controlled by regulation and is a sine qua non. While many of the factors and microorganisms that can present hazards to the consumer are known and have been intensively studied, new and emerging pathogens not previously regarded as problematic continue to be identified. As already discussed, processors must also increasingly balance the need for assurance of food safety against consumer demand for minimally-processed products. For these reasons, emerging technologie such as HP are of considerable interest and potential benefit to the food industry. However, before the implementation of new preservation technologies, several issues need to be addressed, such as the mechanisms of microbial resistance and adaptation to these new technologies, the mechanisms of microbial and enzyme inactivation, the identification of the most resistant and relevant microorganisms in every food habitat, the role of bacterial stress, the robustness of the technologies, the increased safety relative to existing technologies and, last but not least, the legislation needed to implement them (Hugas et al., 2002). Two regulatory attitudes towards commercialization of food products manufactured using HP have emerged, i.e. within the EU or outside. In countries outside the EU, there is currently no specific legislation applicable to HP treatment. In the USA,

16 High Pressure Processing of Foods: An Overview

for example, the traditional health regulations are applied and products treated by HP, such as guacamole and oysters, have already been introduced to the market without any specific regulation. In EU countries, however, national regulations for new products have been replaced, in the application of the precautionary principle, by a community regulation for novel foods and ingredients (Regulation 258/97/EC), which has been in force since 1997. This ‘Novel Foods’ legislation establishes an evaluation and licensing system that is compulsory for new foods and new processes. HP-processed food products are novel foods since they fulfill two conditions: their history of human consumption has so far been negligible and, secondly, a new manufacturing process has produced them. In July 2001, after the last meeting of the EU commission in charge of ‘novel foods’ several decisions were taken to simplify the regulations. Specifically, if it is possible to show that the new product (e.g. the HP-treated food) is substantially equivalent to a product already on the market, then the product can be treated at a national regulation level and will not need to comply with the ‘novel food’ regulation (Hugas et al., 2002). All new pressure vessels to be used in the EU have to comply with the ‘Pressure Equipment Directive’ (PED) regulation, which came into force in 2002. This regulation is an extension of the ‘CE’ safety standard already employed in the EU and now recognized world-wide. As pressure vessels of all types utilize potentially hazardous energy, the PED regulation seeks to identify good design, good manufacturing practices and detailed safety assessment for safe operation and maintenance of the vessels and auxiliary parts.

4 Effects of high pressure on food quality While food safety and shelf-life are often closely related to microbial quality, other phenomena such as biochemical reactions, enzymatic reactions and structural changes can significantly influence consumers’ perception of food quality (Le Bail et al., 2003). Conventional thermal sterilization processes involve extensive slow heat penetration to the core (cold point) of the product and subsequent slow cooling. This thermal process induces changes in product quality to an extent dependent on the product being treated and the temperatures reached; these may include off-flavour generation, textural softening and destruction of colours and vitamins. As stated already, unlike heating, HP treatment at moderate pressures generally does not change the odour, flavour or other sensory characteristics of foods. Therefore, HP processing offers the food industry a technology that can achieve the food safety properties of heat-treated foods while meeting consumer demand for freshertasting foods. In order to select the most suitable processing conditions for a particular food product, sensory characteristics must be taken into account (Polydera et al., 2003). Increasing treatment pressures will generally increase microbial inactivation in shorter times, but higher pressures may also cause greater levels of protein denaturation and other potentially detrimental changes in food quality that could affect the appearance and texture of the food, compared to the unprocessed product.

Effects of high pressure on food quality 17

4.1 Effect of high pressure on food colour The importance of colour in consumer acceptance is well-known. For some food products, depending on the pressure-time exposure, some degree of protein denaturation can take place during HP treatment. This can result in a change in physical functionality and/or changes in colour relative to raw products. For fresh meat, poultry and related products, pressure-induced colour modifications greatly depend on treatment conditions (pressure, time and temperature) and are due to changes in myoglobin, such as denaturation, heme displacement or release and ferrous atom oxidation (Mor-Mor and Yuste, 2003). Therefore, the application of HP to fresh meat products can result in a cooked-like appearance and the products may sometimes develop a rubbery consistency (Hugas et al., 2002). For semi-cooked or cooked products, this effect is not observed, since their proteins have already been denatured (Ting and Marshall, 2002). Due to the effects of HP treatment on raw red meat colour, the final products have a cooked appearance and cannot be sold as fresh meat. Packaging of meat under vacuum with an oxygen scavenger partly protects the meat colour. However, in practice, HP processing of fresh red meat cannot be envisaged unless subsequent (or simultaneous) cooking is done before the final product is presented for sale and consumption. This would be the case if pre-cooked ready meals were to be HP-treated. In contrast, HP processing of cured meats or white meat (or fish) is unlikely to cause any serious colour problems (Cheftel and Culioli, 1997).

4.2 Effect of high pressure on food texture Knowledge of the rheological and/or textural properties of food products is essential for product development, quality control, sensory evaluation and design and evaluation of process equipment (Ahmed et al., 2003). The physical structure of most high-moisture products remains unchanged after exposure to HP, since no shear forces are generated by pressure. For gas-containing products treated under HP, the colour and texture may be changed due to gas displacement and liquid infiltration. Physical shrinkage can occur due to mechanical collapse of air pockets and shape distortion may be related to anisotropic behaviour. For foods not containing air voids, HP frequently results in minimal or no permanent change in textural characteristics (Ting and Marshall, 2002). For certain food products, HP has an enormous potential as a technique to modify the texture. If required, HP treatments can induce desirable changes in product texture and structure and, accordingly, can be used for the development of new products or to increase the functionality of some ingredients (Hugas et al., 2002). For example, it was recently reported that HP treatment of Mozzarella cheese significantly accelerated the development of desirable functional properties on melting (O’Reilly et al., 2002). HP processing is increasingly being used in the production of surimi and kamaboko, traditional Japanese products made from fish mince, due to the superior quality of pressure-induced gels (Okamoto et al., 1990; Ohshima et al., 1993; Ashie and Simpson, 1996). The differences in structure of heat- and HP-treated gels are attributed to the different mechanisms of gelation induced by the differential effects of pressure and

18 High Pressure Processing of Foods: An Overview

heat on various bonds (Angsupanich and Ledward, 1998; Angsupanich et al., 1999; Gudmundsson and Hafsteinsson, 2002). HP-induced fish gels are described as glossy and soft, with a smoother and more uniform texture than gels produced by heat treatment; they also retain the colour and flavour of the untreated fish. In addition, HP can induce the gelation of sarcoplasmic proteins, usually lost during the traditional production process for surimi, providing the opportunity for their inclusion in the preparation of surimi and related products (Ohshima et al., 1993).

4.3 Effect of high pressure on food sensory quality One of the most frequently cited benefits of HP processing relative to other preservation methods is the possibility of increasing shelf-life while still retaining the sensory characteristics of fresh food products. For example, Hugas et al. (2002) stated that panellists could not distinguish between meat products treated with heat and/or HPP and untreated controls in a sensory evaluation. An example is shown in Table 1.4 listing results from a sensory analysis of sausages. Generally speaking, HP-treated sausages were considered more cohesive and less firm than heat-treated sausages. In some cases, the sensory panel did not detect differences between both types of sausages; when differences were detected, HP-treated samples were preferred on the bases of appearance, taste and especially texture. Palou et al. (2002) also reported that HP treatments could preserve delicate sensory attributes of avocado and assure a reasonable safe and stable shelf-life. Avocados are used in the preparation of guacamole, which was one of the first HP-treated food products to be commercialized in the USA. Furthermore, high pressure has also been used to preserve fruit juices. Table 1.5 shows that the orange juice HP-treated at 500 MPa for 5 minutes apparently has an extended shelf-life as compared with products treated by thermal pasteurization at 80°C for 30 seconds. HP treatment of meat and fish can result in increased oxidation, probably due to the release of free metal ions into the tissue; oxidation, if not controlled, can negatively affect the colour and flavour of such products (Cheah and Ledward, 1996, 1997; Angsupanich and Ledward, 1998). Although HP treatment does not substantially alter the taste of fish fillet tissue, pressures above 300 MPa give the product an opacity similar to that obtained by a very light cooking (Hoover et al., 1989).

4.4 Effect of high pressure on food yield HP treatment may affect the yield of certain products, a very important economic issue for food manufacturers (Mor-Mor and Yuste, 2003). While effects on yield depend on the type of product and the intensity of treatment, HP treatment can give a higher yield in food products than heat treatment. For example, Mor-Mor and Yuste (2003) reported that weight loss was significantly higher in heat-treated sausages than in HP-treated control samples; HP treatment also helped to prevent any sour taste, off-flavours, ropiness and colour changes (Hugas et al., 2002).

Effects of high pressure on food quality 19

Table 1.4 Results from a comparative sensory study of heat-treated (80–85°C for 40 min) and high pressure-treated (500 MPa at 65°C) sausages (Mor-Mor and Yuste, 2003) Triangle test

Correct judgements

Subject preferences

Heat-treated versus pressurized for 5 min

16

Heat-treated versus pressurized for 15 min

22

Pressurized ⫽ 8 No preference ⫽ 5 Heat-treated ⫽ 3 Pressurized ⫽ 11 No preference ⫽ 5 Heat-treated ⫽ 6

Table 1.5 Shelf-life comparison of orange juice based on sensory evaluation (Polydera et al., 2003) Storage temperature (°C)

0 5 10 15

Shelf-life (days) High-pressure treated (500 MPa/5 min/35°C)

Thermally pasteurized (80°C for 30 s)

⬎90 ⬎90 47 32

60 47 25 16

HP processing also has an important additional advantage for oyster processors. A large proportion of oysters are sold ‘on the half-shell’ or ready shelled, traditionally necessitating the shucking of oysters by hand. This requires a skilled workforce, as the process is hazardous and inexpert handling can damage the oyster meat, reducing the quality and appearance of the finished product. During HP treatment, the adductor muscle of oysters detaches from the shell; HP treatment at 241 MPa for 2 min caused detachment of adductor muscle in 88 per cent of oysters, while treatment at 310 MPa, with immediate pressure release, resulted in 100 per cent efficiency of shucking (He et al., 2002). The fitting of oysters with heat-shrinkable plastic bands before treatment holds the shells together and reduces the loss of intervalval fluid. Oysters treated in this way obviously do not gape and have proved an attractive alternative to traditional live oysters. In addition to reduced labour cost and risks and increased safety and shelf-life of oysters, yield increases of 25–50 per cent using HP processing are usual (Motivatit Seafoods Inc, personal communication). In conclusion, both heat treatment and HP processing inactivate microorganisms, denature proteins and extend the shelf-life of food products. However, heat treatment often achieves this at the expense of quality attributes, e.g. colour, flavour or nutrient levels. HP treatment, in contrast, maintains the quality of fresh foods, with few direct effects on flavour and little effect on nutritional quality. HP processing can also potentially modify the functional properties of food constituents (e.g. proteins) and even increase the yield of certain food products.

20 High Pressure Processing of Foods: An Overview

5 Other applications of high pressure Water has many physical and chemical properties that are significantly affected by pressure (Otero et al., 2002). Pressure opposes reactions associated with volume increase, such as freezing of water at atmospheric pressure. This forms the basis of a new field of HP food applications, such as high-pressure freezing, thawing and storage of food at temperatures below 0°C without freezing. Recently, the effect of pressure on the water–ice equilibrium has attracted the attention of many food technologists and engineers studying the freezing and thawing of foods. This area was reviewed in detail in Denys et al. (2002).

5.1 High pressure freezing applications Freezing has long been established as an excellent method for preserving food products, with the potential to allow high retention of food quality over long storage periods. Generally, freezing preserves the taste, texture and nutritional value of foods better than any other preservation method; as a result, ever-increasing quantities of food are being frozen throughout the world. The main potential disadvantage of freezing foods is the risk of damage caused by the formation of ice crystals; the formation of ice crystals mechanically damages cell structures in tissue-derived food products (e.g. fruit, vegetables, meat) by puncturing cell walls and distorts the tissue structure, as well as inducing protein denaturation. The size and location of ice crystals formed during freezing depend on the rate of freezing and the final temperature of the process and affect important quality parameters such as texture and colour of the frozen products and exudation of moisture (drip loss) on thawing (Martino et al., 1998). It is well-known that a fast freezing rate results in a fine ice structure, with intensive nucleation and the formation of high numbers of small ice crystals, which causes less damage to the structure of the product than slow freezing rates that favour the formation of large ice crystals; rapid freezing of food is thus preferred (Thiebaud et al., 2002). In the past few years, HP processes in which phase transitions take place, such as pressure shift freezing (PSF), have attracted the attention of many researchers. In traditional freezing processes (such as air blast, plate contact or cryogenic systems), when food comes into contact with the refrigerating medium, ice nucleation occurs in the region next to the refrigerated border and is controlled by the magnitude of supercooling reached in this zone. Supercooling (the difference between the actual temperature of the sample and the expected solid–liquid equilibrium temperature at a given pressure) is the driving force for ice nucleation and is an important parameter that controls the size and number of ice crystals formed (Chevalier et al., 2000). Burke et al. (1975) stated that, for each degree K of supercooling, there is an increase of about tenfold in the ice nucleation rate. The thermal gradient existing between an interior point in a food product and the surface determines to a great extent the local cooling/freezing rate of the sample. The freezing rate decreases towards the centre of the product and is particularly important in large volume products (Sanz et al., 1999). In inner regions of

Other applications of high pressure 21

40

Temperature (⬚C)

30 20 10 0

Liquid

Ice VI

⫺10 Ice V

Ice I

⫺20

Ice III

⫺30 0

100

200

300

400

500

600

700

800

900

1000

Pressure (MPa) Figure 1.3

Phase diagram of water.

products being frozen, because of the thermal gradients, supercooling to produce ice nucleation may not be achieved, resulting in the growth of undesirably large ice crystals. Once phase changes of water under pressure are fully understood, HP can potentially greatly aid the freezing process and improve retention of product quality. The effects of pressure on the phase diagram of water are thus of great potential interest and are shown in Figure 1.3 (Kalichevsky et al., 1995). For example, water can exist in a non-frozen, liquid state at temperatures well below 0°C at certain pressures, e.g. the freezing point of water exhibits a minimum temperature of ⫺22°C at 207.5 MPa. Releasing pressure rapidly can achieve a very high supercooling and, as a result, a very high ice nucleation rate. The type of ice formed during the freezing of foods is a major factor affecting the quality of the product. Today, nine solid ice phases are known, each one prevailing in a distinct area in the pressure–temperature phase diagram of water. When water is frozen at atmospheric pressure, its volume increases, with the formation of ice I which, uniquely among water ice types, has a lower density than that of liquid water, resulting in a volume increase of about 9 per cent on freezing at 0°C and about 13 per cent at ⫺20°C (Kalichevsky et al., 1995). This increase in volume is a primary cause of tissue damage during freezing. However, under pressure, several different ice types (ice II to IX) with different chemical structures and physical properties to those of ice I are formed (Figure 1.3). The densities of the types of ice that are formed under pressure are higher than that of water and these ices do not expand in volume during phase transition, which reduces tissue damage. The principle of pressure shift freezing (PSF) involves reducing the temperature of a food sample, held in an HP vessel whose temperature is regulated at sub-zero temperatures, to below 0°C under pressure. The food sample is typically cooled to ⫺20°C at 200 MPa, at which pressure the water remains in a liquid state. The vessel is then depressurized, the pressure rapidly returns to atmospheric pressure and the sample undergoes a sudden temperature rise to the phase change temperature at the current pressure; partial freezing is initiated during this phase due to high supercooling of the

22 High Pressure Processing of Foods: An Overview

sample. The temperature increases according to the temperature–pressure equilibrium relationship of liquid water and ice I (Chevalier et al., 2000). Sanz et al. (1997) showed that the ratio of ice:water can reach 0.36 for a sample of pure water at the end of the pressure release step, indicating that only partial freezing can be obtained during PSF. Freezing must thereafter be completed at atmospheric pressure. The main advantage of PSF is that ice formation is instantaneous and homogeneous throughout the whole volume of the product because of the high degree of supercooling reached on release of pressure. Therefore, PSF can be especially useful for freezing of foods with large dimensions in which a uniform ice crystal distribution is required and where, in classical freezing processes, thermal gradients would be pronounced and damage during freezing would occur. From a micro-structural viewpoint, damage to cells is minimized because of the small size of ice crystals, resulting in a significant improvement in product quality.

5.2 High pressure thawing Thawing generally occurs more slowly than freezing, potentially allowing further damage to food products. Therefore, the thawing of frozen food products is a particularly important stage of the handling of frozen food products, in particular in terms of minimizing the amount of proteinaceous exudate (drip) lost from many food products (e.g. meat, fish) on thawing. The loss of such fluid generally reduces the eating quality, binding ability and weight of food, all factors contributing to its quality and value. The volume of drip produced on thawing has been shown to be closely related to the rate of freezing (Kalichevsky et al., 1995), which in turn is related to the size and location of ice crystals in frozen foods (Chevalier et al., 2000). To prevent microbial growth, the temperature for this stage should be less than 4°C; under such conditions, thawing times are very long, and processes to reduce this time are consequently of great interest. Recently, a promising route involving, once again, the effect of pressure on the melting point of water, has been identified. This HP method permits not only control of the thawing time, but also control of the ice front propagation dynamics, which presents significant advantages for maintaining food quality. When HP treating a frozen sample to induce thawing (‘high-pressure thawing’), the transition to the non-frozen state occurs at a high pressure and an introduction of a pressure-related latent heat seems necessary (Denys et al., 2000). Preliminary studies have revealed that thawing under HP preserves food quality and reduces thawing times (Denys et al., 2000). Knorr et al. (1998) and Otero and Sanz (2003) distinguished between processes of pressure-assisted and pressure-induced thawing. In the former, the phase transition (ice to water) occurs at constant pressure by increasing the temperature, while in the latter the phase change is initiated by a pressure change and proceeds at constant pressure.

5.3 High pressure non-frozen storage From the phase diagram of water (see Figure 1.3), it is clear that there is a non-frozen region below 0°C under pressure where food products can thus be stored without the

Modelling HP processes 23

formation of ice crystals. As stated earlier, the formation of ice crystals can cause irreversible damage to cell structures and seriously affect the quality of the food; therefore, non-frozen storage under pressure has been examined as a means of preventing the detrimental effects of ice crystal formation. It has been reported that significant energy savings could be made using storage rather than freezing under pressure (Charm et al., 1977). In addition, product deterioration due to freezing and thawing effects can be avoided. For example, raw pork could be stored under pressure, avoiding drip losses occurring after thawing. The counts of most microorganisms in meat samples were reduced by low temperature storage under pressure (200 MPa, ⫺20°C), in some cases more than by freezing (Kalichevsky et al., 1995). Yeasts and some bacteria were inactivated and enzymes that are inactivated by freezing generally had reduced activity after non-frozen storage but were not inactivated (Kalichevsky et al., 1995). Low-temperature non-frozen storage under pressure therefore appears to be a method of prolonging the shelf-life of certain foods, e.g. compared to refrigeration, while avoiding the damage caused by freezing. Product deterioration due to freezing and thawing effects can be avoided and some authors pointed out that a significant energy saving could be made using pressure storage rather than freezing (Charm et al., 1977). To date, very little research has been carried out investigating this application and, as such, no accurate estimation of the cost has been made. However, if this process were to prove economically sound, it seems that the possibilities are great.

6 Modelling HP processes Since the discovery by Pasteur that microorganisms are responsible for food spoilage, scientists have attempted to develop mathematical models to predict the effect of processing and storage on food constituents. Some of these models have been based on thermodynamic principles, while others were strictly empirical. Today, both types of model are in common use by researchers, industry and regulatory agencies. Generally, these models do not provide insight into the actual mechanisms of either microbial inactivation or chemical change (Lund, 2003).

6.1 Modelling high pressure processes From an engineering point of view, theoretically-based heat and mass transfer models allowing the prediction of the physical history of food undergoing a given process are desirable. One of the main difficulties when modelling heat transfer in HP processes is the lack of knowledge of important thermophysical properties of materials being processed, or food constituents (e.g. water) while actually under pressure. Water is of special interest because all raw foods contain high levels of water and water is commonly employed as the pressure-transmitting fluid in the food industry (Otero and Sanz, 2003). However, understanding of such properties of food constituents is increasing rapidly, primarily because of advances in the basic sciences. With these advances, it can be

24 High Pressure Processing of Foods: An Overview

expected that rapid improvements in the ability to predict the impact of processing on food constituents will follow in time. Some critical control points required in order to model treatment of the pressurized product include temperature in the HP vessel chamber prior to processing, product temperature, uniformity of temperature throughout the product, ratio of pressurizing fluid to product and pressurization time. All of these variables affect the heat transfer in the HP vessel in a substantial way. It would thus be desirable to have a model that takes into account all these parameters and allows prediction of the changes in temperature in a sample under pressure in order to better control the process and its effects. Obtaining this type of model is a difficult task because of: 1 the aforementioned lack of data for thermal properties of the materials under pressure 2 the pressure and temperature dependency of thermal properties 3 the calculation of the adiabatic temperature increase/decrease after the pressure build up/release 4 the convective heat transfer that takes place in the pressurizing fluid between the inner wall of the vessel and the sample (Otero and Sanz, 2003). However, simple models have been developed which can predict the pressure and temperature conditions required to achieve a given level of microbial inactivation in specific food products (Polydera et al., 2003). These conditions can then be used to determine the optimum treatment, in terms of sensory quality, suitable for each product.

6.2 Modelling high pressure freezing processes Due to the advantages of PSF over classical atmospheric freezing methods, some attempts to model it have been made (Sanz et al., 1997; Otero and Sanz, 2000; Denys et al., 2000; Otero et al., 2002). Modelling processes that involve a phase change at atmospheric pressure in foods is difficult because of the heterogeneous biological structure of food, i.e. water is not totally available for freezing. The current literature available lacks enough thermophysical data for food and its components to be used in the modelling process in high-pressure domains (Otero et al., 2002). When the phase change is induced under pressure or by fast pressure release, modelling the process is considerably further complicated (Otero and Sanz, 2003). Any model developed for a classical freezing process at atmospheric pressure should be valid to reproduce the process under pressure if appropriate thermophysical properties were considered. Such models might take into account the temperature and pressure dependence of the thermophysical properties of the products, including their latent heats, freezing point and convective heat transfer coefficient and this presents the main difficulty for model development. It is also essential to consider the temperature change that the system undergoes after an adiabatic compression or expansion. A properly-developed model must quantify the amount of ice instantaneously produced after the adiabatic expansion. When modelling HP processes at sub-zero temperatures, pressure/temperature phase transition data and values for latent heats under pressure are needed. Experimental

Outlook for high pressure processing of food 25

determinations of these properties in food models and real food systems are essential to improve models (Otero and Sanz, 2003). Future models should consider all the thermal exchanges involved in the HP freezing process, including those between the pressuretransmitting medium and the steel mass of the vessel. This would allow development of accurate tools to allow real thermal control in HP processes (Otero and Sanz, 2003). Thermal expansion, isothermal compressibility, specific volume and specific isobaric heat capacity are some of the essential thermodynamic properties in modelling phase transition phenomena involved in PSF and HP thawing around the liquid–ice I melting curve of water (Otero et al., 2002). Other interesting thermophysical parameters, such as thermal conductivity and diffusivity and phase change enthalpies, behave more independently in their derivation and measurements (Otero et al., 2002).

7 Outlook for high pressure processing of food Consumers are changing their food consumption and purchasing behaviour; premium product sales, restaurant spending and ready-to-eat food spending are all increasing world-wide, as are health- and nutrition-driven product sales. New technologies such as HP can allow producers to create new markets not possible with old technologies and such benefits are only now being exploited. Consumers are generally willing to pay more for greater perceived value (Ting and Marshall, 2002). Adoption of new technologies for food preservation and processing is traditionally a slow process. However, the food industry is today seeking new technologies to enhance the safety, nutritional quality and sensory quality of food products. In the near future, the range of new non-thermal technologies will very likely complement current technologies, or even replace them for certain food products. The commercial feasibility of any technology depends ultimately on business profitability. The production cost of a process must, of course, be lower than the value added to the product. The value added by HP can be measured in terms of higher product quality, increased safety and longer shelf-life. These issues can further translate into reduced transportation, storage, insurance and labour costs, consumer convenience and enhanced safety. Fundamentally, the strategy for superior products is based on a higher perceived added value rather than on absolute cost (Ting and Marshall, 2002). The use of HP as a possible alternative processing method to thermal treatment has brought about the need to study the pressure–temperature behaviour of macromolecular food ingredients since, for example, the mechanisms of protein denaturation under pressure are far from fully understood. It is well known that HP can modify the activity of some enzymes and the structure of some proteins. Although covalent bonds are not affected, hydrogen bonds as well as hydrophobic and intermolecular interactions may be modified or destroyed. From this perspective, some concern about the potential risks of HP may arise. It is necessary to compile data in order to clarify the role of HP towards toxicity, allergenicity, loss of digestibility and the eating and nutritional quality of foods (Hugas et al., 2002). However, no studies to date have revealed any evidence that this could pose a problem for HPP.

26 High Pressure Processing of Foods: An Overview

There have been many studies of the use of HP as a pre-treatment method to improve the textural properties of food products. As a pre-treatment tool, HP processing appears effective in improving gelation properties of meat, egg or soy proteins, as well as improving the coagulating properties of milk (Galazka et al., 2000). Further studies are also required to understand the potential of the technology for rheological control in food protein systems, as well as to optimize the operating conditions that should be used during actual processing. Before any food product can be produced commercially using HP, optimization of processing conditions is essential to ensure product safety (McClements et al., 2001). The greatest value of HP to producers is related to safety. As microbiological standards become more widely mandated and sensitive assessment techniques become available in food production, the financial cost of unacceptable products will be transferred back to the producer. Large-scale product recalls will provide a quantifiable value to using an active intervention method such as HP. The value of food safety is frequently difficult to quantify prior to an incident. However, as has been observed in recent pathogen contamination events, a producer’s reputation or brand name may never recover from a single food safety related incident. Having a consistently safe product is now essential in the food industry (Ting and Marshall, 2002). The principal disadvantage of HP is capital cost, i.e. initial outlay on equipment, which is very expensive; industrial scale vessels can cost from 0.5 to 4 million Euro. In many sectors of the food industry, for example, the cooked meat and ready meals sectors, HP treatment offers a unique opportunity to produce fresh-tasting products which are safe and have a desirable shelf-life. The growing markets for these sectors and the commercially successful implementation of HP by a number of companies in these sectors suggests that the initial investment costs may be sustainable. It appears that consumers are willing to pay extra for new products, or products which have higher quality and are more convenient than the existing range; HP technology can deliver on both these aspects. It is important, however, that thorough study is carried out in terms of eating quality, safety and cost benefits, before the technology is more widely embraced. Food companies must be able to make a realistic cost-benefit analysis of the potential rewards in investment in HP processing. The value of HP in terms of increasing food safety assurance, in some cases, may alone be sufficient to justify such investment. The actual cost of operating a HP processing plant will depend on many factors, ranging from operating pressure, cycle time and product geometry to labour skills and energy costs. As with all capital equipment, the greater the utilization, the more costeffective it is. As the technology matures and producers gain experience, lower equipment and operations costs can be anticipated (Ting and Marshall, 2002). The high cost of HP treatment currently limits its application to high-value products. However, it can be expected that these costs will decrease as a consequence of further progress in technology. A clear advantage of pressure treatment is the low energy input and the stability of small molecules, such as nutrients, to HP (Smelt, 1998). As HP food processing technology develops further, new products and applications will be discovered and investigated. In many ways, HP food processing is now following the same sequence of events that food irradiation encountered in the 1950s. However, without the negative perceptions associated with irradiation, the relatively

References 27

simple mechanical aspects of pressure use should prove easier to accept by both the consumer and the regulatory agencies. Of course, both the economics and safety of pressureprocessed products must be in agreement, but the prognosis seems to be very good (Hoover, 1993).

8 Conclusions The application of any new technology presents significant challenges to food technologists and food researchers. HP processing offers the food industry a technology that can achieve the food safety of heat pasteurization while meeting consumer demand for fresher-tasting minimally-processed foods. In addition, other favourable organoleptic, nutritional and rheological properties of foods have been demonstrated following HP, in comparison to heat processing. The retention of colour and aroma and the preservation of nutritive components are enormous benefits to both the food processing industry and consumers. Also, from a food processing/engineering perspective, key advantages of high-pressure applications to food systems are the independence of size and geometry of the sample during processing, possibilities for low temperature treatment and the availability of a waste-free, environmentally-friendly technology. Application of HP can inactivate microorganisms and enzymes and modify structures, while having little or no effects on nutritional and sensory quality aspects of foods. HP food processing is today being used on an ever-increasing commercial basis. Opportunities clearly exist for innovative applications and new food product development. HP can affect the functionality of protein and carbohydrate molecules in often unique ways, which may allow the optimization of food manufacturing processes and the production of novel foods. The range of commercially-available HP-processed products is relatively small at present but there are opportunities for further development and the production of a wide range of HP-treated products. The main drawbacks of pressure treatment of solid foods are the use of batch or semicontinuous (the latter for liquids only) processing and the high cost of pressure vessels. HP is an environmentally-friendly, industrially-tested technology that offers a natural alternative for the processing of a wide range of different food products. This method prolongs product shelf-life while at the same time preserving organoleptic qualities, by inactivating microorganisms and enzymes while leaving small molecules such as flavours and vitamins intact. It is a technology with many obvious advantages, especially for food products with a high added value, targeted at a growing group of consumers that demand maximum safety and quality in the products they purchase.

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28 High Pressure Processing of Foods: An Overview

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High-pressure Processing of Salads and Ready Meals Srilatha Pandrangi and V M Balasubramaniam The Ohio State University, Department of Food Science and Technology, Columbus, Ohio, USA

High-pressure processing (HPP) holds the potential for preserving foods by combining elevated pressures (up to 900 MPa or approximately 9000 atmospheres) and moderate temperatures (up to 120°C) over a short period. Though high-pressure processed salads are currently unavailable, HPP has been used as an alternative to heat pasteurization for processing ready meals in the USA and Europe. HPP effects on selected ready meals, salad dressings, dips and sauces are reviewed. Aspects covered include combined pressure-thermal effects on microbial and enzyme inactivation. Pressures up to 600 MPa can inactivate yeasts, moulds and most vegetative bacteria, including pathogens. While HPP leaves small molecules such as flavour compounds and vitamins intact, its influence on colour is product dependent. This varies between a full retention of the fresh colour and colour change similar to thermally processed foods.

1 Introduction High-pressure processing retains food quality and natural freshness and extends shelflife. During high-pressure processing (HPP) the food material is subjected to elevated pressures (up to 900 MPa or approximately 9000 atmospheres) with or without addition of heat to achieve microbial inactivation with minimal damage to the food. Physical compression of the product during the process increases the temperature of the product only during the treatment. This unique compression heating effect helps to reduce the severity of thermal effects encountered with conventional processing techniques such as retorting. Other advantages of the technology include uniform pressure application, minimal heat damage to food and potential for altering functional properties of foods. HPP breaks only hydrogen bonds and disrupts hydrophobic and electrostatic interactions. HPP does not affect covalent bonds and has little effect on chemical constituents associated with desirable food qualities such as flavour, colour or nutritional content. The possibility of extending shelf-life without heating Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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34 High-pressure Processing of Salads and Ready Meals

Table 2.1 World-wide distribution of high-pressure processed salad and ready meal products Product manufacturers

Category

Product

Avomex Incorporated, USA

Ready meals

Avotec International, USA Calavo Growers Inc., USA Hannah International, USA Meidi-ya, Japan Espuna, Spain Hormel, USA Perdue, USA Orchard House, USA Leahy Orchards, Canada Winsoms of Walla Walla, USA

Sauces and dips Sauces and dips Dips Dips Sauces and salad dressings Ready meats Ready meats Ready meats Beverage Snacks Ready meal ingredient

Chicken fajita, chipotle chicken and chipotle beef dinners; smoothies Guacamole and salsa products Salsa and guacamole Guacamole Hummus Salad dressings, fruit sauces Sliced ham Ham Seasoned chicken breasts Smoothies and orange juice Applesauce snacks Chopped speciality onions

the food for prolonged periods greatly helps to satisfy consumer demand for fresher and higher quality heat-sensitive foods that are otherwise difficult to process using conventional food preservation methods. Currently there are no commercially available pressure-processed salads, but limited ready meals are commercially available (Table 2.1). In this chapter, the challenges and opportunities for high-pressure processing of ready meals and salads are highlighted.

2 Importance of salads and ready meals Salads and ready meals comprise a popular and growing segment of the food industry. Sales of pre-cut salad mixes were at $1.94 billion in year 2001 and increased to $2.11 billion in 2003 (Hodge, 2003). In restaurants alone, salads/vegetables comprise 9 per cent of the main menu (Sloan, 2003). Estimated sales of ready meals in Europe alone were $10.4 billion in 2000 (Anon, 2003a). Total growth in the value of ready meals in the global market has increased by 4 per cent in the year 2002. Ready meals are very popular in the USA, the UK and Scandinavian countries. The consumer lifestyle tending towards smaller households and longer working hours has been cited as the main reason for the increase in the consumption of convenient ready meals. In Europe, it is projected that between 2002 and 2007, this segment of the market will record an 18 per cent increase in growth (Anon, 2003b). Salads have evolved from traditional ones consisting of greens and potatoes to ones with widely varied compositions. Caesar salads, tossed green salads, potato salad and salads with pasta/macaroni are some of the most popular salads in USA (Sloan, 2003). Some of the ingredients in salads include but are not limited to lettuce, diced/cherry tomatoes, spinach, onions, shredded carrots, potatoes, coleslaw, cheese, chicken, tuna and mayonnaise. Ready meals include any entrée or snack that requires minimal preparation before consumption. These would include dinner entrées with combinations of meat and rice

Pressure effects on microorganisms 35

or vegetables and snacks such as carrots. A processor may choose to process certain components of the ready meal using high pressure while other components may be thermally processed.

3 Pressure effects on microorganisms The effect of high-pressure processing on the degree and mechanism of microbial inactivation has been extensively studied and reviewed (Cheftel, 1992, 1995; Smelt, 1998). Most vegetative cells can be inactivated at relatively low pressures, typically 200–400 MPa (Cheftel, 1995), while bacterial spores are more resistant and need a combination of higher pressures and temperatures (Rovere et al., 1996; Reddy et al., 1999; Balasubramaniam, 2003). High pressure can be used for both pasteurization and sterilization of food products. High-pressure pasteurization involves the application of moderate pressures (between 400 and 600 MPa) at temperatures up to 60°C. Like conventional thermal treatment, high-pressure pasteurization treatment inactivates harmful vegetative bacteria in foods effectively (Cheftel, 1995; Farkas and Hoover, 2000; Raghubeer et al., 2000) with minimal nutrient degradation. The products pasteurized by high pressure require refrigeration during storage and distribution. High-pressure sterilization is essentially a pressure-accelerated thermal process that requires a combination of elevated pressures (up to 800 MPa) and temperatures (up to 120°C) to produce a shelf-stable food free from harmful bacterial spores (Meyer et al., 2000; Balasubramaniam, 2003). At present, a number of food processors are actively investigating the feasibility of introducing high-pressure sterilized products in commercial markets.

3.1 Efficacy of microbial inactivation in HPP processed ready meals Vegetables and meat are major ingredients in both salads and ready meals. When lettuce, tomato, asparagus, onions, cauliflower and spinach were high-pressure processed between 200 and 400 MPa for 30 min at 5°C, a 2–4 log reduction in mesophilic bacteria, yeast and mould was observed (Arroyo et al., 1999). Significant work has been done to evaluate the effect of high-pressure processing on the microbiological safety of meat products. High pressure has been used not only to reduce spoilage organisms and extend shelf-life, but also to reduce pathogens and spore formers. The physicochemical properties of the food that contribute to the survival of microorganisms have also been studied. Survival of microorganisms in food products depends not only on sensitivity of the microorganism to pressure, but also on the protective effect that a product offers during treatment and subsequent storage. Therefore, microbial reductions in media such as buffers cannot be extended to food systems. Hugas et al. (2002) reported that log reductions in meat model systems are lower compared to phosphate buffer. Furthermore,

36 High-pressure Processing of Salads and Ready Meals

water activity of the food significantly affects survival under high pressure. When vacuum packaged marinated beef (aw 0.99), cooked ham (aw 0.98) and dry cured ham (aw 0.89) were spiked with Staphylococcus aureus and lactic acid bacteria and processed at 600 MPa for 6 min at 31°C, the log reductions in the meat were lower at lower water activity. Other factors such as suboptimal pH and use of antimicrobials in food can enhance the effect of high pressure. It is therefore important to consider all these factors while evaluating the effect of high pressure on microbial reductions in food. Despite the variable effects of food systems, in general, high-pressure treatment seems to be more effective in reducing bacterial numbers in a shorter time compared to thermal processing and it can significantly increase the shelf-life of the product. In tomato puree containing meat balls inoculated with Bacillus stearothermophilus, high-pressure processing the sauce mixture preheated to 80 or 90°C at 700 MPa for 30 s using single or double pulse, reduced the background microflora, which mainly consisted of spores, to undetectable levels. A single pulse at the above-mentioned conditions was sufficient to reduce six logs of the B. stearothermophilus populations to undetectable levels, while conventional sterilization processes reduced the levels of organism by less than two logs (Krebbers et al., 2003). Chicken breast inoculated with Clostridium sporogenes and pressure treated between 400 and 800 MPa for 1–60 min showed up to 3-log reduction in spore populations (Crawford et al., 1996). Hugas et al. (2002) reported that marinated beef loin and dry cured ham processed at 600 MPa for 6 min at 31°C, showed significant reduction in the number of spoilage microflora. Pressure-treated beef loin and cured ham both remained acceptable for 120 days, four times longer than untreated samples. Pressure effect can be enhanced by combining it with mild to moderate heat, as observed in study by Krebbers et al. (2003). This effect was also demonstrated when ground pork patties inoculated with Listeria monocytogenes were pressure treated at 414 MPa for 6 min at a moderate temperature of 50°C (Murano et al., 1999). About a 10-log reduction in the pathogen population was observed by increasing the processing temperature. Furthermore, under refrigerated storage, the shelf-life of the product was increased to 28 days compared to 5 days for the untreated sample. Similarly, minced pork meat inoculated with Streptococcus faecalis, Bacillus subtilis or Bacillus stearothermophilus and treated with pressures between 50 and 400 MPa for 1–60 min between 20 and 80°C, showed synergistic effects of pressure and temperature with higher log reductions observed at 400 MPa pressure at 80°C (Moerman et al., 2001). Pressure application in food processing can be continuous or pulsed. When mechanically recovered poultry meat (MRPM) was subjected to an alternate low (60 MPa) and high pressure (450 MPa) pulse or continuous high pressure at 450 MPa for 15 min at ambient temperature, no decrease in survival of mesophiles was observed because of pulsing. Approximately 3.2 to 3.8 log reduction in mesophiles was observed with both methods. However, a higher reduction in psychrotrophs of up to 5.3 logs was observed with pulsing (Yuste et al., 2001). All the above studies suggest that high-pressure processing can be successfully used for meat products to reduce spoilage and prolong shelf-life. It can also be used for obtaining significant reductions in pathogenic microflora. However, as with thermal processing, some studies showed that the elimination of microorganisms is not complete

Pressure effects on enzyme activity 37

and some portion of the population seems to be pressure resistant. Increasing the pressure levels does not affect this portion of microorganisms (Crawford et al., 1996) and high pressure may have to be combined with other methods such as use of antimicrobials to increase the process lethality.

3.2 Efficacy of microbial inactivation in HPP-processed dips, sauces and salad dressings Commercially processed mayonnaise and salad dressings may have to undergo some form of processing because of the recent emergence of acid-tolerant pathogens and some spoilage microflora. For such products, high-pressure processing may be considered as an attractive alternative processing method. There are a limited number of studies on survival of microorganisms in HPP salad dressings and sauces. In addition, a few studies discuss the safety of high-pressure processed salad or sauce ingredients such as cheeses. In salad dressings (ranch, French and slaw) inoculated with 104 cfu/g of Lactobacillus fructivorans and Zygosachharomyces bailli and treated for 10 min between 500 and 800 MPa at 25 or 50°C, the spoilage organisms were reduced to undetectable levels at the lowest pressure and temperature used (Neinaber et al., 2001). In three types of cheeses inoculated with L. monocytogenes and subjected to pressures of up to 500 MPa for 15 min at room temperature, an approximately 6-log reduction of pathogen was observed (Szczawinski et al., 1997).

4 Pressure effects on enzyme activity Enzyme activity is an important parameter affecting quality, particularly of cut fruits and vegetables. In whole fruits and vegetables, the enzymes are usually confined to compartments. However, in cut products, this compartmentalization is destroyed and the substrates and enzymes mix freely causing undesirable changes in the products. Of particular importance in fruits and vegetables are pectin methyl esterase (PME) and polygalacturonase (PG), which are involved in cell wall breakdown and thus cause reduction in viscosity and changes in colour and other organoleptic properties. Other enzymes such as peroxidase; and polyphenol oxidases and lipoxygenase affect colour and lipid breakdown, respectively. The effect of high pressure on enzyme activity seems to be variable. Pressure affects weak bonds such as hydrophobic interactions, which are primarily responsible for maintaining the tertiary structures of proteins. Since enzymes are proteins, it is expected that application of pressure changes the structural conformation and may sometimes lead to loss of activity. However, it has been observed that though there is a partial or complete inactivation of enzymes in some cases, activation of enzymes has been reported in others. It can be concluded that in general, pressure alone, in most vegetables, is insufficient to inactivate enzymes and hence needs to be combined with heat.

38 High-pressure Processing of Salads and Ready Meals

4.1 Effect of high pressure on enzyme activity of fruits and vegetables Most work related to the effect of high-pressure processing on enzyme activity has been done with respect to PE and PG in intact, cut, and pureed vegetables or juices. Fachin et al. (2002) investigated the suitability of high-pressure processing for tomato products. They studied the inactivation kinetics of the crude polygalacturonase (PG) extract under isothermal and isobaric-isothermal conditions. The authors (Fachin et al., 2002) reported that the pressure–temperature ranges that inactivate tomato PG are between 300 and 600 MPa and 5 and 50°C. Stoforos et al. (2002) studied the inactivation kinetics of tomato pectin methylesterase (PME) under a combination of temperature and high pressure. PME inactivation rates increased with increasing processing temperature. While pressure ⬍700 MPa did not inactivate the enzyme successfully, at pressures ⬎700 MPa higher inactivation was observed. In tomato puree that was preheated to 90°C and high-pressure processed at 700 MPa for 30 s using single or double pulse, pectin methyl esterase and polygalacturonase activity were reduced to undetectable levels (Krebbers et al., 2003). Enzyme inactivation can be best described by a first order kinetic model taking thermal, pressure and their combination effect into consideration. Diced tomatoes processed at 400, 600 and 800 MPa for 1, 3 or 5 min at 25°C or 45°C showed loss of polygalacturonase activity (PG) at pressures above 400 MPa irrespective of temperatures (Shook et al., 2001). However, pectin esterase (PE) activity markedly increased with application of 400 MPa pressure at 45°C. In the same study a loss of lipoxygenase activity was observed with application of pressure ⬎600 MPa at all temperatures. Whole cherry tomatoes processed under high pressure showed a similar trend in PE and PG activity (Tangwongchai et al., 2000). In general, tomato polygalacturonase seems to be sensitive to pressure while tomato pectin methyl esterase seems to be insensitive to pressure. The effect of high pressure on peroxidase activity has also been documented. Among pressure-treated tomato, lettuce, spinach, onion, asparagus and cauliflower (100–400 MPa for 30 min at 5°C), the only vegetable that showed slight browning at higher pressures was cauliflower and this was attributed to incomplete inactivation of peroxidase. The other vegetables did not show a significant change in sensory properties (Arroyo et al., 1999). In green peas processed under high pressure (400–900 MPa) for 5 or 10 min at 20°C, peroxidase activity was significantly reduced because of the pressure treatment. Greatest reduction in enzyme activity (about 88 per cent) was obtained at 900 MPa for 10 min and this reduction, according to the authors (Quaglia et al., 1996), was comparable to blanching. The effect of high pressure on polyphenoloxidase (PPO) activity was studied by Gomes and Ledward (1996). In general, crude extracts of potatoes and mushrooms showed considerable reduction in polyphenoloxidase activity when treated with pressures ranging between 200 and 800 MPa for 10 min, with the exception of mushroom extract at 400 MPa, which showed a marked increase in PPO activity. However, whole tissues subjected to similar pressures showed considerable browning. Potatoes treated with 800 MPa alone showed acceptable colour but had a cooked appearance.

Pressure effects on texture 39

4.2 Effect of high pressure on enzyme activity in meats Loss of colour in meat is an important quality aspect affecting its salability. The mechanism of colour loss is an issue of debate. Some researchers attribute it to certain enzyme systems in the meat, while others attribute this change to denaturation of globin proteins at higher pressures. Colour changes typically occur above 150 MPa, resulting in products that resemble cooked meats (Hugas et al., 2002). In fresh beef, formation of metmyoglobin is responsible for loss of red colour and a decrease in perceived quality. Fresh beef processed for up to 2 days with pressures between 80 and 120 MPa for 20 min showed a reduction in formation of this pigment. However, the effect of pressure processing was quickly lost if the time between slaughter and processing increased to more than 7 days (Cheah and Ledward, 1996). Colour changes are most significant in raw products. In sausages containing various levels of mechanically recovered poultry meat and minced pork meat, and cooked with high pressure (500 MPa for 30 min) at 50, 60, 70, or 75°C, a significant loss of redness and an increase in lightness were observed (Yuste et al., 1999). In products that have already been cooked, the application of high pressure has no additional effect. Secondary pasteurization is routinely performed for meat products where casings have been removed to destroy any microbes that have been transferred during postprocess handling. High pressure treatment of cooked sausages at 500 MPa for 5 or 15 min at 65°C did not significantly affect the colour and therefore high pressure has been suggested as a substitute treatment for pasteurization for this product (Mor-Mur and Yuste, 2003). Lack of colour change due to high-pressure processing has also been observed in cooked ham (Goutefangea et al., 1995).

5 Pressure effects on texture 5.1 Textural changes in pressure treated ready meals The texture of vegetables is an important quality attribute for commercial sale. The effect of high-pressure processing on texture varies with commodity and applied pressure. For example, processing green peas between 400 and 900 MPa for 5–10 min at 20°C did not significantly affect the texture (Quaglia et al., 1996). On the other hand, for carrot, celery and red and green peppers, a ‘dual effect’ of pressure was reported, where an initial loss of texture was seen immediately after pressurization, followed by regaining of texture during a hold time. This effect depended on the type of vegetable and duration and level of pressure applied. The immediate loss of texture on application of pressure was described as ‘instantaneous pressure softening (IPS)’. For carrot, celery and green pepper, application of 100 MPa with extended hold times up to 60 min resulted in a higher firmness compared to untreated products. However, at higher pressures the firmness decreased. At high pressures celery was the most sensitive while red peppers were least sensitive (Basak and Ramaswamy, 1998).

40 High-pressure Processing of Salads and Ready Meals

(a)

(b)

20 ␮m

(c)

100 ␮m

Figure 2.1 Freeze fracture scanning electron micrographs (SEM) of raw spinach leaf before high-pressure processing; (a) parenchyma cells, (b) vascular tissue, (c) magnified vascular tissue with organelles. (Source: Préstamo and Arroyo, 1998 Journal of Food Science.)

In tomatoes and lettuce subjected to pressures ranging between 100 and 600 MPa for 10 or 20 min at 10 or 20°C, changes in sensory qualities were observed above pressures of 300 MPa. In tomatoes, loosening of skin was observed, whereas in lettuce browning was observed, though flavour in both vegetables was unaffected (Arroyo et al., 1997). Cherry tomatoes processed under pressures of 200–600 MPa for 20 min at 20°C showed different degrees of texture loss and cell damage. Between 200 and 400 MPa, higher losses of water from cell tissue and a higher degree of cell rupture were observed, which resulted in a product with less firmness compared to those processed between 500 and 600 MPa. Light microscopy of samples revealed that processing tomatoes at lower pressures resulted in entrapment of bubbles, which seemed to have been expelled under high pressures. Samples processed at 500–600 MPa were visually similar to their unprocessed counterparts and hence were considered more acceptable (Tangwongchai et al., 2000). In spinach leaves, loss of texture was reported after processing at 400 MPa for 30 min at 5°C (Prestamo and Arroyo, 1998). At microscopic level, loss of parenchyma structure, formation of cavities and loss of intercellular space was observed (Figures 2.1 and 2.2). Compared to cauliflower processed under these same conditions, marked changes and degradation were observed in spinach and the researchers (Prestamo and Arroyo, 1998) attributed this to the softer nature of the spinach tissue. It was concluded that spinach is unsuitable for high-pressure processing. High-pressure processing can lead to reversible or irreversible changes in textural properties of meat products. Similar to colour, changes to the texture of meat seem to depend on the time lag between meat slaughter and processing. In fresh meat where rigor had not set in, pressurization leads to shortened muscles accompanied by severe damage to the muscle structure. However, the processed muscle was found to be tender on cooking (MacFarlane, 1973). Hence, it can be inferred that pressure treatment of pre-rigor muscle can increase tenderization.

Pressure effects on texture 41

(a) (b)

(d)

20 ␮m (c)

10 ␮m

10 ␮m

5 ␮m

Figure 2.2 Effect of high-pressure processing (400 MPa, for 30 min at 5°C) on spinach tissues as observed after freeze fracture SEM; (a) collapsed parenchyma cells after HPP, (b) cavity formation, (c) preservation of vascular tissue structure, (d) folding of collapsed membrane after HPP. (Source: Préstamo and Arroyo, 1998 Journal of Food Science.)

High-pressure treatment of cooked sausages at 500 MPa for 5 or 15 min at 65°C did not significantly affect the colour. However, high pressure treated sausages were less firm than their heat pasteurized counterparts. A taste test on both products concluded that for attributes that were significantly different for the two products, high pressure processed sausages were preferred over heat pasteurized samples (Mor-Mur and Yuste, 2003).

5.2 Textural changes in pressure-treated dips, sauces and salad dressings French, ranch and slaw dressings treated with high pressures between 500 and 800 MPa for 10 min at 25 or 50°C differed significantly in their viscoelastic behaviour, which was attributed to compositional differences. French dressing containing egg yolk as the stabilizer was found to be least stable, while ranch dressing containing proteins and xanthan gum was found to be most stable (Neinaber et al., 2001). However, it was concluded that pressure treatment does not significantly change the rheological properties of salad dressings. Further studies on acidified emulsions containing soy lecithin, polysorbate 60 and whey protein isolate as emulsifiers and xanthan gum as stabilizer confirmed that pressure does not significantly affect the rheology of emulsions. Differences in the flow behaviour, viscoelasticity, particle size and stability of the emulsions were attributed to the lipid content and emulsifier used. It was observed that soy lecithin-based emulsions were unstable from the point they were prepared and this instability was increased with the application of high pressure. Emulsions containing whey protein and polysorbate 60 were stable to pressure. Whenever xanthan

42 High-pressure Processing of Salads and Ready Meals

gum was added as stabilizer to polysorbate 60 emulsions, the stability of the emulsion was further enhanced by the application of pressure (Arora et al., 2003). Process temperature, pressure holding time and nature of the surfactant seem to be the important factors affecting the physical stability of emulsion systems. In model emulsion at neutral pH containing sodium caseinate (50 g/kg) and peanut oil (300 g/kg), application of 450 MPa of pressure for 30 min at room temperature did not affect particle size distribution or viscosity. However, when sodium caseinate was substituted with ␤-lactoglobulin, application of pressure increased emulsion viscosity and promoted gelation at 40°C (Dumay et al., 1996). Dickinson and James (1998) further observed that ␤-lactoglobulin-stabilized emulsions showed increases in average droplet diameter when they were subjected to 800 MPa pressure for up to 60 min.

6 Pressure effects on nutrients It has been generally known that high pressure has very little effect on low molecular weight compounds such as flavour compounds, vitamins and pigments compared to thermal processes. This effect is particularly important in salads, as most vegetables are rich sources of antioxidant compounds, pigments and vitamins. Butz et al. (2002) studied the effect of 600 MPa pressure in combination with elevated temperatures on the pigment and vitamin content of three vegetables. Broccoli treated with pressure of 600 MPa for 40 min at 75°C showed no loss of chlorophyll a or b compared to the untreated samples. Similarly, tomatoes treated at a combination of 600 MPa pressure and 25°C temperature for 60 min did not show a change in either lycopene or carotenoid content and were similar to the heat-treated sample (95°C, 60 min). In addition, the loss in antioxidant capacity of a water-soluble portion of pressure treated carrot and tomato (500 and 800 MPa for 5 min) was very little compared to the untreated samples. High retention of ascorbic acid (82 per cent) was observed in green peas treated with 900 MPa pressure for 5–10 min at 20°C (Quaglia et al., 1996). Model multivitamin systems containing varied levels of water-soluble vitamins such as ascorbic acid, thiamin and vitamin B6 (pyridoxal) and food systems containing naturally occurring levels of vitamin C were subjected to pressures ranging between 200 and 600 MPa for 30 min to determine the effect on vitamin retention. In the model systems observed, ascorbic losses were close to 12 per cent while in food material, these losses were insignificant. Compared to conventional sterilization processes, highpressure treatments retained the vitamins better. Thiamin and pyridoxal in the model system were unaffected by high-pressure processing (Sancho et al., 1999). These findings confirm the fact that high pressure has minimal effect on nutrients in foods.

7 Conclusions High-pressure processing of salads and ready meals is an emerging niche market that provides unique opportunities and challenges to the food industry. HPP lends itself for

References 43

processing a variety of novel, convenient, minimally processed ready meals with long shelf-life, fresh-like attributes and natural colours. Identification of commercially viable applications is the likely challenge faced by the food processor. Furthermore, combination processes are likely to reduce the severity of process requirements.

Acknowledgement Reference to commercial product or trade names is made with the understanding that no endorsement or discrimination by The Ohio State University is implied.

References Anon (2003a) The European ready meals market. Ready Meals Information, PO Box 72, Caldicot, Gwent, NP26 3ZG, UK. Anon (2003b) Ready meals benefit from lifestyle and demographic trends. Euromonitor International 122 S Michigan Ave, Chicago, IL USA. Arora A, Chism GW, Shellhammer TH (2003) Rheology and stability of acidified food emulsions treated with high pressure. Journal of Agricultural and Food Chemistry, 51, 2591–2596. Arroyo G, Sanz PD, Prestamo G (1997) Effect of high pressure on the reduction of microbial populations in vegetables. Journal of Applied Microbiology, 82 (6), 735–742. Arroyo G, Sanz PD, Prestamo G (1999) Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy. Journal of Applied Microbiology, 86 (3), 544–566. Balasubramaniam VM (2003) High-pressure processing of foods. In Encyclopedia of Agricultural, Food and Biological Engineering (Heldman D, ed.). New York: Marcel Dekker, Inc., pp. 490–496. Basak S, Ramaswamy HS (1998) Effect of high-pressure processing on the texture of selected fruits and vegetables. Journal of Texture Studies, 29 (5), 587–601. Butz P, Edenharder R, Garcia AF, Fister H, Merkel C, Tauscher B (2002) Changes in functional properties of vegetables induced by high pressure treatment. Food Research International, 35, 295–300. Cheah PB, Ledward DA (1996) Inhibition of metmyoglobin formation in fresh beef by pressure treatment. Meat Science, 45 (3), 411–418. Cheftel J-C (1992) Effects of high hydrostatic pressure on food constituents: an overview. In High Pressure and Biotechnology (Balny C, Hayashi R, Heremans K, Masson P, eds). Montrouge: Colloque Inserm/John Libbey Eurotext Ltd., pp. 195–209. Cheftel J-C (1995) Review: high pressure, microbial inactivation and food preservation. Food Science and Technology International, 1 (2/3), 75–90.

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Crawford YJ, Murano EA, Olson DG, Shenoy K (1996) Use of high hydrostatic pressure and irradiation to eliminate Clostridium sporogenes spores in chicken breast. Journal of Food Protection, 59 (7), 711–715. Dickinson E, James JD (1998) Rheology and flocculation of high pressure-treated ␤-lactoglobulin-stabilized emulsions: Comparison with thermal treatment. Journal of Agricultural and Food Chemistry, 46, 2565–2571. Dumay E, Lambert C, Funtenberger S, Cheftel JC (1996) Effects of high pressure on the physicochemical characteristics of dairy creams and model oil/water emulsions. Lebensmittel-Wissenschaft-und-Technology, 29, 606–625. Fachin D, Van Loey A, Ludikhuyze IL, Hendrickx MEG (2002) Thermal and highpressure inactivation of tomato polygalacturonase: a kinetic study. Journal of Food Science, 67 (5), 1610–1615. Farkas D, Hoover D (2000) High-pressure processing. Kinetics of microbial inactivation for alternative food processing technologies. Journal of Food Science Supplement, pp. 47–64. Gomes MRA, Ledward DA (1996) Effect of high-pressure treatment on the activity of some polyphenoloxidases. Food Chemistry, 56 (1), 1–5. Goutefangea R, Rampon V, Nicolas N, Dumont JP (1995) Meat colour changes under high pressure treatment. In Proceedings of 41st International Congress of Meat Science and Technology San Antonio, Texas, USA, pp. 384–385. Hodge K (2003) Salads still hot after all these years. Fresh-cut magazine. Yakima: Columbia Publishing and Design, July, p. 22. Hugas M., Garriga M, Monfort JM (2002) New mild technologies in meat processing: high pressure as a model technology. Meat Science, 62, 359–371. Krebbers B, Matser AM, Hoogerwerf SW, Moezelaar R, Tomassen MMM, van den Berg RW (2003) Combined high-pressure and thermal treatments for processing of tomato puree: an evaluation of microbial inactivation and quality parameters. Innovative Food Science & Emerging Technologies, 4, 377–385. MacFarlane JJ (1973) Pre-rigor pressurization of muscle: effects on pH (hydrogen-ion concentration), shear value and taste panel assessment. Journal of Food Science, 38 (2), 294–298. Meyer RS, Cooper KL, Knorr D, Lelieveld HLM (2000) High-pressure sterilization of foods. Food Technology, 54 (11), 67–72. Moerman F, Mertens B, Demey L, Hughebaert A (2001) Reduction of Bacillus subtilis, Bacillus stearothermophilus and Streptococcus faecalis in meat batters by temperature-high hydrostatic pressure pasteurization. Meat Science, 59, 115–125. Mor-Mur M, Yuste J (2003) High-pressure processing applied to cooked sausage manufacture: physical properties and sensory analysis. Meat Science, 65 (3), 1187–1191. Murano EA, Murano PS, Brennan RE, Shenoy K, Moriera RG (1999) Application of high hydrostatic pressure to eliminate Listeria monocytogenes from fresh pork sausage. Journal of Food Protection, 62, 480–483. Neinaber U, Arora A, Shellhammer TH (2001) High pressure processed salad dressings: Microbiological and rheological aspects. Annual Meeting of the Institute of Food Technologists, Technical session on Food safety and quality aspects of non thermal processing technologies. Abstract no. 28.4, New Orleans.

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Prestamo G, Arroyo G (1998) High hydrostatic pressure effects on vegetable structure. Journal of Food Science, 63 (5), 878–881. Quaglia GB, Gravina R, Paperi R, Paoletti F (1996) Effect of high pressure treatments on peroxidase activity ascorbic acid content and texture in green peas. LebensmittelWissenschaft-und-Technology, 29, 552–555. Raghubeer EV, Dunne CP, Farkas DF, Ting EY (2000) Evaluation of batch and semicontinuous application of high hydrostatic pressure on foodborne pathogens in salsa. Journal of Food Protection, 63 (12),1713–1718. Reddy NR, Solomon HM, Fingerhut GA, Rhodehamel EJ, Balasubramaniam VM, Palaniappan S (1999) Inactivation of Clostridium botulinum type E spores by high pressure processing. Journal of Food Safety, 19, 277–288. Rovere P, Carpi G, Dall’Aglio G et al. (1996) High-pressure heat treatments: Evaluation of the sterilizing effect and of thermal damage. Industria Conserve, 71 (4), 473–484. Sancho F, Lambert Y, Demazeau G, Largeteau A, Bouvier J-M, Narbonne J-F (1999) Effect of ultra-high hydrostatic pressure on hydrosoluble vitamins. Journal of Food Engineering, 39, 247–253. Shook CM, Shellhammer TH, Schwartz SJ (2001) Polygalacturonase, pectinesterase, and lipoxygenase activities in high-pressure-processed diced tomatoes. Journal of Agricultural and Food Chemistry, 49, 664–668. Sloan AE (2003) Going for the green: super salads. Food Technology, 57 (7), 18. Smelt JPPM (1998) Recent advances in the microbiology of high pressure processing. Trends in Food Science and Technology, 9, 152–158. Stoforos NG, Crelier S, Robert MC, Taoukis PS (2002) Kinetics of tomato pectin methylesterase inactivation by temperature and high pressure. Journal of Food Science, 67 (3), 1026–1031. Szczawinski J, Szczawinska M, Stanczak B, Fonberg-Broczek M, Arabas J, Szczepek J (1997) Effect of high pressure on survival of Listeria monocytogenes in ripened, sliced cheeses at ambient temperature. In High Pressure Research in Biosciences and Biotechnology (Heremans K, ed.). Leuven: Leuven University Press, pp. 295–298. Tangwongchai R, Ledward DA, Ames JM (2000) Effect of high-pressure processing on the texture of cherry tomato. Journal of Agricultural and Food Chemistry, 48, 1434–1441. Yuste J, Mor-Mur M, Capellas M, Guamis B, Pla R (1999) Mechanically recovered poultry meat sausages manufactured with high hydrostatic pressure. Poultry Science, 78 (6), 914–921. Yuste J, Pla R, Capellas M, Sendra E, Beltran E, Mor-Mur M (2001) Oscillatory highpressure processing applied to mechanically recovered poultry meat for bacterial inactivation. Journal of Food Science, 66 (3), 482–484.

Microbiological Aspects of Highpressure Processing Montserrat Mor-Mur and Josep Yuste Universitat Autònoma de Barcelona, Centre Especial de Recerca Planta de Tecnologia dels Aliments (CeRTA, XIT), Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Barcelona, Spain

High-pressure processing is an alternative technology for food safety and preservation. It effectively inactivates pathogenic and spoilage organisms, especially by affecting cell walls, membranes and also key enzymes. Pressure treatment can be performed at room or lower temperatures, resulting in foods with good nutritional and organoleptic qualities, great safety and long shelf-life. Pressurization at mild or high temperatures is very effective, especially for achieving the inactivation of bacterial endospores, which are the most pressure-resistant organisms. Staphylococcus aureus and Escherichia coli O157:H7 are also highly resistant. Low pH and high water activity enhance inactivation, whereas the food structure probably protects microorganisms against pressure. Pressurization is usually more effective in suspensions than in foods. Treatment conditions (pressure, time and temperature) influence the decrease in microbial counts. Oscillatory treatments are generally more effective than continuous treatments. Pressurization induces spore germination and then inactivation of the resulting vegetative cells. Adiabatic heating due to the work of compression should also be considered. High pressure sublethally injures a fraction of the microbial population. These injured microorganisms can recover and develop, which is a risk for safety and preservation of foods. Pressure inactivation of microorganisms is often characterized by first-order kinetics survival curves, but with marked tailing.

1 Introduction High-pressure processing is an alternative technology for food safety and preservation, which makes it possible to pasteurize at room or lower temperatures and so does not markedly change the nutrient content, odour and taste, resulting in foods with good nutritional and organoleptic qualities, great safety and long shelf-life. Likewise, pressurization at mild or high temperatures greatly improves the microbiological Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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48 Microbiological Aspects of High-pressure Processing

quality of foods, with the latter being especially useful for bacterial spore inactivation (Cheftel, 1995; Heremans, 1995). From a microbiological point of view, a wide range of food products (i.e. different compositions) and microorganisms (species, serovars, strains) should be tested to avoid general conclusions that can be erroneous. It is also important to take into account the type and conditions of treatment: continuous or oscillatory pressurization, come-up time and decompression rate, magnitude of pressure, length of holding time and temperature (including adiabatic heating). Research into microbial inactivation should consider all the factors mentioned above as well as the state of vegetative cells (logarithmic growth or stationary phase) and spores and differences between inoculated suspensions and indigenous or inoculated bacterial populations in model food systems and real foods. High pressure, as other physical and chemical treatments, sublethally injures a fraction of the microbial population. These injured microorganisms are usually difficult to detect, but can recover and develop during the storage of processed foods, which is a risk for safety and preservation of those foods. The food structure by itself probably performs a baroprotective effect and so the rate of surviving microorganisms increases. Modifications in cytoplasmic membrane (the primary site of pressure damage) are probably the main cause of sublethal injury (Cheftel, 1995; Heremans, 1995).

2 Factors affecting effectiveness of treatment 2.1 Types of organisms The barosensitivity of different organisms is highly variable. In general, the higher the complexity of an organism, the greater the sensitivity to pressure treatment. Bacterial endospores, especially those of Clostridium botulinum, are the most pressure-resistant life forms (Gould, 1995). Indigenous microbiota grows more easily than inoculated laboratory collection strains. Cells in the logarithmic growth phase are more sensitive than those in the other phases (Cheftel, 1995). Benito et al. (1999) evaluated the resistance of natural isolates of Escherichia coli O157:H7 in phosphate buffer. For exponential-phase cells, 200 MPa for 8 min inactivated 5–5.5 log cfu/ml but for stationary-phase cells, it was necessary to use 500 MPa for 10 min to inactivate 4.5–6.5 log cfu/ml. The maximum temperature reached during pressurization at 500 MPa was 45°C. Pagán and Mackey (2000) observed that loss of viability was correlated with a permanent loss of membrane integrity in logarithmic growth-phase cells, whereas membranes were repaired to a greater or lesser extent following decompression in stationary-phase cells. For a given species or serovar, different strains sometimes have very different sensitivity to high pressure. Frustoli et al. (2003) inoculated Listeria monocytogenes into a broth having the same physicochemical characteristics as those of smoked salmon and observed differences among strains. They point out that the behaviour of given strains should not be generalized to other strains of the same species.

Factors affecting effectiveness of treatment 49

For bacteria, there is a correlation with the Gram stain type and cell morphology. Thus, Gram-positive bacteria are generally assumed to be more resistant than Gram-negative bacteria, with notable exceptions. On the other hand, the most sensitive bacteria are rodshaped and the most resistant are cocci (Cheftel, 1995; Ludwig and Schreck, 1997). Among the pathogenic non-sporeforming bacteria, Staphylococcus aureus is the most pressure-resistant. Noma and Hayakawa (2003) found that barotolerance of the pathogen in sodium chloride solution was even increased by pre-incubation at below 0°C. Escherichia coli O157:H7 has shown high levels of pressure resistance and Yersinia enterocolitica is one of the most sensitive pathogenic bacteria. Heat-resistant bacteria are usually more pressure-resistant than heat-sensitive bacteria (Farkas and Hoover, 2000). Shigehisa et al. (1991) reported ⭓6 log cfu/g reductions of different microorganisms inoculated into pork slurries, by pressurization at different levels for 10 min at 25°C: Campylobacter jejuni, Salmonella typhimurium, Y. enterocolitica and Pseudomonas aeruginosa at 300 MPa; E. coli, Saccharomyces cerevisiae and Candida utilis at 400 MPa; Micrococcus luteus at 500 MPa; and S. aureus at 600 MPa. In contrast, for Bacillus cereus spores, only 1 log cfu/g was inactivated. Berlin et al. (1999) worked on pathogenic Vibrio spp. (Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae and others) in artificial seawater. A treatment at 250 MPa for 10 min at 25°C resulted in all of them dropping below the detection limit. Ulmer et al. (2000) employed pressurization at 15°C in model beer inoculated with Lactobacillus plantarum. For 6.5–7 log cfu/ml reductions, it was necessary to use 400 or 500 MPa for 10 min and 600 MPa for 1 min. Yuste et al. (2003) investigated the effect of pressurization at 20°C on different pathogens inoculated into minced chicken and liquid whole egg. At 400 MPa for 5 min, no growth occurred, except for E. coli (4–5.5 log cfu/g or ml reductions) and S. aureus, which was the most resistant. In general, the sensitivity of pathogens was: Y. enterocolitica ⬎ S. typhimurium ⬎ E. coli ⬎ L. monocytogenes ⬎ S. aureus. On pressurizing suspensions inoculated with a pure culture, there is an initial lag period, followed by a consistent inactivation rate (first-order inactivation kinetics, which is usually predominant) and finally a more reduced inactivation rate (tailing, which is extreme in some cases mainly due to spores). A possible explanation for tailing is that a small fraction of the cell population is more pressure-resistant or adapted to the pressure stress that makes the remaining cells more resistant. Therefore, tailing is an indication of heterogeneity in the microbial population with respect to the high pressure effect. Thus, only using a linear model is not adequate for describing the pressure inactivation. The fact that some food-borne pathogens have a low infective dose may require a zero tolerance for these microorganisms in foods. Therefore, it is important to find ways to reduce or eliminate the tailing effect in high-pressure processing, such as the use of combined treatments or oscillatory instead of continuous pressurization (Farkas and Hoover, 2000; Masschalk et al., 2001; Chen and Hoover, 2003).

2.2 Food products Different food constituents and ingredients can play a protective role against pressure. Carbohydrates are generally more baroprotective than salts (Smelt, 1998). Simpson

50 Microbiological Aspects of High-pressure Processing

and Gilmour (1997a) compared the effect of pressure on L. monocytogenes in phosphate buffer with that in model food systems, to investigate whether survival to high pressure increases in media with nutrients, which are essential for repair or may provide protection against damage. Treatment at 375 MPa for 20 min at 20°C induced decreases of 6–7 log cfu/ml with phosphate buffer. In buffer with an added source of protein (bovine serum albumin), carbohydrates (glucose) or fat (olive oil), counts were reduced by 3.5–5.5, 4.5–6 and 5–6 log cfu/ml, respectively. Gervilla et al. (2000) studied the inactivation of different bacteria inoculated into ewe’s milk at three fat contents (0, 6 and 50 per cent). They found the following pressure sensitivities: Pseudomonas fluorescens ⬎ E. coli ⭓ Listeria innocua ⬎ Lactobacillus helveticus ⬎ S. aureus. Compared to reductions in Ringer’s solution, ewe’s milk showed a baroprotective effect on all microorganisms. However, the effect of fat content was not clear: in some cases, either there was no baroprotection or increasing fat content did not result in increasing baroprotection. On the other hand, the food structure by itself, which determines microbial attachment to food constituents, probably performs a baroprotective effect. Patterson et al. (1995) showed the influence of substrate on survival of the pathogens, with different protective effects depending on the organisms. Tables 3.1 and 3.2 compare the inactivation of E. coli O157:H7 and L. monocytogenes under different pressure treatments.

Table 3.1 High-pressure inactivation (log cfu/ml or g) of Escherichia coli O157:H7 inoculated into foods Food

Treatment

Inactivation

Reference

UHT milk Poultry

600 MPa/30 min/20°C

1.5 2.5

Patterson et al. (1995)

UHT milk Poultry

400 MPa/15 min/50°C

5.0 6.0

Patterson and Kilpatrick (1998)

Orange juice UHT skimmed milk

550 MPa/5 min/30°C 200 MPa/30 min/10°C

⭓6.0 0.3

Linton et al. (1999) Linton et al. (2000)

Table 3.2 High-pressure inactivation (log cfu/ml or g) of Listeria monocytogenes inoculated into foods Food

Treatment

Inactivation

Reference

UHT milk Poultry

375 MPa/30 min/20°C

1.5 5.5

Patterson et al. (1995)

Pork loin

414 MPa/10 min/25°C

6.0

Ananth et al. (1998)

Beef frankfurter

300 MPa/8 min/15°C 500 MPa/3 min/15°C

6.0 6.0

Lucore et al. (2000)

UHT milk

500 MPa/35 min/20°C 500 MPa/1 min/50°C 500 MPa/5 min/50°C

8.0 6.5 8.0

Chen and Hoover (2003)

Liquid whole egg Minced chicken

400 MPa/5 min/20°C

4.5 5.0

Yuste et al. (2003)

Factors affecting effectiveness of treatment 51

Pressurization is generally most effective in inoculated suspensions (buffers or microbiological media) and less effective in real foods than in model food systems. From that point of view, two facts greatly determine food microbiological safety and stability: the effect of the food during treatment and after treatment and during recovery of microorganisms. Therefore, the results of studies in suspensions or model food systems cannot be directly extrapolated to real foods. Pressure treatment damages microbial cells and so sensitizes them to other stresses (treatments, agents or environmental conditions). Low pH enhances inactivation during treatment and also inhibits growth of cells sublethally injured, because they are more acid sensitive than native cells. Although repair proteins possibly are not damaged, acidic conditions may inhibit repair (Farkas and Hoover, 2000). Linton et al. (2000) and Pagán et al. (2001) observed that pressure treatment sensitizes E. coli O157:H7 to acidity and heat. Sensitization may involve loss of protective and repair functions. Therefore, high pressure improves the safety of acidic or heated foods. García-Graells et al. (1998) treated, at 300 MPa for 15 min at 20°C, juices inoculated with pressure-resistant E. coli mutants. Reductions were ⬍1 log cfu/ml in orange (pH 3.8) and mango (pH 4.0) and ⬎2 log cfu/ml in apple (pH 3.3). Low pH also enhanced inactivation during storage at 8°C: counts decreased by 3.5 log cfu/ml after 4 day storage in mango juice, 5 log cfu/ml after 13 day storage in orange juice and 8 log cfu/ml after 2 day storage in apple juice. Compression may shift the pH of foods. The direction and magnitude of pH shift depend on each food treatment process. Usually, lowering pH under pressure increases microbial inactivation (Heremans, 1995). In general, low water activity makes most microorganisms more pressure resistant. However, injured microorganisms can be inhibited by low water activity. Consequently, the net effect of water activity is difficult to predict (Farkas and Hoover, 2000). Butz et al. (1996) employed pressurization in different solutions, buffers and foods inoculated with heat-resistant moulds and found that at 70°C Byssochlamys nivea ascospores in solutions and buffers were reduced by 4 log cfu/ml at 600 MPa for 60 min, 700 MPa for 30 min and 800 MPa for 5 min. Treatment at 700 MPa for 15 min decreased 5 log cfu/ml in grape juice and only 1 log cfu/g in bilberry jam. So, jam is strongly baroprotective due to the low water activity. Oxen and Knorr (1993) studied the behaviour of Rhodotorula rubra in sucrose solutions and found that the higher the sucrose concentration, the lower the inactivation of the yeast. The material and type of packaging, with the resulting redox potential, during and after treatment also play a role in the inactivation for some microorganisms. As microbial inactivation usually continues after high-pressure processing, the intrinsic characteristics of the food as well as the storage conditions greatly influence survival or death of microorganisms that have not been inactivated by the treatment, which determines to a great extent the safety and shelf-life of pressurized foods. Capellas et al. (1996) studied pressurization (400–500 MPa for 5–15 min at 2–25°C) in fresh cheese made with E. coli-inoculated goat’s milk and they did not detect growth throughout 60-day vacuum storage at 4°C, except for samples treated at 400 and 450 MPa for 5 min at 25°C. Yuste et al. (1998, 2002) reported considerable reductions of the microbial population and extension of shelf-life (vacuum storage at 2°C)

52 Microbiological Aspects of High-pressure Processing

of mechanically recovered poultry meat, by combining high pressure (350 and 450 MPa for 5 and 15 min at 2 and 20°C) with nisin (100 and 200 ppm) and nisin was found to be more effective at lower pH (addition of 1 per cent of glucono-delta-lactone). Pressurization at ⫺20°C was also tested, with decreases in counts not so obvious as at the other two treatment temperatures (2 and 20°C). Aerobic psychrotrophs were found to be more sensitive than aerobic mesophiles, probably because a part of the surviving psychrotroph population was sublethally injured and was not able to grow under refrigeration. Yuste et al. (2000a) compared pressurization (500 MPa for 5 and 15 min at 65°C) and heating (80–85°C for 40 min) applied to cooked poultry sausages. Pressure was more effective against lactic acid bacteria and Listeria spp. than heat and both treatments were very effective against enterobacteria. Minimal or no growth of Listeria spp. and enterobacteria occurred in pressurized sausages throughout 18 week chilled vacuum storage. High pressure proved to be a valid treatment for replacement of heat pasteurization of cooked meat and poultry products.

2.3 Conditions of treatments Increases in the magnitude of pressure and the length of holding time generally lead to higher lethalities, but not linearly. There is a minimum critical pressure that depends on the type of organism below which microbial inactivation will not take place regardless of process time (Farkas and Hoover, 2000). Pagán and Mackey (2000) state that the mechanism of inactivation may differ depending on the level of pressure. Mild heat (45–50°C) considerably enhances pressure-induced killing of microorganisms. Patterson and Kilpatrick (1998) reported larger inactivation by pressurization at 50°C than by either treatment alone, in poultry and UHT milk inoculated with E.coli O157:H7 and S. aureus (see Table 3.1). Capellas et al. (2000) evaluated pressurization of fresh cheese made with inoculated goat’s milk. At 500 MPa for 5 min at 50°C, Staphylococcus carnosus was reduced by 7 log cfu/g, whereas at 500 MPa for 30 min at 10 and 25°C, counts did not substantially decrease. For Bacillus subtilis, germination treatment at 60 MPa for 210 min followed by inactivation treatment at 500 MPa for 5 min, both at 40°C, was more effective than the same combination of treatments applied at 25°C. Treatment temperature is closely related to other parameters, such as holding time and pH of the sample. Park et al. (2001) investigated the pressure effect on inoculated Lactobacillus viridescens. Pressurizations for 5 min decreased counts by 8 log cfu/ml at 600 MPa at 25°C and 400 MPa at 50°C in MRS broth (de Man, Rogosa and Sharpe broth). For a 7 log cfu/g reduction in ham, a treatment at 500 MPa for 5 min at 45°C was necessary. So, increasing the process temperature solved the lower level of inactivation in ham. Chen and Hoover (2003) also tested pressurization at 50°C for inactivation of L. monocytogenes inoculated into UHT milk (see Table 3.2). The treatment resulted in an initial rapid drop followed by a long tailing. At 500 MPa, 5 and 35 min of treatment were necessary at 50 and 25°C, respectively, to achieve a 8 log cfu/ml reduction. Yuste et al. (2000b) used pressurization (500 MPa for 10 and 30 min at 50, 60 and 70°C) in Salmonella enteritidis-inoculated poultry sausages. Decreases were

Factors affecting effectiveness of treatment 53

⬎7 log cfu/g for all treatments, whereas heating alone induced ⭓7 log cfu/g reductions only at or above 70°C. Linton et al. (1999) reported a ⭓6 log cfu/ml reduction when orange juice inoculated with a pressure-resistant strain of E. coli O157:H7 was treated at 550 MPa for 5 min (see Table 3.1). At 20°C, the reduction occurred at pH 3.9 and 4.5, but not at pH 5.0, whereas at 30°C, it was at pH 3.9, 4.5 and 5.0. This suggests the importance of the temperature of pressurization for inactivation of pathogens. Decimal reduction time (D value), an important kinetic parameter obtained from inactivation curves, has been studied by many authors. In Table 3.3, where some D values for high-pressure processing are reported, the importance of treatment temperature and the differences in sensitivity between vegetative cells and spores are shown. Oscillatory treatments (several cycles either at high pressure or by alternating medium and high pressures) are generally more effective than equivalent continuous treatments of comparable total holding time, against both vegetative cells and spores. The higher inactivation rate is probably due to greater injury to the cell membrane from rapid changes in intracellular-extracellular differences at the membrane interface (Palou et al., 1998). The pressure-pulse profile (i.e. the number and frequency of cycles) influences microbial inactivation. Palou et al. (1998) evaluated effects on a Zigosaccharomyces bailii-inoculated model food system (Sabouraud broth at aw 0.98 and pH 3.5) treated at 276 MPa for 10, 15 and 20 min (total holding time) at 20°C. They compared continuous with oscillatory pressurizations (2, 3 and 4 cycles of 5 or 10 min each) and found cycles more effective (reductions 1 log cfu/ml higher in treatments for 15 and 20 min of total holding time). Hayakawa et al. (1994) compared continuous and oscillatory (cycles of 5 min each) pressurizations in Bacillus stearothermophilus suspensions. Treatments at 800 MPa for 60 min at 60 and 70°C decreased spore counts by 3.5–4.5 log cfu/ml. Oscillatory pressurization was more effective as at 600 MPa at 70°C reductions were 3 (2 cycles), 4.5 (4 cycles) and 6 log cfu/ml (6 cycles). Ponce et al. (1999) found a large inactivation of S. enteritidis inoculated into liquid whole egg and pressurized at 50°C. They reported an 8 log cfu/ml reduction at 350 and 450 MPa, with oscillatory treatments (2 and 3 cycles of 5 min each) being more effective than continuous treatments for the same total holding time.

Table 3.3 High-pressure-induced D values (min) of inoculated bacteria Substrate

Pressure (MPa)

Microorganism

Pork loin

414

L. monocytogenes S. typhimurium

Ewe’s milk

300 250

E. coli P. fluorescens

Distilled water

400

B. subtilis spores

600

D value (°C)

Reference

1.3 (2) 2.2 (25) 1.5 (2) 0.9 (25)

Ananth et al. (1998)

2.5 (50) 2.8 (50)

Gervilla et al. (1999)

20.0 (51–52) 2.2 (73–75) 2.2 (51–52) 0.4 (73–75)

Gola et al. (2001)

54 Microbiological Aspects of High-pressure Processing

Come-up time and decompression rate are important factors. A slow ramp rate may induce a stress response and so make the process less effective. On the other hand, a fast depressurization may contribute to higher inactivation, due to cavitations in the cells and spores that result in physical disruption (Smelt, 1998). Lucore et al. (2000) demonstrated the influence of come-up time on bacterial inactivation. During the time to achieve 300, 500 and 700 MPa (from 1 to 2 min), at 15°C, L. monocytogenes inoculated into beef frankfurters decreased by 1, ⬎3 and ⬎5 log cfu/g, respectively. Pressure treatment always results in a temperature increase due to the work of compression (adiabatic heating). Depending on the magnitude of pressure, composition of the food and type of pressure-transmitting fluid (compressibility and specific heat), adiabatic heating may be considerable and markedly affect microbial inactivation (Chen and Hoover, 2003).

2.4 Combined treatments Combination of high pressure with other physical treatments or chemical agents based on the hurdle concept has been tested and applied for food safety and preservation, generally with a killing effect greater than that of either treatment alone. Surviving vegetative microorganisms, especially those sublethally injured, are not able to develop and try to restore their homeostasis. In most cases, they use up their energy and finally die because of metabolic exhaustion (Leistner, 2000). As toxigenic microorganisms are harmless if they cannot multiply, sublethal injury of these microbes is enough when combined with suboptimal growth conditions in the food. Pressure-inducible proteins, similar to those described for heat, have been found (Smelt, 1998). Protective stress shock proteins make microorganisms more resistant or virulent. Combined treatments require energy-consuming synthesis of several types of stress shock proteins, which may contribute to metabolic exhaustion. A synergistic killing effect occurs if gentle hurdles with different targets in the microbial cells are applied, because the recovery of homeostasis and the activation of stress shock proteins are more difficult (Leistner, 2000). Table 3.4 shows the inactivation of different pathogens under combinations of high pressure with other physical or chemical treatments. Hauben et al. (1997) evaluated inactivation of pressure-resistant E. coli mutants in phosphate buffer. At 600 MPa for 15 min at 20°C, counts decreased by 0.5 log cfu/ml, whereas the parent E. coli strain was reduced by 8.5 log cfu/ml. Pressure inactivation was higher in cell suspensions supplemented with nisin than in those without nisin, for both barotolerant mutants and the parent strain (Table 3.4). Furthermore, due to a particular stress, microorganisms may acquire cross-tolerance. Cells subjected to stresses other than pressure become more resistant to pressure, due to stabilization of the structure of membrane-bound enzymes (Smelt, 1998). Wuytack et al. (1998) found that pressure treatment resulted in a fraction of spores becoming resistant to pressure and other physical and chemical treatments because of the presence of more small acid-soluble proteins after pressurization. In contrast, Metrick et al. (1989) examined a treatment at 340 MPa for 40 min at 25°C on a heat-sensitive strain of S. typhimurium and Salmonella senftenberg (the most heat-resistant Salmonella), in

Factors affecting effectiveness of treatment 55

Table 3.4 Inactivation (log cfu/ml or g) of inoculated bacteria induced by high pressure combined with other treatments Substrate

Pressure treatment

Other treatment

Microorganism

Inactivation

Reference

Phosphate buffer

250 MPa/ 15 min/20°C

Nisin (100 IU/ml)

E. coli

2–3.5

Hauben et al. (1997)

Peptone solution

345 MPa/ 1 min/22°C

Nisin and/ or pediocin (5000 AU/ml) Pediocin (3000 AU/ml)

E. coli O157:H7 L. monocytogenes S. typhimurium L. monocytogenes E. coli O157:H7 S. typhimurium S. aureus

6.5–8.5 6.5–9.5 8.5–10 3.5 4.5 8 8.5

Kalchayanand et al. (1994)

Bacteriocin (1280 AU/g) Pediodin or sakacin or enterocin (1280 AU/g) Nisin (1280 AU/g)

Salmonella spp.

6

Garriga et al. (2002)

L. monocytogenes

6

E. coli

6

C. sporogenes spores

5–6

345 MPa/ 5 min/25°C

Meat model system

Chicken

400 MPa/ 10 min/17°C

680 MPa/ 10 min/80°C

Ionizing radiation (3 kGy)

Kalchayanand et al. (1998)

Crawford et al. (1996)

inoculated phospate buffer and chicken baby food and demonstrated that heat-resistance does not always imply pressure-resistance. Nakayama et al. (1996) inoculated six Bacillus strains into suspensions and did not find a correlation between pressure- and heat-resistances. Crawford et al. (1996) tested pressurization at 80°C and ionizing radiation for inactivation of spores of Clostridium sporogenes in chicken. At 680 MPa for 20 min, counts decreased by 2 log cfu/g. Radiation at 3 kGy induced a 1.5 log cfu/g reduction. Combination of pressure and ionizing radiation resulted in larger inactivation (see Table 3.4). Chicken treated at 680 MPa for 20 min and 6 kGy showed a decrease ⭓3 log cfu/g higher than pressurized chicken. Masschalk et al. (2001) tested the bactericidal activity of pressurization for 15 min at 20°C in phosphate buffer, with and without lactoferrin (500 ␮g/ml), lactoferricin (20 ␮g/ml) or nisin (100 IU/ml). For inactivations in the range of ⬎0.5–⬎3 log cfu/ml, they evaluated different magnitudes of pressure depending on the microorganism: P. fluorescens ⬍ Shigella sonnei ⬍ S. typhimurium ⫽ Shigella flexneri ⬍ S. enteritidis ⬍ E. coli O157:H7 ⬍ S. aureus. In general, the combined treatments induced larger inactivations than high pressure alone, with nisin being the most effective, followed by lactoferricin. Kalchayanand et al. (1994, 1998) combined pressurization (345 MPa at 25°C) and bacteriocins against several bacteria in peptone solutions. Addition of nisin and/or pediocin enhanced the effect of pressure treatment on E. coli O157:H7, L. monocytogenes and S. typhimurium (see Table 3.4). With pressurization for 5 min, reductions (log cfu/ml) were: 1.3–2.3 for L. monocytogenes and Lactobacillus

56 Microbiological Aspects of High-pressure Processing

sakei, 3.8 for E. coli O157:H7, 6.7–7.2 for S. aureus and P. fluorescens and 8–8.3 for Leuconostoc mesenteroides, S. typhimurium and Serratia liquefaciens. High pressure plus pediocin (3000 AU/ml) increased the killing effect by 0.5 log cfu/ml for L. mesenteroides and E. coli O157:H7 and 1.5–2 log cfu/ml for L. sakei, S. aureus and L. monocytogenes (see Table 3.4). The authors (Kalchayanand et al., 1994, 1998) state that high pressure sublethally injures a fraction of the microbial populations and these cells are more sensitive to bacteriocins. Through nisin addition (5 mg/l), Ponce et al. (1998) improved pressure (450 MPa for 10 min at 20°C) inactivation of L. innocua and E. coli inoculated into liquid whole egg. Garriga et al. (2002) studied pressurization at 400 MPa for 10 min at 17°C in a meat model system, with and without bacteriocins (1280 AU/g), subsequently vacuum stored at 4°C for 60 days. A 6 log cfu/g reduction and no significant changes in counts during storage were observed with all treatments (high pressure alone and combined with any bacteriocins) for Salmonella spp. and with pressure combined with bacteriocins for other organisms: pediocin or sakacin or enterocins for L. monocytogenes, nisin or enterocins for L. sakei (slime-producing organism) and nisin for E. coli and Leuconostoc carnosum (slime-producing organism) (see Table 3.4). Staphylococcus spp. were the most resistant, with the combination of high pressure and nisin giving the best results and the most marked effect (a 4 log cfu/g reduction at the end of storage) occurring in S. carnosus (starter organism). Papineau et al. (1991) investigated high pressure (238 MPa for 30 min) combined with chitosans lactate or hydroglutamate (0.2 mg/ml, 30-min exposures). Reductions were 4–6 and 3–5.5 log cfu/ml for E. coli and S. aureus suspensions, respectively. Adegoke et al. (1997) combined high pressure (180 MPa for 1 h at 25°C) and monoterpenes for inactivation of S. cerevisiae suspensions. Pressurization alone decreased counts by 1.5–2 log cfu/ml, whereas reductions were 4.5 and 6.5 log cfu/ml in pressure-treated suspensions containing 2200 ␮g/ml of D-limonene and 150 ␮g/ml of ␣-terpinene, respectively.

3 Effects of high pressure 3.1 Bacterial and fungal cells Pressurization affects morphology, cell wall and membrane, biochemical reactions and genetic mechanisms, which explains to a great extent the mode of action of high pressure against microorganisms (Hoover et al., 1989). 3.1.1 Morphology

Under high pressure, the following morphological changes have been reported: cell compression, with partial irreversibility upon return to atmospheric pressure; separation of the cell wall from the cytoplasmic membrane; proteins more detached from the membrane; vacuolar regions in the cytoplasm; structural damage of the nuclear membrane, mitochondria, cytoskeleton and lysosomes (with resulting leakage of

Effects of high pressure 57

enzymes into cytoplasm); and collapse of intracellular gas vacuoles (Cheftel, 1995; Knorr, 1995; Smelt, 1998).

3.1.2 Cell wall and membrane

The primary site of pressure damage is the cell membrane. Cell permeability and so ion exchange are affected, mainly due to alteration of carriers of transport systems. Thus, homeostatic and barrier functions are impaired. The pressure-set crystallization of membrane phospholipids probably contributes to the inactivation of several microorganisms. Membrane potential decrease has also been described (Cheftel, 1995; Ritz et al., 2001). At more than 500 MPa, it is possible to observe internal cellular damage, whereas at less than 500 MPa, in addition, disruption of wall and membrane commonly occurs, resulting in leakage of intracellular constituents from the cytoplasm and permeation of extracellular substances into cells (Kato and Hayashi, 1999; Farkas and Hoover, 2000). Compared with untreated cells, pressurized bacteria can be stained with propidium iodide or ethidium bromide, which only penetrate into the cell when the membrane is altered and then bind to nucleic acids. Russell et al. (1995) state that less fluid membranes are more pressure-sensitive. Cells with a relatively high content of diphosphatidylglycerol are more sensitive due to the interaction of this with divalent cations, which causes rigidity in membrane. Unsaturated fatty acids play a role in maintaining the proper fluidity of membrane phospholipids. If cells are grown at a suboptimal temperature, their fatty acids become more unsaturated with the consequent pressure-resistance increase. According to Kato and Hayashi (1999), the fluidity of the membrane decreases under pressure, which results in functional disorders of associated enzymes and, finally, reversible fragmentation of the phospholipid bilayer and denaturation of enzymes and other proteins associated with the membrane. Modifications in the cell membrane are probably the main cause of sublethal injury, which often induces sensitivity to salts and other selective agents and an extension of the lag time. Selective culture media contain agents that may inhibit the repair and subsequent development of injured cells. This has negative implications because these cells are not usually detected, which allows pressure inactivation to be over-evaluated, but the cells can recover and develop during the storage of processed foods. Fluorescent dyes allow discrimination between dead and living cells, but not between viable and non-viable injured cells. Real-time polymerase chain reaction (PCR) is a promising technique for the detection and identification of the latter cells. Yuste et al. (1999) investigated pressurization (500 MPa for 5 and 15 min at 65°C) and heating (80–85°C for 40 min) in cooked poultry sausages and found similar rates of sublethally injured cells. However, they observed that, after several weeks of vacuum storage at 6–8°C, mesophile and psychrotroph counts in heat-treated sausages remained low when submitted to a temperature abuse, whereas in pressurized sausages counts substantially increased. This is because changes induced by heat are different from those induced by pressure, which are reversible and so cells are able to grow and multiply again under favourable conditions. Ritz et al. (2000, 2001) observed

58 Microbiological Aspects of High-pressure Processing

variable degrees of pressure-induced injury and also suggested reversible damage and cellular repair. They reported that the outer membrane (especially the associated proteins) seems to be more damaged. Mackey et al. (1994) also observed that cells are not affected equally. Chilton et al. (2001) found that membrane damage was repaired when cells were incubated at 37°C in tryptone soya broth. They stated that damage in the cytoplasmic membrane probably differs from that in the outer membrane due to the different structures. 3.1.3 Biochemical reactions

Changes in microbial key enzymes are an important reason for the inhibitory effects of pressure. Membrane damage, protein denaturation and decrease of intracellular pH suggest that membrane-bound enzymes associated with efflux of protons may be one of the major targets in pressure inactivation of microorganisms. Ion movements by membrane-bound ATPases are also altered due to inactivation or dislocation of enzymes such as Na⫹-K⫹-ATPase and Ca2⫹-ATPase (Smelt, 1998). Simpson and Gilmour (1997b) observed a wide variation in barotolerance of microbial enzymes. Multimeric enzymes seem to be more sensitive because pressure induces dissociation into subunits. 3.1.4 Genetic mechanisms

Nucleic acids are more resistant to pressure-induced denaturation than proteins. The structure of nucleic acids can remain intact even at 1000 MPa (Heremans, 1995). However, Chilton et al. (1997) observed that DNA and ribosomic RNA chains are ruptured in vivo by pressure. There is also extreme (but reversible) condensation of the nuclear material and cell division slows down (Smelt, 1998). As enzymes are affected by high pressure, mechanisms of DNA replication and transcription, and translation into proteins are inhibited. The inhibition of protein synthesis is also caused by dissociation of ribosomes under pressure (Cheftel, 1995).

3.2 Bacterial spores The great resistance of bacterial endospores is one of the major challenges to the application of high-pressure processing. While most vegetative cells are inactivated at 400–600 MPa, spores of some species can survive at pressures above 1000 MPa (Gould, 1995). Pressure resistance of spores is highly variable, depending on the conditions of sporulation and pressurization. Bacterial endospores can be inactivated at pressures in the range of 500–700 MPa at 90–110°C. Unless pressure is higher than 800 MPa, heat is required for inactivation of sporeforming bacteria in low-acid foods (Farkas and Hoover, 2000). Pressure resistance of spores is attributed to their inner viscous state or to the presence of dipicolinic acid, which protects them against pressure (Knorr, 1995). The greater the dipicolinic acid release, the higher the spore germination. The temperature of sporulation of bacteria determines their pressure resistance, as they are more resistant when sporulated at lower temperatures. Initiation of pressure-induced germination is also affected by sporulation at lower temperatures (Farkas and Hoover, 2000).

Effects of high pressure 59

Spore inactivation can be achieved by applying a number of high pressure cycles, which induces changes in permeability and disruption of the wall (Hayakawa et al., 1994). Spores are killed more rapidly at low pH, but pressure-induced germination is faster at neutral pH (Smelt, 1998). Pressure treatment can induce spore germination under relatively mild conditions (e.g. by combining pressures from 50 to 250 MPa with temperatures above 40°C). Low temperatures and extreme pH values minimize such germination (Gould and Sale, 1970). Under atmospheric pressure, activation of spores prior to germination is necessary by low pH or especially by heat. It is not known whether pressure induces activation in a manner similar to heat. Treatment at medium pressure seems to enhance spore germination more than exposure to heat (Farkas and Hoover, 2000). Germination is usually a prerequisite for pressure inactivation of sporeforming bacteria. Generally, pressurization induces spore germination and then inactivation of the resulting vegetative cells (Gould and Sale, 1970). Therefore, another mechanism to minimize spore survival is by alternating medium pressure (for germination) and high pressure (for inactivation), once or several times. Wuytack et al. (1998) studied pressure-induced germination of B. subtilis in phosphate buffer. The level of germination was similar at 100 and 500 MPa for 30 min at 40°C. However, spores germinated at 100 MPa were more sensitive (⬎3 log cfu/ml difference) to inactivation at 600 MPa for 10 min than spores germinated at 500 MPa, which remained almost unaffected. Spores germinated at 100 MPa were also more sensitive to ultraviolet light and hydrogen peroxide.

3.3 Parasites Ohnishi et al. (1992) studied the effect of high pressure on Trichinella spiralis. At 200 MPa for 10 min at 25°C, the organism was neither motile (larvae recovered from muscle) nor infective (muscle larvae and adult warms recovered from intestine). The authors (Ohnishi et al., 1992) stated that pressurization of meat at ⬎200 MPa can prevent trichinellosis and be considered as an effective substitute for heating. Butz and Tauscher (1995) reported no fruit fly (Ceratitis capitata) egg survival (i.e. no development of larvae and flies) at ⬎125 MPa at 25°C, almost independently of treatment time. As parasites are more pressure-sensitive than bacterial cells, it can be assumed that food-borne parasites do not survive pressure treatments designed for inactivation of pathogenic bacteria.

3.4 Viruses Viruses have a high degree of structural diversity, which results in a wide range of pressure resistances. There are two potential applications of pressure inactivation of viruses: vaccine development and virus sterilization. Pressurization inactivates viruses but preserves their immunogenic properties, because usually it does not markedly change viral structure. Furthermore, the antibodies against pressurized virus particles are as effective as those against untreated viruses. High-pressure processing could be a good

60 Microbiological Aspects of High-pressure Processing

method of vaccine preparation, i.e. with the whole virus particle and lack of infectivity (Pontes et al., 1997). Otake et al. (1997) investigated pressure effects on human immunodeficiency virus suspensions. With pressurization for 10 min at 25°C, two clinical strains decreased by 4–5 log TCID50/ml (50 per cent tissue culture infectious dose/ml) at 550 MPa, whereas a laboratory strain decreased by ⬎5 log TCID50/ml (complete loss of infectivity) at 450 MPa. They observed a decrease in reverse transcriptase activity and rupture of the viral envelope with resulting leakage of core protein. They also stated that the degree of enzyme inactivation and structural changes of viral particles determine the decrease in human immunodeficiency virus infectivity, which is different for different strains. Studies made with vegetable, animal and human viruses show that exposure to pressures below 400 MPa is generally almost ineffective. Most human viruses are probably inactivated at pressures that kill bacteria.

4 Conclusions High-pressure processing kills microorganisms effectively, resulting in foods of very good microbiological quality, with enhanced safety and longer shelf-life. For inactivation of bacterial endospores, the most pressure-resistant organisms, simultaneous application of heat is usually required. High pressure is a valid technology for replacement of heat treatment of some food products. Combination of pressure treatment with other physical or chemical treatments, not only exploiting membrane damage but also having different targets in the microbial cells, is recommended to guarantee food safety and preservation, because a fraction of the microbial population is often only sublethally injured by high pressure. The combined treatment, based on the hurdle concept, has a killing effect greater than that of pressurization alone, with the effect sometimes being synergistic. Some specific remarks:

• Development of barotolerance should cause concern about the safety of high pressure processing of food.

• The marked temperature increase due to adiabatic heating should be considered when pressure treatment conditions are designed. • Tailing should be reduced or avoided, because it is a problem in quantitative studies and the development of predictive models. • Mathematical models describing the pressure inactivation kinetics of microorganisms would be helpful for the food industry to develop safe process conditions.

References Adegoke GO, Iwahashi H, Komatsu Y (1997) Inhibition of Saccharomyces cerevisiae by combination of hydrostatic pressure and monoterpenes. Journal of Food Science, 62, 404–405.

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Ananth V, Dickson JS, Olson DG, Murano EA (1998) Shelf life extension, safety, and quality of fresh pork loin treated with high hydrostatic pressure. Journal of Food Protection, 61, 1649–1656. Benito A, Ventoura G, Casadei M, Robinson T, Mackey BM (1999) Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses. Applied and Environmental Microbiology, 65, 1564–1569. Berlin DL, Herson DS, Hicks DT, Hoover DG (1999) Response of pathogenic Vibrio species to high hydrostatic pressure. Applied and Environmental Microbiology, 65, 2776–2780. Butz P, Tauscher B (1995) Inactivation of fruit fly eggs by high pressure treatment. Journal of Food Processing and Preservation, 19, 161–164. Butz P, Funtenberger S, Haberditzl T, Tauscher B (1996) High pressure inactivation of Byssochlamys nivea ascospores and other heat resistant moulds. LebensmittelWissenschaft und-Technologie, 29, 404–410. Capellas M, Mor-Mur M, Gervilla R, Yuste J, Guamis B (2000) Effect of high pressure combined with mild heat or nisin on inoculated bacteria and mesophiles of goat’s milk fresh cheese. Food Microbiology, 17, 633–641. Capellas M, Mor-Mur M, Sendra E, Pla R, Guamis B (1996) Populations of aerobic mesophils and inoculated E. coli during storage of fresh goat’s milk cheese treated with high pressure. Journal of Food Protection, 59, 582–587. Cheftel JC (1995) Review: High-pressure, microbial inactivation and food preservation. Food Science and Technology International, 1, 75–90. Chen H, Hoover DG (2003) Modeling the combined effect of high hydrostatic pressure and mild heat on the inactivation kinetics of Listeria monocytogenes Scott A in whole milk. Innovative Food Science and Emerging Technologies, 4, 25–34. Chilton P, Isaacs NS, Mackey BM, Stenning R (1997) The effects of high hydrostatic pressure on bacteria. In High Pressure Research in the Biosciences and Biotechnology (Heremans K, ed.). Leuven: Leuven University Press, pp. 225–228. Chilton P, Isaacs NS, Mañas P, Mackey BM (2001) Biosynthetic requirements for the repair of membrane damage in pressure-treated Escherichia coli. International Journal of Food Microbiology, 71, 101–104. Crawford YJ, Murano EA, Olson DG, Shenoy K (1996) Use of high hydrostatic pressure and irradiation to eliminate Clostridium sporogenes spores in chicken breast. Journal of Food Protection, 59, 711–715. Farkas DF, Hoover DG (2000) High pressure processing. Journal of Food Science, 65 (special supplement: Kinetics of Microbial Inactivation for Alternative Food Processing Technologies), 47–64. Frustoli M, Gola S, Miglioli L, Rovere P (2003) Combined heat/high-pressure treatments of Listeria spp. in a model system. Industria Conserve, 78, 169–182. García-Graells C, Hauben KJA, Michiels CW (1998) High-pressure inactivation and sublethal injury of pressure-resistant Escherichia coli mutants in fruit juices. Applied and Environmental Microbiology, 64, 1566–1568. Garriga M, Aymerich MT, Costa S, Monfort JM, Hugas M (2002) Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage. Food Microbiology, 19, 509–518.

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Gervilla R, Ferragut V, Guamis B (2000) High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents. Journal of Dairy Science, 83, 674–682. Gervilla R, Mor-Mur M, Ferragut V, Guamis B (1999) Kinetics of destruction of Escherichia coli and Pseudomonas fluorescens inoculated in ewe’s milk by high hydrostatic pressure. Food Microbiology, 16, 173–184. Gola S, Rovere P, Carpi G (2001) Inactivation of Bacillus subtilis and Bacillus cereus spores subjected to heat treatments under high pressure. Industria Conserve, 76, 365–370. Gould GW (1995) The microbe as a high pressure target. In High Pressure Processing of Foods (Ledward DA, Johnston DE, Earnshaw RG, Hasting APM, eds). Loughborough: Nottingham University Press, pp. 27–36. Gould GW, Sale AJH (1970) Initiation of germination of bacterial spores by hydrostatic pressure. Journal of General Microbiology, 60, 335–346. Hauben KJA, Bartlett DH, Soontjens CCF, Cornelis K, Wuytack EY, Michiels CW (1997) Escherichia coli mutants resistant to inactivation by high hydrostatic pressure. Applied and Environmental Microbiology, 63, 945–950. Hayakawa I, Kanno T, Yoshiyama K, Fujio Y (1994) Oscillatory compared with continuous high pressure sterilization on Bacillus stearothermophilus spores. Journal of Food Science, 59, 164–167. Heremans K (1995) High pressure effects on biomolecules. In High Pressure Processing of Foods (Ledward DA, Johnston DE, Earnshaw RG, Hasting APM, eds). Loughborough: Nottingham University Press, pp. 81–98. Hoover DG, Metrick C, Papineau AM, Farkas DF, Knorr D (1989) Biological effects of high hydrostatic pressure on food microorganisms. Food Technology, 43 (3), 99–107. Kalchayanand N, Sikes A, Dunne CP, Ray B (1994) Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins. Applied and Environmental Microbiology, 60, 4174–4177. Kalchayanand N, Sikes A, Dunne CP, Ray B (1998) Interaction of hydrostatic pressure, time and temperature of pressurization and pediocin AcH on inactivation of foodborne bacteria. Journal of Food Protection, 61, 425–431. Kato M, Hayashi R (1999) Effects of high pressure on lipids and biomembranes for understanding high-pressure-induced biological phenomena. Bioscience, Biotechnology and Biochemistry, 63, 1321–1328. Knorr D (1995) Hydrostatic pressure treatment of food: microbiology. In New Methods of Food Preservation (Gould GW, ed.). Bishopbriggs: Blackie Academic & Professional (Chapman & Hall), pp. 159–175. Leistner L (2000) Basic aspects of food preservation by hurdle technology. International Journal of Food Microbiology, 55, 181–186. Linton M, McClements JMJ, Patterson MF (1999) Inactivation of Escherichia coli O157:H7 in orange juice using a combination of high pressure and mild heat. Journal of Food Protection, 62, 277–279. Linton M, McClements JMJ, Patterson MF (2000) The combined effect of high pressure and storage on the heat sensitivity of Escherichia coli O157:H7. Innovative Food Science and Emerging Technologies, 1, 31–37.

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Overview of Pulsed Electric Field Processing for Food Stefan Toepfl, Volker Heinz and Dietrich Knorr Berlin University of Technology, Department of Food Biotechnology and Food Process Engineering, Berlin, Germany

The application of pulsed electric fields (PEF) as a membrane permeabilization technique has received considerable attention during the last decades due to its potential to enhance or to create alternatives to conventional methods in food processing. Among the emerging non-thermal technologies, the application of PEF is one of the most advanced processing methods, undergoing intensive scientific evaluation. Low processing temperature and short residence times allow a highly effective inactivation of microorganisms while retaining product quality. Its ability to permebealize cellular tissue in a short time can be utilized to replace energy- and time-consuming conventional thermal or mechanical disintegration techniques or enzymatic maceration. Within this chapter the key advantages of this emerging technology will be identified and summarized after a brief introduction of the historical background and mechanisms of action. The design of impulse generation systems and treatment chambers and the impact of the main processing parameters will be discussed. Some applications for the induction of stress reactions, permeabilization of biological tissue and gentle food preservation will be presented to show the tremendous innovative potential of this maturing technique.

1 Introduction Increasing consumer demand for food with a high nutritional value and a ‘fresh-like’ taste led to the development of new mild processes and alternatives to enhance or substitute conventional techniques such as heat treatment for food preservation. Several non-thermal pasteurization methods, including the application of high hydrostatic pressure or pulsed electric fields, have been developed to achieve sufficient microbial reduction while maintaining food quality. The use of an external electrical field for a few microseconds induces local structural changes and a rapid breakdown of the cell membrane. Based on this phenomenon, called electroporation, many applications of high intensity pulsed electric fields (PEF) have been studied in the last decades. In the area of plant and microbial genetics pulsed electric fields are applied to cause an electroporation of cell membranes to Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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70 Overview of Pulsed Electric Field Processing for Food

infuse foreign material such as DNA into the cell (Neumann, 1996; Zimmermann, 1996). This process of reversible pore formation has to be controlled to maintain viability of the organisms during the application of the PEF. Due to the reversible permeabilization the cells repair their membranes through resealing the electropores immediately after the PEF treatment. This principle can also be utilized to induce stress reactions and secondary metabolite biosynthesis, which can be desirable food constituents. At higher treatment intensity PEF can be utilized for the inactivation of microorganisms by an irreversible breakdown of the cell membrane. In food technology this irreversible pore formation by PEF can be applied as a mild preservation technique for liquid food as well as a substitute for conventional cell disintegration methods, such as grinding or enzymatic treatment, as a pre-treatment step for mass transfer improvement prior to dehydration, extraction or pressing.

2 Historical background The bactericidal effect of an electric current had already been tested at the end of the nineteenth century (Prochownick and Spaeth, 1890; Krüger, 1893; Thiele and Wolf, 1899), but the lethal effects found by applying direct or low-frequency alternating current resulted from thermal or electrochemical effects. In the 1920s a process called ‘Electropure’ was introduced in Europe and the USA (Beattie and Lewis, 1925; Fetterman, 1928; Moses, 1938). Being one of the first attempts to use electricity for milk pasteurization, it was performed by the application of a (not pulsed) 220 V alternating current within a carbon electrode treatment chamber. About 50 plants were in operation until the 1950s, but due to rising energy costs and competition with mild novel thermal preservation technologies such as UHT, these (ohmic heating) plants have been replaced (Reitler, 1990). Apart from thermal effects based on the mechanism of ohmic heating, lethal effects of electrochemical reactions such as the hydrolysis of chlorine were found when subjecting food to discharges with a voltage of 3–4 kV (Pareilleux and Sicard, 1970). Pulsed discharges of high voltage electricity across two electrodes for microbial inactivation were first investigated in the 1950s (Allen and Soike, 1966; Edebo and Selin, 1968), resulting in a process called electrohydraulic treatment. The electrodes were submerged in the liquid medium within a pressure vessel, electric arcs were generated by high voltage pulses forming transient pressure shock waves up to 250 MPa and ultraviolet light pulses. Electrochemical reactions, shock waves and ultraviolet light forming free, highly reactive radicals were responsible for the bactericidal effect, but disintegration of food particles and electrodes, leading to food contamination inhibited an industrial application of this process except for wastewater (Jeyamkondan et al., 1999). Experiments conducted by Doevenspeck (1960, 1961, 1984) revealed that pulsed electric fields can be applied for disruption of cells in food material and were further developed and expanded to the inactivation of microorganisms and wastewater treatment. Secondary effects of electrochemical reactions and temperature increase became less relevant when short, homogeneous pulses without arcing were applied. Based on this

Historical background 71

work, the ‘Elcrack’ process for the disintegration of animal material such as fish or meat and the ‘Elsteril’ process for liquid media decontamination were developed by Krupp Maschinentechnik GmbH, Germany (Sitzmann and Münch, 1988). The application of electric fields for electroplasmolysis of apple mash was first reported by Flaumenbaum (1968), in which an increase in juice yield of 10–12 per cent was found and the products were described to be lighter in colour and less oxidized than after a heat or enzymatic pre-treatment (McLellan et al., 1991). The first systematic studies investigating the nonthermal lethal effect of homogeneous pulsed electric fields on microbes were conducted by Sale and Hamilton (Sale and Hamilton, 1967). They showed that electric field strength and total treatment time, the product of pulse width and number, were the most important factors involved in microbial inactivation. By treating microorganisms in a gel impermeable for electrolytic products they showed the insignificance of electrolysis on the lethal effect of direct current (DC) pulses. Damage to the cell membrane, causing an irreversible loss of its function as a semipermeable barrier between the cell and its environment, was proposed as the cause of cell death. After treatment, leakage of ions, loss of cytoplasmic content as well as changes in membrane morphology and cell lysis (Sale and Hamilton, 1968; Kinosita and Tsong, 1997) have been reported. Interest in the study of electroporation of cell membranes surged as its applicability to load exogenous material such as drugs or DNA into the cell had been shown. Since then an unprecedented quantity of research activities has been carried out regarding this novel, non-thermal cell permeabilization technique. In the field of food processing most of the work concentrated on microbial inactivation in different liquid food products (Zhang et al., 1995; Grahl and Märkl, 1996; Wouters and Smelt, 1997; Barbosa-Cánovas et al., 1999) and engineering aspects (Peleg, 1995; Zhang et al., 1995a; Heinz et al., 2002), but the electropermeabilization of plant cells (Brodelius et al., 1988; Eshtiaghi and Knorr, 1999; Fincan, 2003; Lebovka et al., 2004) and the effects on food matrices (Aibara and Esaki, 1998) as well as inactivation of enzymes (Ho et al., 1997; Hodgins et al., 2002; Bendicho et al., 2003) and induction of stress reactions and secondary metabolite production (Dörnenburg and Knorr, 1993; Guderjan et al., 2005) have been investigated. Important early patents in the application of PEF for treatment were applied by Krupp in Germany, developing the ‘Elcrack’ and ‘Elsteril’ processes, for inactivation of vegetative microorganisms in milk and fruit juices with an electric field strength up to 30 kV/cm, but heating due to high energy dissipation and consequently high costs of operation inhibited a successful industrial application. Later patents were applied by PurePulse Technologies, San Diego, USA with electric fields in the range from 10 to 25 kV/cm and the microbial inactivation and effect on fruit juice quality was investigated by Dunn and Pearlman (1987) showing an increase of shelflife of about one week. Operating at higher electric field strengths the system was unstable due to arcing and the project was abandoned (Hodgins and Mittal, 2003). Today about 20 research groups are working in this area worldwide, but still there is no commercial, industrial system available. For liquid food preservation four pilot scale systems are available at present, at Ohio State University (USA), at Stork Food and Diary Systems (The Netherlands), at SIK (Sweden) and at the Berlin University of Technology (Germany). For cell disintegration pilot systems are available at Forschungszentrum Karlsruhe (Germany) and Berlin University of Technology (Germany).

72 Overview of Pulsed Electric Field Processing for Food

3 Mechanisms of action It is generally accepted that the primary effect of PEF on biological cells is related to local structural changes and the breakdown of the cell membrane, which is a highly important component of the biological cell as it acts as a semipermeable barrier responsible for mass transfer and plays an important role in the synthesis of RNA and DNA, protein and cell wall components as well as many other complex metabolic activities (Rogers et al., 1980). Disruption of intracellular organelles and other structural changes have also been described (Harrison et al., 1997). The electrical breakdown of cellular membranes has been explored based on model systems such as phospholipid vesicles and planar bilayers as well as microorganisms (Zimmermann et al., 1974; Chernomordik et al., 1987; Chang et al., 1992; Wouters and Smelt, 1997; Barsotti et al., 1999; Ho and Mittal, 2000) but until now there has been no clear evidence on the underlying mechanism of membrane permeabilization at the cellular level. Elucidation of membrane permeabilization is a difficult task as the time sequence of the formation of pores is in the submicrosecond range and the area of pore formation is only in the range of 0.1 per cent of the total membrane surface. The permeabilization of a cell membrane requires two key steps: first the formation of a pore has to be induced by the electric field applied and, secondly, this pore has to be stable enough to allow interaction of the intra- and extracellular media. Very little information is available regarding the time sequence and the dynamics of the electroporation process as well as on reversible-irreversible structural changes of cells during and after PEF treatments. Sale and Hamilton (1968) developed a theory based on the formation of a transmembrane potential, ⌬⌿, across the cell membrane when exposed to an external electrical field. Crowley (1973) suggested an electromechanical instability theory to explain microbial inactivation by PEF. The cell membrane is considered as a capacitor filled with dielectric material of low electrical conductance and a dielectric constant in the range of 2 (Zimmermann et al., 1974). Accumulation of charges with opposite polarity on both sides of the membrane leads to a naturally occurring, perpendicular transmembrane potential of about 10 mV. By exposure to an external electrical field an additional potential is induced by movement of charges along the electric field lines, resulting in a viscoelastic deformation of the cell membrane (Figure 4.1) When the overall potential exceeds a critical value of about 1 V, depending upon the compressibility, the permittivity and the initial thickness of the membrane (Crowley, 1973; Zimmermann, 1996), the electrocompressive force causes a local dielectric rupture of the membrane inducing the formation of a pore, acting as a conductive channel (Schoenbach et al., 1997). Taking into account a membrane thickness of 5 nm, this translates to a dielectric strength of 2000 V/cm. A drastic increase in permeability re-establishes the equilibrium of the electrochemical and electric potential differences of the cell plasma and the extracellular medium, forming a Donnan-equilibrium (Glaser et al., 1998). The electric breakdown is reversible if the pores induced are small in comparison to the membrane area. Increase of electric field strength and treatment intensity by increasing pulse width and/or number will promote formation of large pores and the reversible damage will turn into irreversible breakdown, associated with mechanical destruction of the cell membrane and cell death. Experimental evidence is supporting

Mechanisms of action 73

E

Cell membrane ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺

⫹ ⫺ ⫹ ⫺ ⫹ ⫺

E ⬍ Ecrit

Intact cell

E ⬎ Ecrit

Reversible

E ⬎⬎ Ecrit

Irreversible

⫹ ⫹ ⫹

Figure 4.1 Schematic depiction of mechanism of membrane permeabilization by electrocompressive forces induced by an external electrical field. Increasing treatment intensity will lead to formation of large, irreversible membrane pores.

this electromechanical compression model. A critical electric field strength was found, which was dependent on the size and geometry of a cell, in the range of 1–2 kV/cm for plant cells and 10–14 kV/cm for microbial cells such as E. coli. However, as subsequent behaviour, such as resealing of pores, membrane conductance course and transport phenomena are not taken into account, several other models have been proposed to predict the mechanisms at a molecular level, such as the fluid mosaic model of a lipid bilayer with protein units embedded (Jacob et al., 1981). Dimitrov (1984) presented an extension of the electromechanical model taking into account the viscoelastic properties of the membrane, membrane surface tension and molecular rearrangements as well as pore expansion to describe the time course of field induced breakdown of membranes. Other alternative concepts are based on molecular reorientation and localized defects within the cell membrane which are expanded and destabilized by exposure to an electric field. The presence of small fluctuating hydrophobic pores in the lipid matrix was suggested to be the initial structural basis of electroporation (Chernomordik, 1992). By external electrical stress these may be transformed into hydrophilic pores by reorientation if the pore radius is increased above the value where the pore energies of both orientations coincide. The pore energy is the change of free energy resulting from the formation of a pore within the lipid bilayer. As long as the pore radius is small, the formation of hydrophobic pores is more favourable, but at a range of 0.5 nm the pore energies of hydrophobic and hydrophilic pores become equal and pore inversion may occur (Glaser et al., 1998). Resealing of pores after turning off the electric field might be inhibited in areas close to membrane proteins.

74 Overview of Pulsed Electric Field Processing for Food

Tsong (1990) related membrane rupture to osmotic imbalances and cell swelling after opening of pores and defined a two-step mechanism: first an initial perforation of the cell membrane after a dielectric breakdown followed by a time-dependent pore expansion. Electroporation could take place both in lipid domains and protein channels and it has been found in artificial lipid bilayer systems as well as in cell membranes, but the mechanisms of action are not necessarily the same in these two systems. Protein channels, sensitive to electric fields due to a very low gating potential and dielectric strength compared to lipid bilayers, can be assumed to be opened before the lipid bilayer when exposed to an electric field. Current flow and associated joule heating might result in denaturation and stabilization of the pores. These pores might also cause a loss of ability to regulate the intracellular pH (Simpson et al., 1999) and the short circuit of protein-pumps (Chernomordik, 1992). Several empirical and phenomenological models to describe the relation between microbial inactivation and electric field strength have been proposed (Hülsheger et al., 1981; Peleg, 1995). The high number of processing and product parameters, differences in treatment systems and experimental conditions and definitions limit the generality of the relations found. Lack of knowledge concerning mechanisms of action at dependence of processing and product parameters prevent development of inactivation models based on the physiological meaning of obtained parameters. Inactivation has been described as basic first order kinetic (Martín-Belloso et al., 1997; Reina et al., 1998) as well non-linear curves (Peleg, 1995; Peleg and Cole, 1998; Barbosa-Cánovas et al., 1999; Rodrigo et al., 2003). Non-linear inactivation has been related to distributions of sensitivity against electroporation, protective effects of food constituents, presence of air bubbles, insulating particles or inhomogeneities in electric field distribution as well as distribution of cell size, geometry and random orientation in the electric field. Yaqub et al. (2004) investigated the presence of sublethal injury of bacteria exposed to pulsed electric fields by fluorescent viability staining, no sublethal damage was found after PEF treatment in contrast to heat treated cells. By investigating growth of Salmonella entericar on different selective media after PEF treatment, Wuytack et al. (2003) observed no significant sublethal damage. Inactivation of microorganisms by PEF was regarded as an ‘all or nothing’ process, i.e. a single target mechanism based on irreversible membrane permeabilization.

4 PEF treatment systems The main components required for a pulsed electric field application are an impulse generation system and a treatment chamber. A crucial prerequisite for an economic and efficient production is continuous operability with high flow rate capacity, which led to the development of continuous treatment chambers, where the food is pumped through while being exposed to the electrical field at ambient or refrigerated as well as elevated temperatures. Before treatment, heat exchangers might be used to preheat the media and after the treatment, the dissipated electrical energy resulting in a temperature increase has to be removed before aseptic packaging. Aseptic packaging is

PEF treatment systems 75

required to prevent recontamination. One of the main advantages of PEF treatment is its continuous operability with very short processing times, therefore a system can easily be implemented into existing processing lines.

4.1 Generation of pulsed electric fields The impulse generation system transforms the electric power from a low utility level voltage to pulsed high intensity electric fields. A simplified circuit for generation of exponential decay pulses is shown in Figure 4.2, consisting of a charging and a discharging unit. In the first one, an energy storage device is charged across a charging resistor by a DC high voltage power supply. The generation of pulsed electric fields requires slow charging and a fast discharging of the energy, as the pulse width is short in comparison to the time between pulses. The charging voltage, U0, required to generate pulses of sufficient electric field strength is highly dependent on the electrode distance. For two parallel plate electrodes the electric field strength E is given by: E⫽

U d

(1)

where U is the voltage (kV) and d (m) the gap between the electrodes. Voltages in the range of 10–60 kV have been used for food treatment. Increasing the gap d to obtain high flow rate capacity imposes increasing the charging voltage and therefore stress on the switching system. The electric power is most commonly stored in a bank of capacitors connected in series or parallel and discharged into the treatment chamber across a high voltage switch and protective resistors within microseconds. As for the discharge switch, many devices are applicable, including vacuum or gas spark gaps, thyratron, high power transistors or semiconductor switches, each with its drawbacks and limitations. The type of switch will determine the maximum repetition rate and the maximum current and voltage rating that it can withstand. The current is highly dependent on the resistance of the treatment chamber and can reach several kA when operating with a low resistivity treatment chamber configuration. Increasing resistive load by appropriate chamber design, e.g. a co-linear configuration, will result in currents far below kA range. A spark gap, which is basically a two electrode system with arc-over when the breakdown voltage is exceeded, provides a very simple and resistant pulse generation which can handle high voltage and current ratings but is limited to repetition rates below 100 Hz and a maximum lifetime of 106 shots. Whereas a spark gap cannot be triggered and the repetition rate is controlled by the charging current, the other types of switches allow generation of pulses with a defined frequency. Implementation of a trigger electrode to a spark gap to promote a controlled arc-over leads to a trigatron switch. Thyratron switches have found wide application for generation of pulses, providing high, triggerable repetition rates up to kHz range. Solid state switches such as SCR (Silicone Controlled Rectifier) GTO (Gate Turn-off) or IGBT (Insulated Gate Bipolar Transistor) switches, require less complex driving circuits and are easy to handle and control by external triggering and optimum

76 Overview of Pulsed Electric Field Processing for Food

HV power supply

HV power supply

Charging resistor

Charging resistor

Storage capacitors Storage capacitor Inductors Protective resistor HV-switch

HV-switch

Treatment chamber

Voltage

Voltage

Treatment chamber

Time

Time

Figure 4.2 Simplified electrical circuits of impulse generation systems and ideal voltage patterns of exponential decay and square wave pulses.

variability of pulse parameters. Limitations have been found in the reliability in longterm use, in particular when exposed to reversal current in case of arcing, treatment chamber blocking or electromagnetic interference. For high voltage pulses greater than 100 kV a ‘Marx’-generator is commonly used, invented by Marx in 1923. In this configuration, capacitors are charged in parallel and switched into series before discharge to increase the output voltage above the charging voltage. An overview of different pulse generator systems was given by Mankovski (2000). In a capacitive storage system, when the high voltage switch is closed by a trigger signal or after its breakdown voltage has been achieved, the energy stored is discharged

PEF treatment systems 77

to ground across the discharge circuit with a protective resistor and the treatment chamber with the food material. Dependent on the type of switch and the configuration of the discharge circuit several pulse waveforms are possible, but two have mainly been used: exponential decay and square wave pulses. Generation of an exponential decay pulse requires a switch with turn on capability only, as the total energy stored in the capacitor bank will be discharged. Square wave pulses can be realized either by an incomplete discharge of a capacitor with high capacity by a switch with on/off capability or a more complex pulse forming network, providing a relatively constant voltage during the pulse width. As switches with turn off capability are not readily available for high power application systems (Mohan et al., 1995), serial or parallel connections of switches (Gaudreau et al., 2001) or lumped or distributed pulse forming networks with several sections of capacitors and inductive elements have to be used. In this case the pulse generation system, in particular the impedance of the line, has to be adapted to the resistive load of the treatment chamber, which is difficult to realize in practice (Zhang et al., 1995a) and could lead to an inflexible system with regard to treatment media which may have a wide range of conductivity. Square wave pulses maintain a high voltage level for the total impulse width, whereas exponential pulses have a long tail with low electric field. Studies comparing the efficacy of different pulse waveforms for PEF inactivation have been conducted by Góngora-Nieto et al. (2002), Kotnik et al. (2003) and De Haan and Willcock (2002) who concluded that both are effective for microbial inactivation, but square wave pulses save energy and require less cooling effort. For an industrial exploitation with high flow rates, high pulse repetition rates up to several kHz are required at high voltage (40–100 kV) and current levels (⬎100 A), which are operating conditions that are hard to handle, in particular if the lifetime of the impulse generation system is taken into account. Lifetime ratings vary from 106 for spark gaps, 108 for thyratrons, up to approximately 1012 pulses for semiconductor switches when operated under nominal conditions. Arcing or operation under nonoptimal conditions will drastically reduce the lifetime. At repetition rates of 1 kHz a lifetime of 109 pulses translates into a continuous operation of 11 days. The reliability in case of malfunction, pump failure, arcing and protection against short circuit will be a crucial parameter for the applicability of a switch.

4.2 Treatment chamber design The treatment chamber, wherein the food is exposed to the electric field pulses, consists of at least two electrodes, one on high voltage and the other on ground potential, separated by insulating material in different geometric configurations. Parallel plates, coaxial or co-linear cylinders have commonly been used. A large number of studies have been performed with parallel plate systems in batchwise and later in continuous flow operation. Batch chambers provide many advantages for laboratory use; small volumes of treatment media are required and the treatment temperature is easy to maintain by cooling the electrodes and by slow repetition rates. Above all, the pulse number for each volume element is well known. Apart from niche products for an industrial application, continuous chambers will be necessary to achieve high volume capacity and easy integration into already existing food processing lines.

78 Overview of Pulsed Electric Field Processing for Food

Among the different electrode configurations (Figure 4.3), parallel plates provide the most uniform electric field in a large usable area between the plates, but treatment intensity is reduced in boundary regions. In batch chambers without mixing or product flow leading to changes of position, a considerable part of the volume may remain under-processed, noticeable as tailing effects in microbial inactivation kinetics. In continuous treatment chambers, this can be prevented by adding multiple treatment zones in line or baffled flow channels (Zhang et al., 1995a). To achieve the high flow rates required for industrial applications, the pulses must be applied with a high repetition rate, leading to a fast temperature increase of the media. Maintaining a constant temperature may require high cooling efforts or intermediate cooling between multiple treatment zones. The electrode and insulator material have to be food grade and autoclavable. Furthermore, the electrochemical properties have to be taken into account. Bushnell (1996) suggested gold, platinum, carbon and metal oxides as alternative to commonly used stainless steel electrodes. To avoid product exposure to the electrode surface, Lubicki and Jayaram (1997) developed a system consisting of a glass coil surrounding the anode and confirmed that microbial inactivation can be obtained even without direct contact. PEF treatment of packaged food without direct contact to the electrodes has been discussed in the last few years and questions, such as how a sufficient (and homogeneous) electric field strength can be induced or if the required pulse energy can be transferred into the product, will have to be addressed.

⫹

⫹

⫹

Product flow (a)

Product flow (b)

Product flow (c)

Figure 4.3 Configurations of treatment chambers for continuous PEF treatment: (a) parallel plate, (b) coaxial and (c) co-linear configuration.

Main processing parameters 79

A co-linear chamber consists of a set of hollow, cylindrical electrodes separated by insulators, in which the product is pumped through the drilling and the flow is not disturbed by any impediments. The geometry of the treatment chamber (together with medium conductivity) has a decisive impact on its total resistance and therefore on the discharge circuit. The ratio of the total resistance of the treatment chamber and the protective resistor is highly important, as the applied charging voltage is divided between them. At the protective resistor, which is necessary to prevent breakdown of the switching system in case of short circuit, a considerable amount of energy might be lost if its resistance is in the same range as that of the treatment chamber.

5 Main processing parameters 5.1 Electric field strength Applying an external electrical field with sufficient strength to cells suspended in an electrically conductive medium will induce the accumulation of charges at the nonconductive microbial membranes. Pore formation will occur when a certain threshold value of the transmembrane potential formed is exceeded, which was found to be in the range of 1 V (Zimmermann, 1996). It was shown that the critical external field strength is highly dependent on cell size as well as cell orientation in the field (Heinz et al., 2002). With decreasing cell size the required field strength sharply increases and variations in cell shape can cause considerable increase of E. This is shown in Figure 4.4, where the critical electric field strength for three microbial strains differing in size and geometry has been calculated for all spatial cell orientations within the electric field. The fraction of cells where the electric field is not sufficient to induce a transmembrane potential for an electropermeabilization, E y

1

AF

A1 0.1

x

1

Listeria

0.01

Characteristical dimensions (µm): E. coli

Saccharomyces

Fraction of intact cells

A2

10 Field strength (kV/cm)

Saccharomyces cerevisiae (A1: 4; A2: 3; A3: 2) Escherichia coli (A1: 2.0; A2: 0.7; 100

A3: 0.55)

Listeria innocua (A1: 0.625; A2: 0.255;

A3: 0.255)

Figure 4.4 Impact of cell size and geometry on transmembrane potential of three microorganisms different in size and geometry (Toepfl et al., 2004).

80 Overview of Pulsed Electric Field Processing for Food

which is highly dependent on the length of the semi-axis in field direction AF, is shown (Toepfl et al., 2004). It can be seen that larger cells are more susceptible to electrical fields. Whereas Saccharomyces cerevisiae is affected at field strengths as low 2–4 kV/cm, smaller cells like Listeria innocua require a minimum of 15 kV/cm. Dependent on orientation of the rod-shaped cells along or across the electric field lines more than 35 kV/cm will be required to bring about extensive microbial inactivation. Increasing the electric field strength was reported to lead to a further increase in treatment efficiency (Hülsheger et al., 1983; Boyko et al., 1998; Heinz et al., 1999, 2003; Heinz and Knorr, 2000; McDonald et al., 2000; Alvarez et al., 2003), but is limited to the dielectric strength of the food material (Ho and Mittal, 2000). Breakdown and associated arcing will cause current flow in a narrow channel and promote undesired electrochemical reactions, bubble formation and electrode erosion. This can be prevented by optimizing the field distribution in the chamber excluding hot spots as well as avoiding the presence of air bubbles.

5.2 Treatment time, specific energy and pulse geometry Apart from the peak electric field strength the product of the pulse width and the average number of pulses applied has often been used to evaluate treatment intensity. Increasing treatment time results in higher microbial inactivation (Sale and Hamilton, 1967). The pulse width is defined as the time where the peak field is maintained for square wave pulses or the time until decay to 37 per cent for exponential decay pulses. Typically, increasing the number of pulses causes an increase in treatment time, as the pulse width is fixed by the impulse generation setup. In general, increasing inactivation has been found when treatment time is increased, but in some cases saturation has been reported after a certain amount of pulses. While for batch chambers the number of pulses per volume element is well defined, for continuous flow chambers the average number has to be considered. In many cases the treatment zone with an electric field above the critical electric field strength is different as a result of the treatment chamber’s geometry. This can in particular be found in the case of co-linear configuration where the distribution of the field intensity has to be taken into account when calculating the medium residence time in the treatment zone. Therefore, the specific energy input was suggested as an intensity parameter and it can be estimated by product temperature increase and specific heat capacity of the medium by assuming an adiabatic system where the energy delivered to the treatment medium is totally converted to heat. The energy input can also be estimated by calculation of the stored energy, taking into account voltage and power division in the discharge system, or most accurately, based on voltage and current signals determined close to the electrodes: WPulse ⫽ ∫

U(t) dt (t)

(2)

Based on media conductivity and electric field strength measured, the specific energy input can also be calculated by: 1 WSpecific ⫽ m. ⭈ m

∞

∫0 ␬(T) ⭈ E(t)2dt

(3)

Main processing parameters 81

. where E, ␬(T), f and m denote the electric field strength, the medium conductivity, the repetition rate and the mass flow rate, respectively. The temperature increase due to energy dissipation can be utilized for process evaluation by comparing energy input with bulk product temperature after treatment. From a processing point of view the energy input required to achieve a given microbial inactivation rate or cell matrices disintegration seems to be advantageous to be used as a treatment intensity parameter, as it can indicate the costs of operation. However, neither treatment time nor specific energy are adequate to describe processing parameters sufficiently as no information is given about the number of energy portions per pulse or the number of pulses per volume element. The impact of pulse rise times from 2 to 100 ␮s was investigated by Kotnik et al. (2003). No significant difference was found and treatment efficacy was correlated to the time with above-critical pulse amplitude. The impulse characteristics of an exponential decay pulse are highly dependent on the parameters of the charging and the discharging circuit. Peak voltage and capacity of the energy storage determine the energy input per pulse, resistance and inductivity of the discharge circuit and influence pulse rise time and pulse width. The impact of impulse characteristics on microbial inactivation has been discussed extensively but, since they are not independent parameters, their influence has not been fully elucidated up to now. Future work will have to focus on the underlying mechanisms to determine the requirements for an efficient electropermeabilization of biological cells.

5.3 Treatment temperature Treatment temperature has a highly synergetic effect on treatment efficacy, as it has a significant influence on cell membrane fluidity and stability. Whereas at low temperatures the phospholipid structure is packed in a gel-like structure, their order decreases with increasing temperature. The temperature dependent phase shift from gel to a liquid crystalline structure affects cell membrane stability (Stanley, 1991). Dunn and Pearlman (1987) observed an increase of inactivation of S. dublin in milk from 1 to 4 log cycles when increasing treatment temperature from 40 to 50°C. Jayaram et al. (1993) reported an enhanced inactivation of L. brevis when increasing treatment temperature from 24 to 60°C, assuming the phase transition of the phospholipids to be responsible for this effect. The effect of treatment temperature on the textural properties of apple tissue has been investigated by Lebovka et al. (2004), showing that preheating to 50°C resulted in more effective tissue damage than PEF treatment alone and better juice extraction by pressing.

5.4 Treatment medium factors Product properties and constitution have a significant influence on microbial growth as well as resistivity against different inactivation techniques. Similar to heat treatment, a strong dependency of susceptibility against pulsed electric fields on product parameters has been reported and physical and chemical parameters such as pH or water activity of the product strongly influence microbial inactivation.

82 Overview of Pulsed Electric Field Processing for Food

5.4.1 Conductivity

One of the key parameters for pulsed electric field processing is the medium conductivity, which is also a function of medium temperature (Reitler, 1990). Media rich in ionic species such as tomato juice present problems in achieving a significant voltage for a supercritical field strength, since a smaller peak field strength is generated across the treatment chamber. This effect is important for the treatment of plant or animal cells as well as to achieve microbial inactivation for liquid food preservation. Conductivity is the inverse of resistivity and is measured in Siemens per unit length (S/m). As the conductivity of most food materials is fixed by its intrinsic properties or recipes only the choice of an electrode configuration and geometry with high load resistivity helps to diminish this effect and to improve voltage division in the discharge circuit. Nevertheless, the temperature increase will lead to changes in conductivity during treatment. Apart from its influence on field strength, the conductivity is supposed to determine the difference between ionic strength in the medium and the cytoplasm (Jayaram et al., 1993). The membrane will be weakened and more susceptible to an electric pulse in media with higher ionic strength, causing higher permeability and structural changes. The relation between inactivation rate and medium conductivity was investigated by Hülsheger et al. (1981) and Vega-Mercado et al. (1996), showing a significant increase in inactivation at lower ionic strength and conductivity, whereas Alvarez et al. (2000) did not confirm this. Heinz et al. (2002) calculated the influence of medium conductivity on charging time constant and showed a negligible influence of the medium conductivity on transmembrane potential build up. 5.4.2 Effect of air bubbles and particles

Apart from electric conductivity, the dielectric strength of the food matrix has a significant influence on the applicability of PEF, as a dielectric breakdown has to be prevented. Air bubbles, which cannot withstand high electric field strengths may be present in the case of sparkling products or be released due to temperature increase or electrochemical reactions. In particular, for microbial inactivation, where an electric field strength in a range of 30–50 kV/cm is required, air has to be removed from the product. Apart from dielectric breakthrough, where a high current will flow within a narrow channel within the bubble instead of the liquid, the different dielectric properties of air will influence treatment efficacy. The perturbating effect of air bubbles present within the treatment chamber has been reported by Góngora-Nieto et al. (2003), indicating that in boundary regions of bubbles a significant drop in field strength will cause food safety problems. A similar effect has been found when agglomerations of microorganisms and/or particles with different dielectric properties such as fat globules are present (Toepfl et al., 2004). Therefore product constitution has to be taken into account in the choice of processing parameters for a certain product. For treatment of solid foods such as plant or animal material or fruit mashes air encapsulations have to be removed to avoid electric discharges. Foam forming products might be unsuitable for PEF treatment.

5.5 Cell characteristics As early as the 1960s, Sale and Hamilton (1967, 1968) observed that yeasts were more susceptible to a pulsed electric field treatment than bacteria. Hülsheger et al. (1983)

Applications 83

showed a broad variety of inactivation in different microbes. Apart from the effect of electric field strength as described above, the membrane constitution of different microbes influences their resistivity. In general, Gram-positive organisms seem to be less sensitive. An influence of cells at different growth phases on PEF treatment was not observed by Sale and Hamilton, but Jacob et al. (1981), Pothakamury (1995), Gaskova et al. (1996) and Alvarez et al. (2000) reported higher efficacy for cells in logarithmic growth phase. Even if some research groups described a slight reduction in some types of spores after PEF treatment (Raso et al., 1998), it is important to note that PEF is not an efficient tool for inactivation of endo- or ascopsores and that germination is not induced after a treatment. Hence it may not be possible to sterilize food products unless a combination with other techniques such as heat or previous germination and inactivation of vegetative cells is performed. It is noteworthy that resistance of cells against PEF does not correlate to its resistance against other thermal or non-thermal treatments, for example strains of Listeria, which are highly sensitive to heat, were shown to be highly resistant against PEF treatment (Toepfl et al., 2004). Lado and Yousef (2003) compared inactivation of nine different strains of Listeria monocytogenes after a treatment at 25 kV/cm and 25°C, which ranged from less than 1 up to 3.5 log cycles. No correlation to heat sensitivity or genotype was found. This variety emphasizes that still there is a need to identify resistant target strains to evaluate and prove process efficacy and safety.

6 Applications Many applications of PEF have been investigated in food as well as in biotechnology or medicine within the last decades, utilizing its impact on biological cell membranes. Dependent on treatment intensity, electropermeabilization of membranes leads to reversible or irreversible pore formation and cell disintegration, which is often a key processing step in food and bioengineering operations. Since after a high intensity treatment cell vitality is lost, a non-thermal inactivation of microbes can be achieved. The following sections will give an overview of different applications of this novel, non-thermal and short-time technique.

6.1 Stress induction The application of a low intensity treatment at low electric field strength and/or pulse number, though initiating a conductive channel across the membrane, does not necessarily cause irreversible cell rupture. For example, for potato tissue after a time of 0.7 ␮s for membrane charging and a membrane potential of 1.7 V a pore is formed, but electrically insulating properties can be recovered within seconds, restoring vitality and metabolic activity (Angersbach et al., 2000). This provides a potential to induce stress reactions in plant systems or cell cultures as previously described for high pressure techniques (Dörnenburg and Knorr, 1999). An airlift bioreactor with a coaxial electrode configuration has been developed to investigate sublethal stress on cultures of

84 Overview of Pulsed Electric Field Processing for Food

Vitis vinifera for recovery of resveratrol. It was shown that metabolic activity can be stimulated and extractability of intracellular compounds was improved. A PEF treatment at low temperatures does not damage enzymes or proteins, thus the potential to extract valuable components is provided. For example, mild sublethal treatment of maize germs increased oil yield and phytosterol production, resulting in plant oil with a higher phytosterol concentration. In subsequent studies with soy beans and olives increased oil yield and isoflavonoid content were found (Guderjan et al., 2005), providing an eminent potential to develop processing concepts activating cells as ‘bioreactors’ to produce high quality food with a high concentration of functional constituents.

6.2 Disintegration of biological material Many operations in food and bioengineering such as extraction, pressing or drying include an enzymatic or thermal treatment or mechanical grinding for disruption of cellular material. These techniques may require a high amount of mechanical or thermal energy as well as holding times and storage tanks for an enzymatic maceration. Furthermore, side activities of natural or added enzymes and thermal degradation lead to significant losses of nutritionally and physiologically valuable substances. When applying PEF to cellular tissues an increase in mass transfer coefficients could be observed (Flaumenbaum, 1968; Knorr et al., 1994; Knorr and Angersbach, 1998; Bazhal and Vorobiev, 2000; Fincan et al., 2004). Based on this possibility, conventional processing can be supported or replaced. As a PEF treatment allows a defined degree of tissue permeabilization, biological systems can be stimulated in terms of metabolic activity and the recovery of valuable components can be improved. Operating at ambient temperatures, a treatment with sufficient electric field strength, pulse repetition rate and/or energy input results in formation of large, permanent pores, while retaining product quality and fresh-like character in contrast to thermal or enzymatic treatments. In juice processing, a similar juice yield with fresh-like quality and a higher concentration of functional components was found. Short-time and continuous operability allows a continuous liquid–solid separation, e.g. by a decanter centrifuge (Knorr et al., 2001). For carrot juice, an increase of juice yield from 60.1 to 66.4 per cent was found in comparison to an untreated sample; in the same way the dry matter of the pomace was increased from 13 to 15 per cent, resulting in less efforts for drying. For grapes a juice yield of 87 per cent, similar to that after an enzymatic maceration and an increased content of soluble solids and pigments was reported after cell disintegration by PEF (Eshtiaghi and Knorr, 2000). A flow chart of such a process is shown Figure 4.5. In the context of consumer demand for functional food with composition and mineral content close to fresh products and a high content of physiologically valuable compounds, an increased extractability of anthocyans from grapes or phenolic substances provides an enormous potential for product development. Increase in extractability of black tea and mint leaves by moderate electric fields was investigated by Sensoy and Sastry (2004), showing an increased leaching of solutes. The applicability of PEF to enhance pressing and extraction rate from beet cossettes

Applications 85

Size reduction

Size reduction

Enzymatic treatment

PEF treatment

Mixing holding

Separation (pressing)

Separation (decanter)

Pomace

Pasteurization (thermal)

Pasteurization (thermal/PEF)

Extraction

Juice

Juice

Pectin

Figure 4.5 Flow chart for fruit juice processing with conventional or PEF pre-treatment prior to liquid–solid separation and pectin extraction from PEF-treated pomace.

has been shown by Bouzrara and Vorobiev (2000). It was shown that high temperature thermal degradation (70–120°C, 10–20 min) became obsolete after a PEF treatment of sugar beet while maintaining sugar quality and yield (Eshtiaghi and Knorr, 1999). Textural changes such as tissue softening for apple, potato and carrot by PEF have been reported by Lebovka et al. (2004). For apple tissue a disintegration similar to freeze-thawed tissue was found after a treatment with 0.5 kV/cm for 10 ms. A low temperature PEF treatment led to almost complete membrane destruction, while the effective stress relaxation time for carrot and apple tissue was still much higher than for freeze-thawed tissue (Lebovka et al., 2002). Dependent on process requirements and parameters a cell membrane permeabilization and loss of turgor can be achieved with or without additional softening of the tissue when significant modification of the tissue structure is required. The tissue softening effect of PEF, based on cell membrane electropermeabilization and loss of turgor (Fincan and Dejmek, 2003), can be utilized to reduce the energy required for cutting of plant material. With a continuous, short time and low energy (⬃10 kJ/kg) PEF treatment of potato tissue, a reduction of grinding energy similar to that of thermal or enzymatic treatment can be achieved (Figure 4.6), indicating its potential to replace or enhance conventional processing techniques. Drying is one of the most energy consuming steps in food and bioengineering, as a large amount of water has to be transported from inside the product to its surface for

86 Overview of Pulsed Electric Field Processing for Food

300

Freezingthawing

Energy (kJ/kg)

Heating

200

100

Enzymatic

Mechanical PEF 0 Time scale

min-h

min

h

s

s

Figure 4.6 Energy required for cell disintegration of potato tissue with different techniques.

removal. A PEF treatment can be utilized to improve mechanical water removal by pressing if applicable and facilitate subsequent air drying by enhancing mass transport. For fluidized bed drying of red pepper slices a reduction of drying time from 360 to 220 min was found (Ade-Omowaye et al., 2001). Osmotic drying rates and diffusion coefficients of carrots were found to be increased (Rastogi et al., 1999). For apple slices, an increased osmotic drying rate and improved rehydration capacity and reduced rehydration times were reported (Taiwo et al., 2002). Drying time or drying temperature reduction will result in a reduction of costs of operation and increase in production capacity.

6.3 Preservation of liquid media Microbial inactivation by PEF has been extensively investigated within the last few decades (Sale and Hamilton, 1967; Qin et al., 1994; Zhang et al., 1995b; Gaskova et al., 1996; Grahl and Märkl, 1996; Jeyamkondan et al., 1999; Ho and Mittal, 2000; Cserhalmi et al., 2002), initially in batch treatment systems and model foods which are free of fat or proteins. Even if the underlying mechanisms of action have not been fully elucidated up to now, key processing parameters have been identified and inactivation of a broad variety of vegetative cells has been shown. In general, yeasts have shown to be very sensitive to PEF treatment (Barbosa-Cánovas et al., 1999), as cell size seems to play an important role in addition to cell membrane constitution. Effective inactivation for most of the spoilage and pathogenic microorganisms has been shown, but it has to be emphasized that, in comparison to the treatment of plant or animal cells, the treatment intensity in terms of field strength and energy input is much higher. The high field strength required (20–40 kV/cm) and the energy input of up to 100 kJ/kg lead to the cost of investment estimated to be in the range of €2 million for systems on an industrial scale of 5 t/h. As a treatment with this field strength will destroy the

Log reduction (N/N0)

Applications 87

0

0

0

0

⫺1

⫺1

⫺1

⫺1

⫺2

⫺2

⫺2

⫺2

⫺3

⫺3

⫺3

⫺3

⫺4

⫺4

⫺4

⫺4

⫺5

⫺5

⫺5

⫺5

⫺6

⫺6 L. innocua

⫺7

S. cerevisiae

⫺7 0

40

⫺6

⫺6

E. coli

80

120

40

80

120

0

40

80

Detection limit B. megaterium

⫺7

⫺7 0

35°C 45°C 55°C

120

0

40

80

120

Specific energy (kJ/kg)

Figure 4.7 Inactivation of four different microbial strains in Ringer’s solution dependent on specific energy input and treatment temperature at a field strength of 23 kV/cm (Toepfl et al., 2004).

structure of solid food, PEF treatment for preservation therefore seems to be virtually impossible for solid food and is limited to liquid media. The potential to achieve sufficient reduction of microbes has been proven in a broad variety of food products, fruit or vegetable juices (Zhang et al., 1994; Qui et al., 1998; Evrendilek et al., 2000; McDonald et al., 2000; Hodgins et al., 2002; Heinz et al., 2003; Molinari et al., 2004), model beer (Ulmer et al., 2002) as well as milk (Reina et al., 1998; Bendicho et al., 2002), liquid egg (Martín-Belloso et al., 1997) and nutrient broth (Selma et al., 2004). Apart from microbial inactivation, the reduction of enzymatic activity is critical in food processing and preservation, but there are only limited reports about the effects of PEF on enzymes. Due to different experimental setups and processing parameters it is sometimes difficult to compare the results from different research groups, as conclusions drawn are often inconsistent. Yang et al. (2004) investigated the inactivation of five different enzymes after a PEF treatment and the results varied from enzyme to enzyme. Whereas lysozyme was not affected by an electric field strength below 38 kV/cm, a significant reduction of pepsin activity was achieved. It was concluded that PEF and PEF-induced heat contribute to enzyme inactivation. Van Loey et al. (2001) reported that lipoxygenase, polyphenoloxidase, pectin-methylesterase (PME) and peroxidase are resistant towards a PEF-treatment in distilled water and inactivation found in more complex products was based on temperature effects. Schuten et al. (2004) found a reduction of PME activity of 24 per cent, which was sufficient to increase shelf-life from 5 to 21 days in refrigerated storage. Inactivation of enzymes by PEF is discussed in detail elsewhere in this book (see Chapter 7), but it has to be taken into account that the higher resistivity of enzymes might be a restriction for preservation of liquid food by PEF, unless utilizing thermal effects. On the other hand, it provides a high potential for the development of new processes and products, as many enzymes are positively used in food processing. The inactivation of four microbial strains different in size and geometry as affected by specific energy input is shown in Figure 4.7. Differences in susceptibility can be

88 Overview of Pulsed Electric Field Processing for Food

seen as well as the influence of initial treatment temperature and specific energy input. The impact of pulse energy dissipation has to be taken into account, as the medium temperature will increase. Dependent on the system setup this energy might be removed by cooling or lead to a temperature increase of the medium. A study of temperature impact on treatment efficacy on Escherichia coli in apple juice has been performed by Heinz et al. (2003) indicating the potential of a combined treatment of PEF and mild heat for microbial inactivation. It was found that increasing treatment temperature from ambient to a range of 35–55°C can reduce the electrical

Tin,1 10°C; 38 kJ/kg Enthalpy in

40 kJ/kg Electrical energy

Tin,2 55°C; 210 kJ/kg Heat exchanger

Treatment chamber Tmax 66°C; 250 kJ/kg

Tout 17°C; 64 kJ/kg Enthalpy out

14 kJ/kg Heat loss

100 HTST-treatment

Temperature (°C)

80

PEF-treatment

60

Preheating

40

Cooling

20

0 0

20

40

60

80

Time (s) Figure 4.8 Enthalpy flow diagram and temperature–time profile of a PEF preservation of fruit juice at elevated treatment temperature in comparison to an HTST-treatment.

Problems and challenges 89

energy required for an inactivation of 6 log-cycles of Escherichia coli from far above 100 to 40 kJ/kg when operating at an initial treatment temperature of 55°C. An energy input of 40 kJ/kg will result in a temperature increase of 11°C in orange juice, showing that with a maximum temperature of 66°C the preservation process is still operating at lower maximum temperature and shorter residence times than during conventional heat preservation (Figure 4.8). Apart from the reduction of energy input required to achieve microbial inactivation when operating at elevated temperatures, the need to preheat the medium to the initial treatment temperature provides a potential to recover the electrical energy dissipated into the product in a heat exchanger. When operating at ambient temperatures there is no need for preheating and therefore high cooling efforts are required. A combination of mild heat and pulsed electric field might also be helpful to achieve sufficient enzyme inactivation to avoid the necessity of refrigerated storage.

7 Problems and challenges Before an industrial exploitation of this novel technique to preserve food on an industrial scale is attempted, it will have to be demonstrated that the process is indeed economically interesting in comparison to existing pasteurization methods, in terms of costs of operation and investment as well as product quality and, in particular, consumer acceptance. There is a clear need for development of impulse generation systems with sufficient electrical field strength, power and repetition rate at reasonable prices. After scaling up of experimental systems more reliable data will be available to estimate the specific requirements and costs for different applications. Apart from consumer acceptance, the legislative situation is still unclear. Development of novel processing techniques at least requires proof of conformity of products to conventionally processed ones. In the last few years problems due to electrochemical reactions at the electrode/medium interfaces have been discussed (Morren et al., 2003; Roodenburg et al., 2003; Toepfl et al., 2004), indicating that there is a challenge to replace commonly used stainless steel electrodes by other materials or to modify pulse generator systems to reduce the amount of electrochemical reactions. An overview of possible electrochemical reactions at a steel electrode is given in Table 4.1. Application of carbon electrodes may be one solution to overcome this problem (Toepfl et al., 2004) and application of shorter pulses or switching systems without leak

Table 4.1 Possible electrochemical reactions at the electrode/medium interface 2H2O l H⫹ ⫹ OH⫺ 2H⫹ ⫹ 2e⫺ l H2(g) 4OH⫺ l O2 (g) ⫹ 2H2O ⫹ 4e 2Cl⫺ (aq) l Cl2 (g) ⫹ 2e⫺ Fe(s) l Fe2⫹ (aq) ⫹ 2e⫺

90 Overview of Pulsed Electric Field Processing for Food

current have also been discussed (Mastwijk, 2004) to avoid electrochemical reactions. Apart from a reduction in electrode life time the release of particles and heavy metals from the electrode may cause toxicity problems. Reyns et al. (2004) reported the generation of bactericidal and mutagenic compounds by a PEF treatment, even if they operated with 300 pulses at a pulse width of 2 ␮s and 26.7 kV/cm, a treatment intensity much higher than required for liquid food preservation. These issues have to be addressed prior to an industrial application.

8 Conclusions Some of the possible applications of PEF as a non-thermal cell membrane permeabilization technique have been highlighted in this overview. The low energy consumption (1–2 kJ/kg for stress induction and 5–10 kJ/kg for plant cell permeabilization) and the continuous operability of this short-time, waste free membrane permeabilization technique are key advantages and allow the development of innovative, costeffective and sustainable processing concepts in the food and drink industry as well as in the biotechnology and pharmaceutical industry. An application of PEF for food preservation provides the tremendous potential to preserve high quality products at lower temperatures and short residence times to retain the fresh-like character and nutritional value of the products. Future work will have to focus on understanding the underlying mechanisms and kinetics of recovery after treatment for the correct choice of processing parameters and development of equipment. A task still challenging for electro-engineers is the development of equipment with reliable, industrial scale generation of high-strength electric field pulses. The availability of these pulse generation systems will be a prerequisite for industrial application. The uniformity of treatment intensity distribution has to be improved by optimization of treatment chamber geometry. The data acquisition and measurement for process evaluation needs to be standardized for better comparison of results from different research groups. Application of PEF in wastewater treatment revealing its applicability to reduce the amount of excess sludge, which is an issue of high importance considering tightened ecological measures, shows the tremendous potential of this emerging technique beyond the few examples presented in this overview.

Acknowledgements The authors wish to acknowledge that part of this work has been supported by EC projects (FAIR CT96-1175, FAIR CT97-3044, W.I.R.E.S. EVK1-CT-2000-00050), the German Research Foundation (DFG Kn 260/6-1 and KN 260/11-3) and by the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn), the AiF and the German Ministry of Economics (Project. No. 10611 N).

References 91

Nomenclature A AF d E f I ⭈ m t U U0 WPulse wSpecific ␬ ⌬⌿

semi-axis of ellipsoid (m) semi-axis in field direction (m) gap (m) electric field strength (V/cm) frequency (Hz) current (A) mass flow (kg/h) time (s) voltage (V) charging voltage (V) energy per pulse (kJ/kg) specific energy input (kJ/kg) electric conductivity (S/cm) transmembrane potential (V)

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Pulsed Electric Field Processing of Liquid Foods and Beverages Gauri S Mittal and Mansel W Griffiths University of Guelph, Ontario, Canada

Basics of the non-thermal processing of pulsed electric field (PEF) of pumpable foods and beverages are provided. PEF has demonstrated a moderate to significant microbicidal decay. The process parameters used for batch processing had a very wide range: DC voltage 2.5–43 kV; Ef 0.6–100 kV/cm; electrode distance 3–77 mm; pulse width 1 ␮s–10 ms; pulse frequency 0.2–50 Hz; number of applied pulses 1–120; and process volume 0.5 ml–1.6 l. Microbial reduction rate could be classified as moderate (1–3 log cycle microbial reduction) to significant (⬎6 log cycles). PEF pasteurization variables can be grouped into three categories: system, medium and subject parameters. However, the varying conditions and equipment used by different groups mean that not all results are in agreement and comparisons between experiments may not always be valid. In some instances, it is possible to identify key characteristics of PEF processing and to observe some generally agreed-upon trends in the three variable divisions. The combination of PEF, mild heat and antimicrobials resulted in a much higher microbial inactivation than the sum of the individual reductions achieved from each treatment alone, indicating synergy. It appears that PEF is most effective when applied to stressed cells, especially if the stress imposed has an effect on cell membrane integrity.

1 Introduction Presently, most liquid foods are preserved commercially by ultra high temperature (UHT) or high temperature short time (HTST) processes. Although heating inactivates enzymes and microorganisms, the organoleptic and nutritional properties of the food suffer because of protein denaturation and the loss of vitamins and volatile flavours. Thus, extending the shelf-life of food by heat treatment is not only energy intensive but, in many cases, adversely affects the flavour, chemical composition and nutritional quality of the treated food. There is a great need for a non-thermal method for inactivating microorganisms that is economical, compact, energy efficient, safe, socially and environmentally acceptable and which does not adversely affect nutrition, texture and flavour of the treated food. Consumers are also increasingly demanding high quality, minimally processed foods. Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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100 Pulsed Electric Field Processing of Liquid Foods and Beverages

The term ‘non-thermal processing’ is more apt for novel non-thermal technologies such as high-pressure processing (HPP), pulsed electric field (PEF), high-intensity ultrasound, ultraviolet light, pulsed light, ionizing radiation and oscillating magnetic fields, intended for microbial inactivation. Certain non-thermal processes can also affect food properties. For example, HPP by affecting the structures of proteins and polysaccharides, changes the texture, physical appearance and functionality of foods (Knorr, 1993; Williams, 1994). Ultrasound also can denature proteins and produce free radicals that can adversely affect the flavour of fruit-based or high-fat foods (Williams, 1994; Sala et al., 1996). High irradiation doses may cause slight colour changes in beef, pork and poultry (Wood and Bruhn, 2000). PEF has been investigated as a potential non-thermal technique for food preservation (Ho and Mittal, 2000) and exhibits a moderate to significant microbicidal effect by discharging high voltage electric short pulses through foods, e.g. milk, fruit juices. The use of PEF for microbial inactivation has been the subject of many reviews (Barbosa-Canovas et al., 1999; Jeyamkondan et al., 1999; Ho and Mittal, 2000; BarbosaCanovas and Zhang, 2001; Wouters et al., 2001a; Yeom et al., 2002; Rastogi, 2003; Ross et al., 2003, Hodgins and Mittal, 2003). Mittal (1993) reviewed earlier work in this area before proper equipment for the technology had been developed. Castro et al. (1993) reviewed the application of PEF mainly based on the work conducted at Washington State University and PurePulse, San-Diego. Zhang et al. (1995a) reviewed engineering aspects and Qin et al. (1995c) emphasized equipment development and shelf-life studies. Sale and Hamilton (1967) were among the first to document microbial inactivation by PEF. They applied 10 pulses of varying electric fields to a variety of bacteria and yeasts, including Bacillus cereus, B. megaterium, B. subtilis, Candida utilis, Clostridium welchii, Escherichia coli, Micrococcus lysodeikticus, Saccharomyces cerevisiae and Sarcina lutea, suspended in a 0.1 per cent sodium chloride solution. One to two log cfu reductions were found, dependent on several variables. Microbial death increased with increasing electric field strength (Ef) from 5 to 25 kV/cm; a smaller effect was noted when the pulse width was increased from 2 to 20 ␮s. However, not all organisms responded in the same way to the Ef. The two yeast species, S. cerevisiae and C. utilis, experienced the greatest reduction at moderate Ef, while M. lysodeikticus underwent less than 1 log reduction at even the harshest conditions. There were some drawbacks to the methods tested. For example, the combination of large currents and voltages led to a temperature rise of 10°C in the test medium and, due to the greater pulse durations used (20 ␮s), electrolysis of the water in the medium could have resulted in the formation of hydrogen peroxide radicals; both of which would have enhanced the killing effect. The success of this early research prompted scientists to continue exploration in the field and further to refine the process. Patents on PEF technology first appeared in Germany, where the ‘Elsteril’ process was developed. Using Ef up to 30 kV/cm, the process was shown to be effective for inactivation of organisms in liquid foods. Reduction of vegetative cell counts of up to 4 log cycles was achieved, but the spores of B. cereus and Byssochlamys nivea remained unaffected (Vega-Mercado et al., 1997b). Heating of the test medium was also severe; tests performed using this equipment added 562.5 J/pulse, which caused a 5.6°C/pulse increase in the fluid temperature (Grahl and Markl, 1996). Use of this equipment would require several cooling periods to

PEF technology 101

achieve the desired reduction in microbial population while maintaining temperatures low enough to retain product quality. A second patent was assigned to Foodco Corp., a subsidiary of Maxwell Laboratories in San Diego, CA (now PurePulse Technologies). The treatment involved combining PEF with heating samples to temperatures of 63–75°C. Using Ef in the range of 12–25 kV/cm and pulse widths of 1–100 ␮s, pumpable food products could be effectively treated (Dunn and Pearlman, 1987). There was no attempt to distinguish PEF effects from other parameter effects, particularly temperature. At 50–65°C, it is more likely that death of the natural microflora was due to thermal shock than the PEF. The system was also unstable at applied fields greater than 35 kV/cm, as arcing was observed. Although several other patents were filed on behalf of PurePulse, unsatisfactory results have led to the abandonment of the project. Nevertheless, interest in the technology has been maintained and several researchers have developed their own PEF equipment. While it is difficult to compare and contrast results between research teams, the general trends from one may be applicable to another. Summaries of more recent experiments in the field are given by Barsotti et al. (1999), Barsotti and Cheftel (1999), Ho and Mittal (2000), Ross et al. (2003) and Hodgins and Mittal (2003).

2 PEF technology A typical PEF generation schematic is shown in Figure 5.1. When the switch is thrown to position A, current flows into the capacitor. The electric field within the dielectric is displaced and the bound electric charges are polarized from their normal position of equilibrium. Then, as the switch is thrown to position B, current flows out of the capacitor. The most important components of a pulsed power generator include: 1 a power supply – either a high voltage, low current supply for a capacitive circuit or a low voltage, high current supply for an inductive circuit 2 an energy storage element – either capacitive or magnetic (inductive) 3 a switch – either closing or opening and 4 a pulse shaping and triggering circuit. A PEF food processing system consists of a pulsed power generator, a treatment chamber and a pump to supply a food through the treatment chamber. An oscilloscope is used C

A B Vs ⫽ 1V

R

T ⫽ RC Figure 5.1

R-C circuit to generate electrical pulses.

Vr

102 Pulsed Electric Field Processing of Liquid Foods and Beverages

to observe the pulse waveform. The power source, a high voltage DC generator, converts voltage from a utility line (110 or 220 V) into high voltage AC, then rectifies to a high voltage DC. Energy from the power source is stored in the capacitor and is discharged through the treatment chamber to generate an electric field in the food. The energy stored in a capacitor (Q, J) is given by Q ⫽ CV2/2, where C is the capacitance in F and V is the charging voltage in V. The effective capacitance, C, can be calculated by C ⫽ ␶␴A/d, where ␶ is the pulse duration in s, ␴ is the conductivity of the food in S/m, A is the area of the electrode surface in m2 and d is the distance between electrodes in m. Assuming the food material has homogeneous dielectric and electrical properties, the effective capacitance can also be calculated using C ⫽ ⑀o⑀rA/d, where ⑀o is the permittivity of free space and ⑀r is the relative permittivity, i.e. dielectric constant of the food. The energy stored in the capacitor is discharged using a high-voltage switch, which must be able to operate at a high power and repetition rate (see Figure 5.1). The main role of a closing switch is to hold off the high voltage during charging and to close and transfer the high intensity energy to the load much more rapidly during discharging. The types of switches that may be used include gas spark gaps, thyristors, thyratrons, ignitrons and vacuum tubes. The switch must be able to resist the maximum voltage present across the capacitor, as well as the peak current, resulting primarily from the food sample’s electrical resistivity. The principle types of arc-discharge switches are excitron, ignitron and thyratron. A thyratron is a hot cathode rectifier with grids to control current flow. It is comprised of essentially a cathode, an anode and a control grid enclosed in an envelope (usually glass, but metal or ceramic are possible) from which the air has been removed and replaced by a gas (usually hydrogen) at a fairly low pressure (Ho and Mittal, 2000). Opening switches are important in an inductive storage circuit as these serve to ‘break’ the charging current in the circuit. A concise review of various opening switches, such as mechanical, solid state, fuse, plasma gun, superconducting, thermal, plasma erosion, etc. is given by Schoenbach et al. (1984). A pseudo-spark switch, which has been tested at high repetition rates of up to 2 kHz, has also been described (Frank et al., 1992). The maximum peak current, Imax, is defined by Imax ⫽ V/R ⫽ VA␴/d. The actual peak current will be smaller than Imax due to inductance in the circuit. Commercial capacitors can store energy in the electric field of either solid or liquid dielectrics. Energy density varies from 10 to 200 J/kg. On the other hand, generators based on induction voltage adders have been identified as an important development in this field as the energy density is greatly enhanced. These systems use linear induction accelerator modules and self-magnetically insulated vacuum transmission lines to generate high voltages with nanosecond output impulses (Ho and Mittal, 2000). Treatment chambers are designed to contain the food material during PEF processing and to house the discharging electrodes. They can be either batch-type or continuous, with batch being more suitable for lab-scale experimentation. A typical treatment chamber consists of two parallel electrodes encased in an insulating material. The chamber volume can be adjusted using spacers made with insulating material of varying thickness. Sale and Hamilton (1967) designed one of the first chambers for PEF treatment using carbon electrodes supported on brass blocks, with a U-shaped polythene spacer placed between the two electrodes. Because of the electrical breakdown

Mechanisms of microbial inactivation 103

of air above the food, the maximum field strength for their design was limited to 30 kV/cm. The chamber used by Dunn and Pearlman (1987) consisted of two stainless steel electrodes, with an effective electrode area of 7800 mm2 and a 20 mm thick cylindrical nylon spacer. Barbosa-Canovas et al. (1998) reviewed other treatment chambers based on these two designs. Sample filling and removal add to the complexity in chamber design since the chamber needs to be degassed before treatment to prevent the dielectric breakdown of the food.

3 Mechanisms of microbial inactivation A microbial cell subjected to a PEF of sufficient strength undergoes membrane permeabilization. The magnitude of the transmembrane potential (TMP) determines whether the formation of pores is reversible (electrical) or irreversible (mechanical). A correlation between PEF inhibition and membrane permeabilization of L. plantarum LA 10-11 cells was demonstrated by Wouters et al. (2001b), whereas no relationship was observed between membrane permeabilization and heat inactivation. Results indicated that the ability of PEF treatment to cause membrane permeabilization was the most important factor in determining inactivation. The exact mode of action for pore formation is unclear. Some theories suggest that a large population of pores is always present, expanding rapidly in response to large potentials, while others hypothesize that pores are rapidly created by large potentials, followed immediately by rapid pore expansion (Ho and Mittal, 1996). PEF treatments are responsible for cell death by two key steps. First, the applied field induces electropore formation in the cell membrane. Secondly, if the pore formation is stable enough, interaction with the surroundings causes leakage of cellular contents, organelle destruction or lysis, resulting in death. Tests on artificial lipid bilayers show that natural analogues present in the cell membranes of organisms are the most likely target for PEFs (Chernomirdik and Chizmadzhev, 1989). Sale and Hamilton (1968) considered the effect of the potential difference developed across the cell membrane. They proposed that damage to the membrane occurs when the TMP is greater than 1 V, resulting in the loss of intrinsic properties, such as electrical resistance, membrane potential and barrier function. Hulsheger et al. (1983) calculated the induced potential (Vm) for spherical cells surrounded by nonconducting membranes by Vm ⫽ 1.5aEc, where a is the cell radius (m) and Ec is the external field strength (V). Zimmerman et al. (1974) expanded this to include nonspherical cells using the assumption that the cell shape can be considered as two hemispheres on either end of a cylinder: Vm ⫽ L(1 ⫺ 0.66a)aEc

(1)

where L is the cell length (m). Coster and Zimmerman (1975) described the electromechanical instability theory on electroporation as a consequence of electrocompressive forces which decrease cell membrane thickness. Since the bilayer volume is incompressible, the membrane undergoes an increase in area per lipid. This results in

104 Pulsed Electric Field Processing of Liquid Foods and Beverages

a destabilized bilayer. The critical potential difference for electromechanical breakdown of the membrane (Vc) is given by: Vc ⫽ [0.3679Gh2/(⑀⑀o)]0.5

(2)

where G is the membrane shear elastic modulus, h is the unstrained membrane thickness, ⑀ is the relative electric permittivity, and ⑀o is the electric permittivity of free space. This theory fails to distinguish between non-reversible membrane rupture and reversible membrane discharge (Ho and Mittal, 1996). Dimitrov (1984) developed an equation to express Vm based on a simple viscoelastic film model which was developed using experimental data. The Vc is given by: Vc ⫽ [24␥Gh3/(⑀2⑀2o)]0.25

(3)

where ␥ is the surface tension. Unlike the model proposed by Zimmerman et al. (1974), this model takes into account the surface tension and viscosity of the membrane. Later work (Neumann, 1989; Tsong, 1989) quantified the relationship between field-induced TMP (V), Ef (V/m) and the cell orientation (N), which is the angle (rad) between the field line and normal to the point of interest in the membrane, as follows: TMP ⫽ 1.5aEf cos ⌽

(4)

TM ⫽ [1.5aEf cos ⌽]/[1 ⫹ (␻RxC)2]

(5)

For AC electric field,

where ␻ ⫽ angular frequency of the field, rad/s; and RxC ⫽ dielectric relaxation constant of the membrane, s/rad. In most cases, the value ␻RxC is much smaller than unity, and Equation (5) reduces to Equation (4) (Tsong, 1989). Thus, the ease of developing the critical potential across the membrane increases with field intensity and cell size. Sale and Hamilton (1967) investigated the effects of high Ef on bacteria and yeasts and described membrane damage in the same way as the lysis of erythrocytes and protoplasts, as well as the leakage of intracellular contents. They also observed the loss of the ability of E. coli to plasmolyse in a hypertonic medium (20 mM phosphate buffer, pH 7.2 ⫹ 10 per cent sucrose) and the release of ␤-galactosidase activity in a permease-negative mutant of E. coli. The value for the field to cause rupture was dependent on several factors, so models were also created to predict them based on cell size and shape, strength and duration of the Ef, as well as biomembrane and medium characteristics (Teissie and Rols, 1993). With exposure to the field between 10⫺7 and 10⫺4 s, the rupture reseals quickly. At times greater than 10⫺4 s, the effects were permanent (Sale and Hamilton, 1967). TMP measurements show that cell death from PEFs is a function of the voltage level and exposure. In contrast, molecular reorientation theories suggest an outward expansion of the membrane. These views are more popular and are supported by experimental evidence which indicates the formation of volcano-like pores on the surface of cells. Rather than emphasizing cycles of stress and relaxation, a greater importance is placed on the reorganization of cell membrane components due to the applied voltage. Some suggest that there are always microfine pores randomly distributed throughout the matrix

Equipment 105

of the membrane due to the fluctuation of molecule-free sites (Sugar, 1989). Application of the voltage field causes immediate widening of these pores. If the applied voltage is great enough, the pore diameter increases to a point where normal attractive forces cannot reseal the breach when the field is removed (Weaver and Powell, 1989). In some instances, no destruction of the membrane bilayer was found by visual microscopic scans, although reductions in cell populations were observed after PEF treatment (Sale and Hamilton, 1967). However, the majority of studies have revealed physical evidence of damage caused by PEF. Unal et al. (2002) have shown that PEF causes cell injury and that fluorescence staining with propidium iodide is a suitable technique for detecting sublethal and lethal membrane damage by PEF. Microscopy has revealed perforated cells (Marquez et al., 1997), increased roughness of cell walls (Calderon-Miranda et al., 1999c), shrinkage of cytoplasm and thinning of cell walls (Pothakamury et al., 1995) and leakage of cellular contents (Simpson et al., 1999) and morphological changes have also been demonstrated by atomic force microscopy (Picart et al., 2002). In other cases, observed damage to the cells did not correspond to resulting lethality and, in yet other studies on S. typhimurium and L. monocytogenes, it has been suggested that PEF is an ‘all or nothing’ effect that kills cells and leaves none that are sublethally injured (Russell et al., 2000). However, this ‘all or nothing’ membrane damage does not involve membrane-bound H⫹-ATPase activity (Simpson et al., 1999). It was observed using transmission electron microscopy that some S. cerevisiae cells subjected to 64 pulses of a 40 V/cm field experienced notable damage. Cytoplasm shrank away from the cell walls, organelles were destroyed and rupture was evident in the cell wall. However, these cells accounted for only 0.1 per cent of the total, while a 99.95 per cent reduction in cell counts was noted (Harrison et al., 1997). This would suggest alternate pathways to inactivation other than those previously proposed. Tsong (1991) suggested that protein channels, not lipid bilayer pores may also be responsible. As they require a smaller voltage to open and last for greater duration than the pores, they may be responsible for longer-term leakage of cellular contents. Holes formed in the barrier to external medium may also impede the ability of the organism to control internal pH (Simpson et al., 1999); uptake of molecules may cause swelling and lysis or proton pumps could be short-circuited, all of which would result in cell death (Chernomirdik and Chizmadzhev, 1989). While both types of theories are backed by experimental evidence and models, neither can fully explain all observed results. The actual sequence of steps may be a function of the equipment used, or be explained by a combination of theories. Initial results suggest that rotavirus was resistant to PEF treatments at 20–29 kV/cm (Khadre and Yousef, 2002).

4 Equipment Orientation and assembly of some high voltage pulse generators and treatment chambers for batch processing were summarized by Ho and Mittal (2000).

106 Pulsed Electric Field Processing of Liquid Foods and Beverages

4.1 Batch treatment system A typical batch treatment system consists of a high voltage pulse generator and a treatment chamber. Other auxiliary devices may also be used for degassing, vacuuming, pre-heating and cooling of the treatment medium. To generate an exponential decay pulse from a conventional high voltage DC power supply, a resistor-capacitor circuit was usually employed with a rotational spark gap (Mizuno and Hori, 1988; Sato et al., 1994) or with a mercury ignitron spark gap (Zhang et al., 1994a). A three-stage Marx bank was designed (Gupta and Murray, 1990) which was immersed in oil to prevent arcing and corona. Four resistors and a bank of six capacitors in parallel and a spark gap for pulse transmission were also used (Dunn and Pearlman, 1989). Electric pulses with other waveforms such as square, oscillatory decay and bipolar were also developed by manipulating the charging-discharging sequence in a pulse forming/feeding network (Qin et al., 1994). A unique low-energy pulsed electric field treatment system, developed by the authors (Ho et al., 1995), consists of a 30 kV DC high voltage pulse generator and a circular treatment chamber. Figure 5.2 shows the block diagram of the unit. The 110 V AC supply is raised in voltage through a high voltage transformer and then rectified. The DC high voltage supply then charges up the 0.12 ␮F capacitor through 6 M⍀ resistors. The generation of high voltage pulses relies on the discharge of the 0.12 ␮F capacitor through the thyratron. The batch unit can generate short duration pulses (2 ␮s width) with Ef up to 100 kV/cm. The pulse waveform is a patented instant charge reversal. The circular treatment chamber (250 mm diameter) has two circular and parallel stainless steel electrodes (165 mm diameter). The insulation, Delrin, was constructed to

AC 110 V Transformer

Rectifier

DC high voltage

Capacitor 0.12 ␮F

Resistor 6 M⍀

Food cell

Trigger circuit

Thyratron

Resistor 40 k⍀

Inductor varies with application

Pulse generator Ground Figure 5.2

Oscilloscope

Block diagram of a high voltage pulse generator.

Equipment 107

have close physical contact with the electrodes. The distance between the electrodes could be adjusted by inserting Delrin circular plates (145 mm diameter) with thickness 3, 6, or 9 mm. Thus, the process volume could be varied between 49.5 ml, 99.1 ml and 148.6 ml, respectively. The effects of these pulses on microbial inactivation in an aqueous solution were studied under different operating conditions (Ef, pulse period and n) and different fluid properties (electrical conductivity, density and rheological characteristics). Ef at 10 kV/cm for 10 pulses (2 s pulse period and 2 ␮s pulse width) was found to deliver significant microbial inactivation (Ho et al., 1995). P. fluorescens in various aqueous solutions were reduced in population by ⬎6 log cycles. Configuration of the chamber and electrodes should minimize sparking or dielectric breakdown (the breakdown of the microbial cell membrane is desired, not the food matrix). This can be achieved by developing a uniform electric field in the chamber; using a smooth electrode surface (bead-blasting); using round edged electrodes, especially at the contact area of electrodes and insulation; and by eliminating air in the food. The process parameters used for batch processing had a very wide range (Ho and Mittal, 2000): DC voltage 2.5–43 kV; Ef 0.6–100 kV/cm; electrode distance 3–77 mm; pulse width 1 ␮s–10 ms; pulse frequency 0.2–50 Hz; number of applied pulses 1–120; and process volume 0.5 ml–1.6 l. Although properties of the suspending media (electrical conductivity, pH and compositions), process temperature and microbiological conditions were sometimes not reported, based on all the studies available, the microbial reduction rate was found to range from a moderate 1–3 log cycles to a highly significant kill of 6–9 log cycles. The efficacy of the treatment was a function of the various process parameters, conditions and procedures. Details of the high voltage electric pulse experiments for batch processing are summarized in detail by Ho and Mittal (2000).

4.2 Continuous treatment system For most research work, the high voltage pulse generator employed was conceptually the same as the batch operation. Qin et al. (1994, 1995a) described two chambers for continuous processing. The first one was a parallel plate chamber based on their batch chamber. The sample would flow through the horizontal test chamber in a series of U-shaped channels. The electrode gap was 5.1 or 9.5 mm giving a volume of 8 or 20 ml. The operating parameters were 35–70 kV/cm, 2–15 ␮s pulse width, 1 Hz pulse frequency and a flow rate of 600–120 ml/min. The chamber was cooled by the circulation of water in jackets around the circular stainless steel electrodes. However, the Ef and flow profile might have been difficult to monitor with the use of U-shaped channels. The second design involved a coaxial treatment chamber. An electric field optimization technique based on finite element method was used to modify the assembly by changing the electrode gap along the chamber. The operating parameters were 50–80 kV/cm field, 2–6 mm electrode gap, 2–15 ␮s pulse width, 1 Hz pulse frequency and a flow rate of 2–10 l/min. Cooling jackets were attached to both electrodes. Using the pulser developed by Physics International, Inc., San Diego (Qin et al., 1994), microbial inactivation experiments were performed. For the parallel plate continuous

108 Pulsed Electric Field Processing of Liquid Foods and Beverages

system, a reduction of 4 log cfu/ml was reported for skimmed milk and simulated milk ultrafiltrate (SMUF) inoculated with an initial count of 8 ⫻ 108 cfu/ml E. coli. The process parameters employed were square wave pulses, 50 kV/cm field, 2 ␮s pulse width, 5.1 mm electrode gap (8 ml volume) and six pulses. For the continuous coaxial chamber system, a reduction of 2.5–3.5 log cycles in SMUF and skimmed milk was reported (Qin et al., 1995a, b). A 6–7 log cycle reduction in count was also obtained with commercial apple juice inoculated with S. cerevisiae. The process parameters used were exponential decay pulses, 40 V/cm field, 2.5 ␮s pulse width, 1 Hz pulse frequency, 6 mm electrode gap (30 ml volume) and two pulses. The flow rate was not reported. A unique low energy pulsed power treatment system was developed at the authors’ laboratory (Mittal et al., 2000) to process fluid foods in continuous mode. The unit consists of a 30 kV DC high voltage pulse generator, a coaxial treatment chamber and devices for pumping, flow rate control and recording. The maximum process capacity used in the research was 180 l/h. The 110 V AC supply is raised in voltage through a step-up transformer and then rectified (Figure 5.3). The DC low voltage supply then charges up the low voltage pulse capacitor (C1) through an electrolytic capacitor bank (Co) and an inductor (L1). The generation of high voltage pulses relies on a chain of synchronized circuit actions. First, the low voltage capacitor bank discharges through the thyristor. The voltage is then raised through a high voltage transformer and the high voltage capacitor (C2) is charged. The discharge of the high voltage capacitor through the thyratron then produces a PEF between the electrodes in the treatment chamber. The high voltage transformer and diode chain are immersed in oil to prevent corona and arcing. By controlling the variac, the unit can provide up to 30 kV of high voltage pulses. The thyratron driver provides a train of 600 V, 1 ␮s square wave pulses with adjustable frequencies to trigger the grid-cathode gap of the thyratron and to provide a synchronized output for triggering the delay timer. The delay timer delivers a triggering pulse for the thyristor with a 500 ␮s delay with respect to thyratron triggering. The pulse transformer

Variac

Step-up transformer

HV pulse transformer

C2 ⫽ 2E ⫺ 9 F

D2

C0 ⫽ 1E ⫺ 3 F 110 V AC

Rectifier

L1 ⫽ 15E ⫺ 3 H

D1

R2 ⫽ 400 ⍀

Thyristor

Pulse transformer

Thyratron driver

Delay timer Figure 5.3

Load

Thyratron

C1 ⫽ 15E ⫺ 6 F

Block diagram of the continuous pulsed power treatment system (Mittal et al., 2000).

Digital oscilloscope

PEF treatment variables 109

serves to separate the thyristor from the triggering delay timer. In this configuration, the pulse rise time is in the range of nanoseconds and the pulse frequency can be adjusted between manual operation or an automatic sequence of 1–200 Hz. The coaxial treatment chamber was designed based on the batch chamber prototype. The electrode area was equal to 1.61 cm2 (Mittal et al., 2000). The materials for electrodes and insulation are stainless steel and Delrin, respectively. The inner electrode (14.3 mm outer diameter) is in the form of a circular pipe with closed, rounded ends. At the centre and around the pipe is the high voltage electrode (20.3 mm inner diameter), which is in the form of a disc with an empty centre and at 3 mm thickness. Thus, the treatment length and the electrode distance are both 3 mm. The inner electrode is held in place by an insulated support leg at each end of the pipe to allow fluid to flow in between. The ground wire goes through one of the legs for electrical connection.

5 PEF treatment variables The process of PEF pasteurization is complex in the number of variables involved, which can generally be grouped into three categories: system, medium and subject parameters. These parameters have been tested by several research groups. However, the varying conditions and equipment used by different groups means that not all results are in agreement and comparisons between experiments may not always be valid. In some instances it is possible to identify key characteristics of PEF processing and to observe some generally agreed-upon trends in the three variable divisions (Hodgins and Mittal, 2003).

5.1 PEF system variables Most significantly, there are multiple approaches to the design of PEF equipment and chambers (Hodgins and Mittal, 2003). Although some work has been conducted using equipment employed for biotechnology such as electroporators from Bio-Rad or Kodak (Genezapper™) (Kalchayanand et al., 1994; Liu et al., 1997), these approaches are inappropriate for food processing. While the peak voltages are quite small, in the range of 1–2.5 kV, the pulse widths are substantial (200–300 ␮s). This large pulse width puts some of the claims of the experimenters in doubt. As water electrolysis can occur when the pulse width is greater than 10 ␮s, these extreme pulse widths may cause the observed microbial deaths to be partially attributed to electrolytic by-products rather than solely due to the PEF treatment (Ho and Mittal, 2000). Poisoning due to the aluminium electrodes and high energy input resulting in temperature increases are other factors which make such equipment ill-suited for PEF research. Consequently, the majority of researchers have designed their own equipment. Most groups have coupled a novel treatment chamber design with commercially available pulser equipment, which commonly has a high energy requirement. When an electric field is generated between two parallel-plate electrodes, the Ef (V/m) is defined as Ef ⫽ U/d, where U (V) is the electric potential difference. Field

110 Pulsed Electric Field Processing of Liquid Foods and Beverages

intensity has a much greater effect on microbial inactivation than either pulse number (n) or pulse duration (Ho et al., 1995). When subjected to a PEF, polarization of the dipoles and the bulk movement of ions induce capacitive and resistive currents. Assuming the food has homogeneous dielectric and electric properties, the resistance, R (⍀) of the food sample is defined by: R ⫽ d/(␴A) ⫽ ␳d/A

(6)

where ␳ is the resistivity of the food (⍀ m). The dielectric constant of a food increases with increasing water content and decreases with increasing temperature. Food conductivity increases with an increase in temperature. The treatment time (t) is given by: t ⫽ n␶

(7)

where n is the number of pulses and ␶ is the pulse width. While the value of critical electric field (Ec) was very specific to the conditions used, exceeding Ec was critical to inducing death in the cells. When E. coli and Bacillus subtilis were suspended in pea soup and treated with ⬍28 kV/cm, it resulted in no more than a 1.5 log cfu reduction. However, when Ef was increased beyond 30 kV/cm, cell death increased dramatically to a maximum of 4.8 log cycles (Vega-Mercado et al., 1997a). The Ec was a function of cell diameter and larger cells were more insulated from the effects of PEF (Grahl and Markl, 1996). However, other factors such as the suspending medium and its conductivity must also play a role. Cells subjected to PEF may also lose their capacity to regulate pH differences between internal and external values, which corresponded with higher death rates at increased Ef (Simpson et al., 1999). In virtually all PEF studies, once some Ec was exceeded, there was a proportional increase in the log colony reduction as the Ef was raised. The ratio of Ef increase to increased log colony reduction varied between experiments, as log reduction is also dependent on microbial and medium parameters. Although infinitely increasing Ef would likely result in a higher death rate, applied fields are limited by the equipment used to ⬍100 kV/cm. Jeantet et al. (2003) have described a pilot-scale PEF system for continuous sterilization of liquids which generates rectangular electrical pulses. The equipment can process up to 25 l/h, at pulse amplitudes of 5–15 kV, frequencies of 1–815 Hz and pulse widths of 50, 100, 250, 500, 1000, 2000 or 3000 ns. Efficacy was tested on a model solution containing sodium sulphate (28 mM) and glucose (28 mM) inoculated with 107 cfu/ml S. enteritidis. A 3–4 log cycle reduction in count was achieved in a single pass through the treatment chamber and the degree of inactivation was linearly related to energy influx between 0 and 90 kJ/kg. On average, 30 ⫾ 1.8 kJ/kg was needed to achieve 1 log cycle reduction in bacterial count. Another significant factor in PEF processing is the treatment time, t, which is the length of time the fluid is exposed to PEF. Commonly, this variable may also be expressed as the product of two others, n and the pulse width (␶). In most scenarios, the ␶ is fixed, as the duration is set by both the system circuitry and the resistivity of the material being treated. The n is then increased for longer treatment times. As with the Ef, the effects of varying n are also dependent on other factors. Nonetheless, research has shown that increasing n increases the extent of cellular injury (Table 5.1) (Hodgins and Mittal, 2003). All studies show an increase in killing with greater n, yet

PEF treatment variables 111

Table 5.1 Rates of microbial inactivation per pulse applied for selected trials (adapted from Hodgins and Mittal, 2003) Source

Organism

Medium

Ef (kV/cm)

Q ( J/ml)

N

D

D/n

Simpson et al. (1999)

S. typhimurium

15

0–6

6E-4

E. coli E.coli, B. subtilis E. coli Natural microflora S. cerevisiae E. coli E. coli

20–60 0–20 0–100 0–35

2–3.5 0–1 0–6 0–5

0.04 0.05 0.06 0.14

Apple juice Simulated milk ultra filtrate Simulated milk ultra filtrate

12 36 55

Insufficient Information Up to 3024 Up to 49 Up to 1025 Insufficient Information Up to 222 Up to 2.4 Up to 121

0–10000

Pothakamury et al. (1995) Vega-Mercado et al. (1997a) Martin-Belloso et al. (1997) Dunn and Pearlman (1987)

Distilled water, model beef broth Simulated milk ultra filtrate Pea soup Liquid egg Orange juice

1–20 1–10 0–8

1–4 0–2.5 0–3

0.16 0.28 0.38

Zhang et al. (1994b) Zhang et al. (1995b) Vega-Mercado et al. (1997b)

12 28 25.8 35.7

Ef ⫽ electric field (kV/cm); Q ⫽ energy applied per ml ( J/ml); n ⫽ number of pulses; D ⫽ log cycle reductions in colony counts; D/n ⫽ log cycle reductions per pulse applied.

those with large ‘n’ generally had smaller log microbial reductions per pulse. The most significant damage occurred during application of the first few pulses, while additional pulses were effective, but less so. The natural microflora in orange juice also behaved similarly, as increasing the treatment time from 240 to 480 ␮s (120 to 240 pulses) did not yield a decreased count (Jia et al., 1999). Similarly, Ho et al. (1995) found that when n ⬎ 10 there was a statistically insignificant effect on the inactivation of Ps. fluorescens. Collectively, these results seem to imply both types of electroporation theories are at work. The initial pulses may cause catastrophic failure of the cell due to micropore formation or loss of pH gradient and subsequent pulses may produce additional stress cycles on cell membranes, similar to those described in the electromechanical rupture models. Additionally, the highest death rates do not correlate as well with n as they do with Ef. Furthermore, in limited cases involving enzyme activities, n did not have any effect. Thus, Ef is the key to inactivation, while the number of pulses has a secondary and limited effect. In addition to n, pulse frequency may also have a small role in determining the lethality of the treatment, although there is contradictory evidence on this point. All other conditions constant, neither a doubling of pulse period from 2 to 4 s (Ho et al., 1995) nor a doubling of pulse frequency from 1.25 to 2.5 s⫺1 resulted in significant changes in microbial death rates (Martin-Belloso et al., 1997). On a larger time scale, (Hulsheger et al., 1981) decreased the frequency of pulses from 300 to 0.1 min⫺1, using the same n (5) and found no difference in the microbial counts. Accordingly, pulse polarity effects, which increase electropermeabilization, are magnified as a result of increased pulse period. Different circuits for the creation of these pulses have also been assembled, leading to several pulse waveforms. Commonly, pulses are monopolar and exhibit either square wave or exponential decay functions. Several groups have compared the two and have decided overwhelmingly in favour of square pulses. For 20 pulses of the same total energy, square waves yielded a 0.5 greater log colony reduction of S. cerevisiae in

112 Pulsed Electric Field Processing of Liquid Foods and Beverages

apple juice than exponential waves (Zhang et al., 1994b). Similar findings were reported for natural microflora in orange juice (Qiu et al., 1998) and E. coli in simulated milk (Pothakamury et al., 1996). Although they have the same total energies and the exponential waves would have a higher peak and greater width, square waves both switch from the off state to the applied level quickly and this Ef is maintained over the pulse width to effect cell destruction. An instant-charge-reversal pulse waveform has also been developed which generates no heat due to the low energy applied per pulse (Mittal et al., 2000). Unlike a bipolar pulse, where the polarity of the pulses is reversed alternately, the charge reversal is instantaneous with no time lag and, therefore, an oscillating field is applied. The amplitude of the negative peak is not large enough to provide cell membrane breakdown, but a high alternating stress on the cell membrane is produced resulting in structural fatigue (Ho et al., 1995). Correspondingly, using the same system, n and Ef, instant charge reversal pulses yielded a ⬎6 log reduction of Ps. fluorescens in aqueous solutions, while exponential decay pulses produced less than 0.5 log reductions. Pothakamury et al. (1996) reported that square wave pulses were 9 per cent more effective than exponential decaying pulses against E. coli and Staph. aureus inoculated in SMUF. The use of alternating polarity and very short duration pulses reduces the risk of undesirable electrochemical reactions, as well as the formation of deposits at the electrodes. Bipolar pulses have simultaneous positive and negative amplitude per pulse, whereas reverse polarity has each successive pulse alternating between positive and negative poles. Bipolar pulses were reported to double microbial killing over monopolar pulses of the same energy and to reduce the amount of deposits on the electrode surfaces (Evrendilek et al., 1999).

5.2 Medium parameters PEF processing has been performed on many different media (Hodgins and Mittal, 2003). In their simplest forms, the media can be distilled water (Simpson et al., 1999), or a solution containing ions (Kalchayanand et al., 1994; Liu et al., 1997). The suspending medium and its associated qualities also influence the efficacy of a PEF treatment. Some characteristics, such as conductivity or the presence of solids, may affect the manner in which the treatment is delivered, by resisting the voltage or altering the pulse width and shape, while other characteristics, such as pH or the presence of antimicrobials increase the lethality of the PEF treatment. 5.2.1 pH Effect

PEF treatments of E. coli, Saccharomyces cerevisiae, Lactobacillus plantarum and L. innocua were more effective at low pH (Wouters et al., 1999). The PEF inactivation of E. coli O157:H7 in a 10 per cent glycerol solution was enhanced synergistically by lowering the pH from 6.4 to 3.4 using benzoic or sorbic acid (Liu et al., 1997). Similarly, adjusting the pH of skimmed milk and liquid eggs with organic acids produced additive or synergistic inactivation of bacteria when combined with PEF treatment (Gongora-Nieto et al., 1999; Fernandez-Molina et al., 2001a, b). Other work has shown that PEF combined with acidification resulted in no extra inactivation of the

PEF treatment variables 113

microflora of raw milk when compared with PEF alone (Smith et al., 2002). The synergism between acetic acid and PEF might be due to the fact that both treatments target the cell membrane (Fernandez-Molina et al., 2001a). In this way, PEF could increase the permeability of the cell wall and membrane, enhancing the entry of undissociated acids into the bacterial cell. Lowered pH as a sublethal hurdle may also jeopardize the efficacy of other antimicrobial processes. For example, Evrendilek and Zhang (2003) found that exposing E. coli O157:H7 to pH 3.6 prior to PEF treatment caused lower inactivation than exposure to pH 5.2 or 7.0. It was concluded that adaptation of E. coli to the imposing acid stress resulted in greater survivability during PEF treatment. 5.2.2 Temperature effect

Altering the temperature before treatment will have an effect on many temperaturedependent variables and the viability/activity of both microorganisms and enzymes are specific to given temperature ranges. However, it was suggested that as temperature increased, resistivity would decrease and therefore so would pulse width, resulting in a smaller treatment time and a lower reduction in colony counts during pasteurization (Hulsheger et al., 1981). On the other hand, high temperatures were also thought to cause membrane phospholipids to become more fluid, rendering them more susceptible to PEF treatments (Pothakamury et al., 1996). Assorted trials from different researchers have shown the latter to be more important. For temperatures ⬍43°C, PEF treatment of E. coli and B. subtilis at Ef ⫽ 30 kV/cm and 15 to 30 pulses resulted in ⬍2 log cycle reduction in counts. When the temperature was increased to 53–55°C under the same PEF conditions, up to 5 log cycle reductions in count were achieved (Vega-Mercado et al., 1997a). Coster and Zimmerman (1975) suggested that the increase in the rate of inactivation with increasing temperature may be due to the increase in the electric breakdown potential of the bacterial cell membrane. At low temperatures, the phospholipids in the cell membrane are closely packed in a rigid gel structure, while at high temperatures they are less ordered and the membrane has a liquid-crystalline structure. Sensoy et al. (1997) studied the temperature effect on Salmonella Dublin in skimmed milk between 10 and 50°C and found that increasing the temperature increased the sensitivity of microorganisms to PEF treatment. Also, increasing the treatment temperature from 20–30°C to 55–65°C reduced the energy consumption required to produce a 6 log cycle inactivation of E. coli in apple juice from ⬎100 to ⬍40 kJ/kg (Heinz et al., 2003). Combinations of PEF (5–50 pulses at field strengths of 15–30 kV/cm) and thermal treatments (0–60°C) for the inactivation of Listeria monocytogenes in media and skimmed milk have been investigated (Fleischman et al., 2004). At temperatures ⬍50°C, a maximal reduction in Listeria count of 1 log cycle was observed. However, at 55°C, a significant synergistic effect was observed between thermal and PEF energy, leading to count reductions of up to 4.5 log cycles. The authors concluded that PEF alone contributed only minimally to the observed reductions in counts of L. monocytogenes, but thermal energy both contributed to the reduction and increased the susceptibility of the pathogen to PEF.

114 Pulsed Electric Field Processing of Liquid Foods and Beverages

5.2.3 Composition effect

With regards to medium composition, there are also several factors involving ions which are interrelated that may change the outcome of a PEF treatment. A large amount of positive or negative ions present would create a high ionic strength, which is associated with high conductivity or low resistivity. This condition would facilitate electrical conduction through the material. The presence of ions generally enhances PEF treatments to some degree, but large concentrations may hinder the process. For example, contrasting treatments of ionic media containing sodium chloride or phosphate ions with non-ionic media such as glycerol and sucrose solutions at identical conditions of 25 kV/cm and 25°C showed that reductions in microbial counts were higher in the ionic medium (ionic strength 1.5–2) than the non-ionic (ionic strength ⭐1) (Liu et al., 1997) and the rate of inactivation was determined primarily by the conductivity of the medium (Dutreux et al., 2000). However, other studies have shown that the inactivation of L. monocytogenes by PEF was not affected by medium conductivity (Alvarez et al., 2003a). The presence of ions alters the TMP so that cell death is more likely (Bruhn et al., 1997). Water activity (aw) would also be changed which would affect microbial decay. It has been shown that the aw of the PEF treatment medium influenced inactivation of Yersinia enterocolitica; a decrease in aw from ⬎0.99 to 0.93 increased resistance of the organism to PEF by 3.5 log cycles (Alvarez et al., 2003b). When the aw of the treatment medium was initially low, an increase in aw resulted in greater microbial inactivation. For example, inactivation of Enterobacter cloacae in chocolate liquor by PEF increased by over 1 log cycle as the aw increased from 0.48 to 0.89 (Min et al., 2002). Cells of E. cloacae that survived in a low aw environment had high resistance to PEF. Generally, PEF pasteurization is most lethal under conditions of low ionic strength, low conductivity and high resistivity. For E. coli in simulated milk with ionic strengths of 28, 56 and 156 mM, 16 pulses at 40 kV/cm and 10°C yielded approximately 3, 2 and 1 log cycle reductions in count, respectively (Vega-Mercado et al., 1997b). The same general trend was also observed for PEF treatment of Byssochlamys fulva spores in fruit juices, with lower conductance generally resulting in greater killing. The order of juice conductance from lowest to highest was cranberry ⬎ apple ⬎ grape ⬎ pineapple ⬎ orange ⬎ tomato and the order of effectiveness of the applied PEF decreased in a similar manner as cranberry ⬎ grape ⬎ pineapple ⬎ orange ⬎ apple ⬎ tomato (Raso et al., 1998b). Multiple ions serve to protect the cells, as extracellular concentrations are closer to intracellular levels (Vega-Mercado et al., 1997b).

5.2.4 Antimicrobials

Among the most widely investigated antimicrobials are nisin and lysozyme. Nisin, produced by Lactococcus lactis subsp. lactis is a member of the class of bacteriocins known as lantibiotics, which contain the amino acid lanthionine. Lysozyme, an enzyme found in foods of animal origin, occurs naturally in milk at a concentration approximating 0.13 ␮g/ml (Reiter, 1978). Lysozyme lyses bacteria by hydrolysing the ␤ (1,4) linkages between N-acetylmuramic acid and the N-acetyl glucosamine of the peptidoglycan layer of cell walls. Nisin and lysozyme have both been granted GRAS

PEF treatment variables 115

(generally regarded as safe) status by the US Food and Drug Administration (Luck and Jager, 1995). Nisin and lysozyme are normally only effective against Gram-positive organisms, as the outer lipopolysaccharide component of the membrane of Gramnegative bacteria prevents such antimicrobial compounds accessing the cytoplasmic membrane or peptidoglycan layer (Hauben et al., 1996). However, lysozyme, nisin and other bacteriocins have been shown to act on several species of Gram-negative bacteria, provided that the barrier properties of the outer membrane are first disrupted (Masschalck et al., 2001; Smith et al., 2002). Under conditions of low treatment intensity, PEF application followed by nisin exposure caused additive inactivation of Listeria innocua in liquid egg, but a synergistic interaction was observed as the intensity of PEF or concentration of nisin was increased (Calderon-Miranda et al., 1999a). Numerous other researchers (Pol et al., 2000; Iu et al., 2001; Liang et al., 2002; Smith et al., 2002) have also observed synergistic inactivation of Gram-negative and Gram-positive bacteria when PEF treatments are combined with nisin and/or lysozyme addition. While the mechanism of synergy is not yet fully understood, the additional stress of PEF probably facilitates the antimicrobial agent’s access to the cytoplasmic membrane. Both treatments may inflict membrane damage, resulting in more or larger pores, or pores with greater stability (Pol et al., 2000; Smith et al., 2002). The bactericidal effects of nisin/lysozyme mixtures on PEF-treated cells were more pronounced than addition of either nisin or lysozyme alone (Liang et al., 2002). Pol and Smid (1999) subjected Bacillus cereus to low doses of nisin (0.06 ␮g/ml) and PEF treatment. The combination of treatments resulted in a reduction of 1.8 log units more than the sum of the individual treatments, which indicates that PEF is able to enhance the bactericidal action of nisin. Calderon-Miranda et al. (1999b) investigated the inactivation of Listeria innocua in skimmed milk by PEF and nisin and noticed a synergistic effect with the addition of nisin following PEF treatment. They obtained a 3.8 log reduction for 32 pulses of 50 kV/cm followed by exposure of the microorganism to 100 U nisin/ml. Kalchayanand et al. (1994) observed that nisin was able to enhance the PEF inactivation of L. monocytogenes, E. coli and S. typhimurium. Using transmission electron microscopy, Calderon-Miranda et al. (1999a) observed that the combination of PEF and nisin exhibited an additive effect on the morphological damage to Listeria innocua. Other membrane active antimicrobial agents have been shown to act synergistically with nisin and PEF. Carvacrol is a natural plant constituent occurring in oregano and thyme whose bactericidal activity is the result of increased permeability of cell membranes leading to dissipation of pH gradients and leakage of inorganic ions (Lambert et al., 2001). Activity against vegetative cells of B. cereus was enhanced when carvacrol (0.5 mM) was applied simultaneously with nisin (0.04 ␮g/ml) and PEF treatment (16.7 kV/cm, 30 pulses) in HEPES buffer, pH 7 (Pol et al., 2001b). In the presence of carvacrol, the level of inactivation increased by over 2 log cycles. Carvacrol was not able to enhance the synergy between nisin and PEF treatment in a 20 per cent aqueous dilution of milk unless it was present at a high concentration (1.2 mM). The combination of carvacrol, nisin and PEF had no effect on spores of B. cereus, but germinating spores were susceptible to the treatment (Pol et al., 2001a).

116 Pulsed Electric Field Processing of Liquid Foods and Beverages

The potential synergistic antibacterial effect of combined ozone (0.25–1.00 ␮g/ml) and PEF treatments (10–30 kV/cm) against Lactobacillus leichmannii ATCC 4797, Escherichia coli O157:H7 ATCC 35150 and Listeria monocytogenes Scott A in 0.1 per cent NaCl has been investigated (Unal et al., 2001). Synergy between ozone and PEF varied with the treatment level and the bacterium treated. Counts of L. leichmannii were reduced by 7.1 and 7.2 log cfu/ml after treatment with PEF (20 kV/cm) and exposure to 0.75 and 1 ␮g/ml of ozone, respectively. This represents a 3.5–6 log cycle greater reduction in counts than produced by application of the treatments individually. Similarly, PEF (15 kV/cm) in conjunction with ozone at 0.5 and 0.75 ␮g/ml reduced counts of E. coli O157 by 2.9 and 3.6 log cfu/ml, respectively. When populations of L. monocytogenes were treated with PEF (15 kV/cm) after exposure to 0.25, 0.5 and 0.75 ␮g/ml of ozone, 1.7, 2.0 and 3.9 log cfu/ml were killed, respectively. 5.2.5 Ionic effect

The presence of additives or other ingredients in the suspending medium may also change the outcome of a PEF treatment. Bivalent cations such as Mg2⫹ or Ca2⫹ paired with the same anions as other monovalent cations (K⫹, Na⫹) showed a protective effect on membranes and thus less killing was observed after PEF treatment (Hulsheger et al., 1981). Other substances such as lipids (Grahl and Markl, 1996) may serve as an insulating barrier against the applied voltage and exert a protective effect on the cells. On the other hand, there are substances which can improve microbial killing. The presence of various carboxylic acids such as benzoates or sorbates in a 10 per cent glycerol solution pulsed at 12.5 kV/cm decreased the count of E. coli O157:H7 by 100 to 1000 times over PEF treatments performed in the absence of the acids (Liu et al., 1997).

6 Target differences Even if the characteristics of the medium are exactly constant, the substances suspended in the medium (i.e. microorganisms, enzymes) will not react in an identical manner to the applied field (Hodgins and Mittal, 2003). Some bacteria are more susceptible to PEF treatments than others. For example, Wouters et al. (2001b) showed that there were differences between susceptibility of two species of Lactobacillus to PEF. Lactobacillus fermentum strain PW7 was more PEF resistant and exhibited less membrane permeabilization than L. plantarum strain LA 10-11. These researchers also showed that for E. coli, Saccharomyces cerevisiae, Lactobacillus plantarum and L. innocua the efficiency of PEF inactivation is dependent on organism and strain. There may even be variation within the same organism at different growth stages with cells being most resistant to PEF in the stationary phase of growth (Wouters et al., 1999). Thus, the microbial survival rate depends more on the physiological properties of the cell population than on the microorganism type (Hulsheger et al., 1983). These include the microbial growth stage, initial inoculum size, growth history, ionic concentration and conductivity of the suspension fluid (Wouters and Smelt, 1997).

Target differences 117

Using flow cytometry, cells of L. plantarum were sorted on the basis of cell size and shape and results were confirmed by image analysis (Wouters et al., 2001b). An apparent effect of morphology on membrane permeabilization was observed, with larger cells being more easily permeabilized than smaller cells and thus more susceptible to PEF. Hulsheger et al. (1983) also observed that Gram-positive bacteria and yeasts are less sensitive to PEF treatment than Gram-negative bacteria when low pulse numbers are applied. Zhang et al. (1994a) showed that, for identical Ef, the log population reduction of the yeast S. cerevisiae was more than one cycle greater than the Gram-negative bacterium, E. coli. Comparing the main groups of bacteria, under the same conditions (simulated milk, Ef ⫽ 12–16 kV/cm, 20 ⬍ n ⬍ 50), the Gram-positive bacterium Staph. aureus underwent 1–3 log cycle reductions in count while the process was more lethal to the Gram-negative E. coli, whose cell numbers were reduced by 2–5 log cycles (Pothakamury et al., 1995). Similarly, at 15 kV/cm and 10 000 pulses, L. monocytogenes was less sensitive to PEF than S. typhi. The treatment achieved a 4 log cycle reduction in count for the Gram-positive organism, whereas the corresponding value was 6 log cycles for the Gram-negative organism (Simpson et al., 1999). The cause behind the difference in resistance is likely due to cell size and structure. Based on observations, predictor models have shown that the effective Ef for a given bacterium is partially a function of cell diameter (Grahl and Markl, 1996). Smaller cells would experience a more uniform field and, thus, cell death would be more likely. Differences also arise on account of the cell wall arrangement. Typically, Gram-positive cells have thick cell walls (20–80 nm), with rigidity enhanced by low lipid content (0–3 per cent), while Gram-negative organisms have a second lipopolysaccharide layer, thinner cell walls (10 nm) and are more flexible (lipid content 11–22 per cent). It appears that the strength imparted by strong cell walls has a direct bearing on the effectiveness of PEF treatment. This conclusion is supported by the work of Hulsheger et al. (1983), which included measurements of microbes with uncommon features. Of the Gram-negative bacteria tested, Klebsiella pneumoniae required the largest Ef, which was attributed to its formation of protective capsules. While one phenotype of L. monocytogenes was difficult to kill with PEF, a second phenotype tested with a physiological defect in the cell membrane assembly was more sensitive to the process. In mixed cultures, the pattern still applies in most instances. Treatment of the natural microflora in orange juice at 30 kV/cm revealed a more significant impact on the yeast and mould plate counts than the overall bacterial count; thus, the bacteria were more resistant (Jia et al., 1999). Likewise, for B. subtilis and E. coli pulsed at 28 kV/cm in pea soup, the Gram-positive bacterial population was reduced by 5.3 log cycles, but the less resistant Gram-negative cells were reduced by 6.5 log cycles. Additionally, the combination of more than one cell type was thought to have a mutually protective effect on the microbes. Both the overall killing rate for the pair was less than that expected for single species and the number of survivors for both bacteria increased in a mixed culture (Vega-Mercado et al., 1997a). Even though vegetative cells are easier to kill, sensitivity to PEF is also dependent on the growth stage of the organism. Cells are more likely to be affected by PEF treatments in the exponential growth phase than any other. Trials using E. coli have shown that for a Ef of 36 kV/cm and 4 pulses, lag phase cells showed the most resistance,

118 Pulsed Electric Field Processing of Liquid Foods and Beverages

with ⬍1 log cycle reduction, followed by stationary phase cells averaging one cycle and then exponential growth phase cells, which were about 100 times more susceptible to the treatment (Pothakamury et al., 1996). Similarly, statistical analysis of exponential growth versus stationary growth phase cells showed that, while the treatment time remained the same, the critical Ef required for PEF processing increased tenfold for the latter (Hulsheger et al., 1983). While this has been conclusively proven for E. coli cells, it is assumed that the relationship likely holds for other types as well. For some bacteria there are also differences in the way that planktonic cells and those attached to surfaces respond to PEF. PEF treatment (electric field intensity and pulse frequency of 41 kV/cm and 3 Hz, respectively) of E. coli attached to polystyrene beads gave higher inactivation rates than PEF treatment of free-living bacteria, whereas inactivation rates of attached L. innocua were almost identical to the inactivation rates of free-living L. innocua (Dutreux et al., 2000). Finally, the initial colony count (N0) has also been suggested to play a role in cell death resulting from Ef, although there are several contradictory studies in the literature. With 105–109 E. coli/ml pulsed at 12 kV/cm, the survival rate decreased from 6 to 8 per cent at the lower inoculum levels to 4 per cent at high initial cell numbers (Hulsheger et al., 1981). Although the differences were slight in most trials, a discernable trend was observed, but this was not repeatable. When treated in simulated milk at 70 kV/cm and 16 pulses, the same bacteria experienced log reductions of 3–3.5 cycles randomly and independently of the initial levels of inoculation (103–108 cfu/ml) (Zhang et al., 1994a). Yet, repudiating both findings, a third study found that, when the initial count of S. cerevisiae was greater than 106 cfu/ml in apple juice, pulsing once at 25 kV/cm yielded ⬍1 log cycle reduction in count, while at inoculum levels less than 104 cfu/ml the treatment resulted in ⬎2 log cycle reductions (Zhang et al., 1994b). The effect of initial microbial concentration on PEF inactivation appears to depend on the type of organism. Alvarez et al. (2000) studied the inactivation of S. senftenberg and concluded that microbial inactivation was not a function of N0, whereas for E. coli O157:H7 more inactivation occurred at lower initial cell numbers (Damar et al., 2002).

7 High-pressure processing (HPP) and PEF Pressurization may reduce the effectiveness of a PEF treatment. Knorr (2001) found that PEF treatment under HPP (200 MPa) exerted a protective effect against permeabilization of bacterial cell membranes, although each treatment alone caused major damage at this site. Pagan et al. (1998) studied the possibility of germinating Bacillus spores using HPP, then inactivating the germinated cells with a PEF treatment. They found that germination of ⬎5 log cycles of spores was initiated by pressurization and, while the germinated cells did become sensitive to a subsequent heat treatment, they were not sensitized to PEF application below 40°C. Spore inactivation by these combined processes could be improved by adding an intermediate holding step, to allow germinated spores to outgrow into vegetative cells. However, it is likely that this will be highly strain dependent and of limited practical use.

Specific results on liquid foods 119

Pulsed electric field treatments have also been used in combination with supercritical CO2 and inclusion of the latter treatment produced a synergistic effect that led to full inhibition of E. coli and Staph. aureus and a decrease in B. cereus spore count of ⭓3 log cycles, depending on the exposure time (Spilimbergo et al., 2003).

8 Specific results on liquid foods 8.1 Milk According to Martin et al. (1997), the inactivation of E. coli is less effective in skimmed milk than in a buffer solution when exposed to the same PEF treatment conditions. This is probably due to the complex composition of skimmed milk and the presence of proteins. The influence of milk fat on the PEF inactivation of microorganisms is unclear. Reina et al. (1998) observed no significant differences in the inactivation of L. monocytogenes in whole milk, 2 per cent milk and skimmed milk. Less than 1 log cycle difference between inactivation of E. coli in milk and in phosphate buffer was reported by Dutreux et al. (2000). However, experiments conducted by Grahl and Markl (1996) indicate that the fat content of the medium is inversely related to microbial inactivation. They performed sensory evaluations on milk treated by PEF at temperatures ranging from 45 to 50°C and concluded that there was no significant organoleptic deterioration. Raso et al. (1998c) achieved only a 2 log cycle microbial reduction in raw skimmed milk. Similar reductions in count (0.3–3 log cfu/ml) were achieved for Ps. fluorescens, Lactococcus lactis and Bacillus cereus in raw and UHT skimmed milk using a continuous PEF bench scale system to deliver 35 kV/cm field strength with 64 pulses of bipolar square wave for 188 ␮s and a flow rate of 1 ml/s (Michalac et al., 2003). The milk temperature was raised to 52°C and then cooled to 22°C during PEF treatment. Almost all of the research on PEF treatment of milk has been performed on skimmed milk or simulated milk ultrafiltrate (SMUF) which has been inoculated with one or more microorganisms. This is useful in determining the susceptibility of microorganisms to PEF treatment relative to other microorganisms, but it does not indicate the effectiveness of this process for milk with a naturally occurring microbial population. The natural microflora of milk would be much more resilient than the inoculated microorganisms which would be under much greater stress due to the changing environments associated with growing, harvesting and inoculating the milk samples. Smith et al. (2002) investigated the combined effect of heat treatment (52°C), the addition of antimicrobials and PEF treatment on naturally occurring microorganisms in milk. Heating raw skimmed milk to temperatures of 52°C or lower had no significant effect on the microbial population. Raw skimmed milk subjected to PEF treatment at 52°C resulted in 1.3 log reduction of microorganisms. A PEF treatment consisting of peak Ef of 80 kV/cm and 50 pulses was applied to each sample. At a temperature of 4°C, PEF treatment had no effect on pH adjusted skimmed milk samples. There was no discernible difference in microbial inactivation between milk at a pH of 5.0 or at its natural pH of 6.7. The antimicrobials, lysozyme and nisin, either alone

120 Pulsed Electric Field Processing of Liquid Foods and Beverages

or in combination did not inactivate a significant number of microorganisms when added to raw skimmed milk at 4°C. When the temperature was increased to 52°C, microbial inactivation was still low, with a 1:3 combination of lysozyme and nisin having the greatest effect (1.2 log cycle reductions in count). Addition of antimicrobials to raw skimmed milk had little effect on the microorganisms. The addition of lysozyme and nisin, both alone and in combination at 4 and 52°C resulted in significantly different effects on microbial inactivation. When combined with PEF treatment using a peak Ef of 80 kV/cm and 50 pulses, the net effect was a significant increase in microbial inactivation. The addition of lysozyme resulted in a 3.2 log reduction while nisin reduced the initial microbial level by 5.9 log cycles. The combination of lysozyme and nisin provided the highest inactivation (a reduction in count of 7.0 log cycles). The microbial reductions achieved by combining lysozyme, and/or nisin, with PEF treatment (80 kV, 50 pulses) were significantly different (Smith et al., 2002). The initial microbial levels were in the range of 107–108 cfu/ml. The final counts in the PEF-treated milk containing nisin or nisin/lysozyme were all less than 250 cfu/ml with approximately half of the treated samples having counts ⬍10 cfu/ml. This indicates that nisin alone is almost as effective as a combination of lysozyme and nisin. The combination of PEF, mild heat and antimicrobials resulted in a much higher microbial inactivation than the sum of the individual reductions achieved from each treatment alone, indicating synergy. The optimal combination of PEF (80 kV/cm, 50 pulses), mild heat (52°C), 1000 ␮g lysozyme/ml and 3000 U nisin/ml was also evaluated by applying 20 and 80 pulses, respectively. Increasing the n during PEF treatment from 20 to 50 had a significant effect on microbial inactivation. Increasing the n to 80 did not have much of an effect, since 50 pulses were sufficient for an almost complete inactivation of the initial microbial population. PEF pasteurization of milk has been reviewed by Bendicho et al. (2002).

8.2 Liquid whole egg and egg white PEF (10.6, 21.3 or 32 pulses at 3.5 Hz for 2 ␮s and Ef of 30, 40 or 50 kV/cm) was applied to liquid whole egg containing L. innocua (Calderon-Miranda et al., 1999a). After PEF treatment of 32 pulses at 50 kV/cm L. innocua counts were reduced by 3.5 log cycles. PEF (32 pulses, 50 kV/cm) followed by nisin (100 U nisin/ml) exposure decreased numbers of L. innocua by a further 2 log cycles. High-intensity electric fields (Ef, 20–35 kV/cm; pulse frequency, 100–900 Hz; pulse number, 2–8; temperature, 4–30°C; pH, 7–9) have been successfully applied to the destruction of Salmonella enteritidis inoculated at levels of 103–107 cfu/ml in diaultrafiltered egg white (Jeantet et al., 1999). Results indicated that Ef is mainly responsible for inactivation of Salmonella but the effect is strengthened by a positive interaction with pulse number. An optimal PEF treatment resulted in a 3.5 log cycle reduction in viable salmonellae. Unlike with a heat treatment of 55°C for 15 min which produces a comparable reduction in Salmonella count, no protein denaturation in the PEF treated egg white was observed.

Specific results on liquid foods 121

8.3 Apple cider and juice Zhang et al. (1994b) reported a 4 log cycle reduction of yeast (S. cerevisiae) inoculated in pasteurized commercial apple juice after application of 20 square-wave pulses of 260 J/pulse energy in a parallel electrode chamber with a batch volume of 25.7 ml. Due to this high energy input, considerable cooling was required and the process temperature was not reported. No data on juice quality were provided. On the other hand, Qin et al. (1995b) reported a 6 log cycle reduction in counts of S. cerevisiae after PEF treatment of a commercial apple juice. The process parameters used were: exponential decay pulses, 45 kV/cm field; 2.5 ␮s pulse width; 1 Hz pulse frequency; and 0.6 cm electrode gap (coaxial chamber, 30 ml volume). The treatment temperature was controlled at 30°C by circulating chilled water. The flow rate was not reported and a suitable control treatment was not conducted to allow for comparison with PEF effects. Due to the large energy input and removal of heat by circulating chilled water, it is difficult to distinguish between the effect of PEF and the thermal effect. A non-pathogenic strain of E. coli O157:H7 (stx gene knockout mutant) was used after comparing its acid and temperature tolerance with the parent strain (Iu et al., 2001). Freshly squeezed, unpasteurized and preservative-free apple cider (average pH ⫽ 4.22 ⫾ 0.03) was subjected to PEF under various conditions, including Ef of 0 (control), 60, 70, 80 kV/cm and 90 kV/cm, each with 10, 20 and 30 pulses and at 20, 30 and 42°C. In addition, cinnamon powder (2 per cent wt/vol) or nisin (2 per cent wt/vol) was added to the cider before PEF treatment. When the treatment temperature was increased from 20 to 42°C, the population of E. coli O157:H7 decreased significantly by ⬎5 log cfu/ml. As the Ef increased from 60 to 80 kV/cm, cell death also increased by 0.1 to 1 log cfu/ml (Iu et al., 2001). However, changing the pulse number (n) had no significant effect on E. coli counts. Therefore, the combination of an elevated temperature, i.e. 42°C and high Ef, i.e. 80 kV/cm, with any n ranging from 10 to 30 should result in significant cell death of E. coli. Similar results were reported by Evrendilek et al. (2000). Significantly greater cell inactivation (5.91 log cfu/ml) was achieved when the Ef was raised to 90 kV/cm with a treatment temperature of 42°C (Iu et al., 2001). PEF treatment had a significantly greater effect on E. coli, coliforms and lactic acid bacteria, than on yeasts and moulds. This could be because yeasts and moulds survive better in an acidic environment and they often require a higher temperature to be destroyed (Ray, 2004). It is generally recognized that Gram-negative bacteria, such as E. coli and coliforms, are more susceptible to the PEF process than Gram-positive species and yeasts and moulds (Janda and Abbott, 1998; Ray, 2004). The addition of cinnamon to apple cider in combination with a PEF treatment of 10 pulses and 80 kV/cm at 42°C induced further cell death by only 1 log cfu/ml (Iu et al., 2001). However, the addition of nisin together with PEF treatment caused a significantly greater reduction in E. coli cells of ⬎3 log cfu/ml. These results suggest that nisin acts synergistically with PEF treatments to reduce E. coli levels in apple cider. The combination of PEF treatment and cinnamon or nisin induced cell death of about 6–8 log cfu/ml when compared to the control conditions. The effectiveness of PEF for inactivation of a generic E. coli strain in apple juice has also been demonstrated (Sen-Gupta et al., 2003). In this case a

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⬎5 log cycle reduction in count was achieved using a 40 kV/cm applied voltage and 100 pulses. The combination of different hurdles, such as moderately high temperatures (⬍55°C), antimicrobial compounds and PEF, to reduce naturally-occurring microbes in freshly squeezed apple cider was explored by Liang et al. (2002). The pH of the cider was 3.7 ⫾ 0.2, specific gravity was 1.03 ⫾ 0.005 and its electrical conductivity was 2.5 ⫾ 0.1 mS/cm. The temperature increase due to energy absorbed from pulses was from 1 to 3°C at 20, 30 and 40 pulses respectively. At all temperatures tested, as the n increased from 20 to 40, the number of microorganisms decreased in the cider. Compared with counts obtained in the untreated juice at 23°C, PEF treatments of 90 kV/cm, 40 pulses, 44°C and 90 kV/cm, 40 pulses, 52°C produced a 3 and ⬎6 log cycle reduction in count, respectively. When reductions due to PEF alone were calculated (by subtracting log N values at 0 and 40 pulses) at 44, 48, 50 and 52°C they were found to be 2.22, 1.45, 1.73 and 1.72 log cycles, respectively. Lower microbial inactivation due to PEF at higher temperatures (⬎44°C) was due to the reduction in the microbial population prior to the PEF treatment as a result of exposure to the elevated temperatures. There was a significant difference in microbial reduction for process temperatures between 44 and 52°C. This indicated that PEF and temperature acted synergistically. At 46°C, an additive effect was observed when nisin (100 U/ml of apple cider) or a mixture of nisin-lysozyme (27.5 U/ml of nisin and 690 U/ml of lysozyme) was added. However, there was no significant difference in microbial reduction between treatments carried out at temperatures below 46°C in the presence and absence of antimicrobial agents. PEF treatment at 87 kV/cm, 40 instant-charge-reversal pulses and 50°C process temperature resulted in no loss of added vitamin C (0.1 mg/ml) in the cider.

8.4 Orange juice Three kinds of orange juice were investigated – already pasteurized without pulp (pH 3.8) and freshly squeezed with and without pulp (pH 4.1). A luminescent strain of Salmonella typhimurium was added to the juices so that the initial cell count was about 107 cfu/ml (Liang et al., 2002). For freshly squeezed orange juice (with and without pulp) cell injury increased with pulse number. The population of luminescent S. typhimurium decreased with increasing pulse number and treatment temperature. At 90 kV/cm, 20 pulses and 45°C, PEF treatment did not greatly enhance viability loss and injury to the cells. At and above 46°C, however, cell death and injury were greatly increased. A maximum reduction in count of 5.9 log cycles was achieved when the juice was treated with 90 kV/cm and 50 pulses at 55°C. A PEF treatment in the presence of nisin, lysozyme and a mixture of nisin and lysozyme, resulted in an additional 0.04 to 2.75 log cycle reduction in cell numbers. The combination of nisin and lysozyme had a greater bactericidal effect than either of them alone, with at least 1.37 log cfu/ml additional viability loss. No further cell death resulted from lowering the pH, but the number of injured cells increased. Inactivation of Listeria innocua, E. coli, Leuconostoc mesenteroides and Saccharomyces cerevisiae in orange juice was investigated in a 100 l/h flowing PEF system at

Specific results on liquid foods 123

Ef levels of 30 kV/cm and 50 kV/cm (McDonald et al., 2000). L. mesenteroides, E. coli and L. innocua were inactivated by as much as 5 log cycles at 30 kV/cm and 50°C, but a maximum reduction of only 2.5 log cycles was achieved for S. cerevisiae ascospores even when an Ef of 50 kV/cm and 50°C was applied. Both electric field levels were effective in inactivating microorganisms at temperatures below those used for thermal pasteurization of orange juice. To investigate the effect of PEF on the inactivation of naturally occurring spoilage microorganisms in orange juice, key parameters, such as temperature, acidity, number of pulses and initial colony count, were varied (Hodgins et al., 2002) to produce the desired level of pasteurization using the ‘hurdle approach’. Freshly squeezed orange juice with spoilage (mostly aciduric) microorganisms at the level of 107 cfu/ml was used. The Ef was 80 kV/cm, n were either 20, 50 or 80 and the treatment temperature was 42–44°C. Pectinmethylesterase (PME) activity was measured with spectrophotometrically at 650 nm (Parish, 1998). Vitamin C was measured using the 2,6-dichloroindophenol titration method (AOAC, 2000). Individual treatments consisting of a temperature of 44°C, a pH of 3.5 and a PEF treatment of 80 pulses at 80 kV/cm were all ineffective at reducing microbial counts; the combination of all three led to a synergistic increase in microbial inactivation. Reductions in colony counts were 1.2 log cycles greater for the combined treatment than for the sum of all three treatments individually. The difference between the storage and processing temperature had a significant effect on the process and the treatment efficiency was also dependent on the initial microbial count, changes in pH and n; cross-effects between the changes in acidity and temperature were also important. To improve further the efficacy of the PEF treatment, the use of the antimicrobials nisin, lysozyme and a combination of the two was investigated. Nisin addition to the juice at a rate of 100 U/ml in conjunction with a PEF treatment consisting of 20 pulses with Ef of 80 kV/cm; 44°C and pH 3.5 resulted in a significant decrease in microbial count (Hodgins et al., 2002). An average reduction in count of 6.83 log cycles was achieved using this process, as compared to processing without nisin, when only a 1.26 log cycle reduction in count was observed. This procedure was also shown to be dosedependent and most effective at low pH. PEF processed orange juice retained many of the characteristics of fresh juice, while still achieving the low microbial counts associated with thermal pasteurization. PME activity was reduced by more than 92 per cent of that found in the untreated juice. Such a level would provide sufficient cloud stability. Vitamin C losses were minimal, with over 97 per cent retention of the initial level. This was a marked improvement over heat pasteurization, where about 18 per cent losses have been documented. The juice shelf-life after processing was also extended so that the beverage would not spoil after a one month storage period at 4°C. If aseptically packed, the juice can be stored for a much longer time at room temperature. In another study, Min et al. (2003) determined the effects of a commercial-scale PEF system on microbial stability, vitamin C, flavour compounds, colour, Brix, pH and sensory properties of orange juice in comparison with those of thermally processed orange juice. Freshly squeezed orange juice was thermally processed at 90°C for 90 s or processed using PEF at 40 kV/cm for 97 ms. Juices processed by both methods had a shelf-life of 196 days at 4°C, but PEF-processed juice retained more vitamin C,

124 Pulsed Electric Field Processing of Liquid Foods and Beverages

flavour and colour than thermally processed juice. The texture, flavour and overall acceptability were ranked highest for untreated juice, followed by PEF-processed juice and then thermally processed juice. PEF processing resulted in significant increases in the hydrocarbon flavour compounds D-limonene, ␣-pinene, myrcene and valencene (Ayhan et al., 2002).

8.5 Tomato juice The population of Byssochlamys fulva conidiospores in tomato juice decreased less than 1 log cycle when 2 pulses of 30 kV/cm were applied and juice temperature was kept below 23°C by circulating chilled water around the treatment chamber (Raso et al., 1998a). Slightly less than 4 log cycle reductions in count were obtained when 15 pulses were applied. The inactivation of naturally occurring microorganisms (mostly yeast and moulds) in tomato juice was investigated by applying PEF (authors’ unpublished work). At an Ef of 80 kV/cm, 20 pulses, 50°C and in the presence of nisin (100 U/ml), the microbial population in the juice was reduced by about 4.4 log cycles. However, only a 0.55 log cycle reduction in count was obtained when juice containing 100 U nisin/ml was heated to 50°C without PEF. There was a significant increase in the microbial inactivation when the temperature was increased from 45 to 50°C. Treated juice, without aseptic filling, could be stored at 4°C for 28 days without any significant microbial growth. The enzyme polygalacturonase was unaffected by PEF, but the activity of PME was reduced by 55 per cent. There was no vitamin C reduction due to the treatment. Sensory analysis has shown that the flavour of PEF-processed juice was preferred to that of thermally processed juice (Min and Zhang, 2003).

8.6 Red grape juice Zygosaccharomyces bailii ascospores and vegetative cells were inactivated in grape juice by heat, high hydrostatic pressure and high energy PEF (Raso et al., 1998a). The Z. bailii ascospore population was more resistant to PEF than vegetative cells. Two pulses with an Ef of 35 kV/cm reduced the population of vegetative cells by 5.0 log cycles and ascospores by 3.5 log cycles at 20°C. A continuous coaxial treatment chamber with a 23 ml capacity and 6 mm electrode gap was used. Exponential decay pulses (2 Hz frequency, 0.5 ␮F charging/discharging capacitor) of 2.3 ␮s width were applied. The effect of PEF treatment on grape juice inoculated with B. fulva conidiospores and Neosartoria fischeri ascospores has been reported (Raso et al., 1998b). Juice temperature was controlled by passing the juice through a cooling coil immersed in iced water. The extent of inactivation of N. fischeri ascospores by PEF was negligible even after 20 pulses each with Ef of 44.8 kV/cm. After a PEF treatment of 8 pulses at 35 kV/cm and 20°C, the population of B. fulva conidiospores in grape juice decreased by 5 log cycles. The combination of different hurdles, such as moderately high temperatures (⬍50°C), antimicrobial compounds and PEF, to reduce naturally-occurring microbes in red and white grape juices was explored (authors’ unpublished work). To study the effect of

Specific results on liquid foods 125

antimicrobials on spoilage microorganisms in grape juice, nisin (2.5 per cent nisin, 97.5 per cent NaCl and lactose, activity of 106 U/g nisin) and two mixtures of nisin and lysozyme (A – 23.75 per cent (w/w of total) pure lysozyme with an activity of 6900 U/mg, 1.69 per cent (w/w of total) pure nisin with an activity of 275 U/mg and B – 23.75 per cent (w/w of total) pure lysozyme with an activity of 6900 U/mg, 0.56 per cent (w/w of total) pure nisin with an activity of 275 U/mg) were used. When 20 pulses, each of 80 V/cm, were applied, a greater reduction in counts was observed at all the temperatures tested (42–50°C). The reductions in counts attributable only to PEF were 0.8, 1.0, 1.2, 2.0 and 2.2 log cfu/ml at 42, 44, 46, 48 and 50°C, respectively. About a 4 log cycle reduction in count was achieved at 50°C and 20 pulses of 80 kV/cm. When PEF was applied after addition of nisin (400 U/ml) to red grape juice at 42°C, a greater reduction in count than found with PEF alone was observed. However, no significant effect was observed by the addition of nisin at 50°C. A log reduction in microbial count of 6.2 in red grape juice was achieved at 51°C when 20 pulses, each of 80 kV/cm, were applied in combination with nisin addition. A mixture of nisin (0.4 g/100 ml) and lysozyme (0.4 g/100 ml) did not improve the efficacy of the treatment at 50°C over that observed with nisin alone. However, the addition of lysozyme (0.4 g/100 ml) alone produced a small, but significant increase in microbial reduction compared to those obtained with nisin and lysozyme. Moreover, when lysozyme and nisin together (mixture A; 0.4 g/100 ml) were added to the juice and mixed for 1 or 2 h, a subsequent PEF treatment of 20 pulses of 65 kV/cm at 50°C produced a reduction in count of between 5.0 and 5.9 log cycles. A reduction in count of 4.53 log cfu/ml was achieved when 30 pulses, each of 87 kV/cm, were applied at 50°C. The greatest reduction in count (4.4 log cfu/ml) was achieved for white grape juice when 20 pulses of 65 kV/cm were applied at 50°C in the presence of lysozyme and nisin together (mixture A; 0.4 g/100 ml).

8.7 Mango juice The combination of various hurdles, such as mild heat (52°C), antimicrobials and PEF, to inactivate naturally-occurring microbes in mango juice was investigated (authors’ unpublished work). The microbial count decreased with an increase in process temperature above 47°C and addition of nisin and lysozyme (0.1 mg/ml; 1.87 U/ml nisin and 1.1 U/ml lysozyme). PEF treatment for 20 and 50 pulses of 87 kV/cm at room temperature reduced the microbial count by 1.2 and 1.33 log cfu/ml, respectively, compared with the control. PEF treatments for 20 and 50 pulses at 47°C reduced yeast counts by 1.40 and 1.49 log cfu/ml, respectively, compared to a control at 24°C. The addition of nisin and lysozyme to mango juice alone did not affect the microbial count. However, treatment of the juice with PEF for 20 or 50 pulses at 47°C in the presence of nisin and lysozyme significantly reduced the microbial count by 2.54 or 2.73 log cfu/ml, respectively, compared with the control at 24°C. The corresponding values when the treatment was applied at 50°C were 3.07 and 3.38 log cfu/ml, respectively. When the processing temperature was increased to 52°C, the microbial count was reduced by 4.40 log cfu/ml. Very low levels of nisin and lysozyme (0.1 mg/ml) were used in this study and greater microbial inactivation may have been achieved by increasing their concentrations.

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8.8 Cranberry juice Cranberry juice was exposed to PEF and PEF ⫹ heat (60°C) and microbial analysis and colour measurements were performed on untreated and treated juice (Evrendilek et al., 2001). The PEF or PEF ⫹ heat treatments did not result in any significant differences in colour retention of cranberry juice and were very effective methods to increase the shelf-life of the juice.

8.9 Beer Inactivation and sublethal injury of the beer spoilage organism Lactobacillus plantarum in a model system using different pulsed electric field (PEF) strengths (10–19 kV/cm) and total energy inputs (13–42 kJ/kg) were investigated (Ulmer et al., 2002). Below the critical Ef value of 13.8 kV/cm, metabolic activity and membrane integrity were reduced but no loss of viability was observed. Above the critical value, however, irreversible cell damage occurred. The presence of nisin or hop extract during PEF treatment resulted in an additional reduction of cell viability by 1.5 log cycles. Addition of hop extract also resulted in an additional 2 log cycles of sublethal injury. Using propidium iodide uptake and staining, membrane damage was shown to be partially reversible. Model beer containing hops was inoculated with Lactobacillus plantarum and stored after PEF treatment to evaluate its efficacy for beer preservation. Cells were inactivated only above critical values of 13 kV/cm and 64 kJ/kg; below these values, cell damage was reversible. Storage experiments revealed that surviving cells were killed after 15 h storage in model beer containing hops. The combination of PEF and hop addition may be an effective non-thermal method for preservation of beer.

8.10 Rice wine (yakju) Yakju (rice wine) was sterilized using high voltage pulses (exponential wave pulses at Ef of 12.5–25 kV/cm) and analysed as to pH, acidity, microbiological quality and electrical conductivity (Su et al., 1999). Log survival rates were shown to decrease linearly at low pulse number, but curvilinearly at high pulse number and a mathematical model of pulsed electric field sterilization kinetics of yakju was developed, in which the sterilization rate constant increased with electric field strength and size of target microorganisms. Pulsed electric field treatment stabilized pH and acidity of yakju stored at 4 and 30°C for 6 weeks and prevented microbial growth during the storage period.

9 Process models 9.1 Energy and power Voltage across the treatment chamber (c), V ⫽ VsC/(C ⫹ Cc). Power demand per pulse, Pc (W): Pc ⫽ Qc/(␶p ⫹ ␶)

(8)

Process models 127

where Qc is the energy per pulse, ␶p is the pulse period and ␶ is the pulse period. Thus, if Cc ⬍⬍ C, then V 艐 Vs and maximum energy efficiency can be achieved.

9.2 Microbial inactivation models A function was developed (Hulsheger and Niemann, 1980) to relate the survival rate of E. coli K12 to Ef (exponential decay pulses) by using experimental data and regression technique: Log (S%) ⫽ A⬘ ⫺ BEmax

(9)

where S% ⫽ surviving organisms, %, Emax ⫽ maximum electric field strength, kV/cm, and A⬘ and B are regression constants, which in turn, are functions of cell concentration and the type of medium used. Unfortunately, the accuracy of the model was affected by the bactericidal action from chlorine produced by electrolysis. The electrolyte was modified (Hulsheger et al., 1981) using sulphate or phosphate rather than chloride salt and described the microbial survival rate of E. coli K12 by another experimentally and statistically determined equation: S% ⫽ [t/tcr]⫺((Ef⫺Ecr)/k)

(10)

where t ⫽ treatment time applied, s, tcr ⫽ critical treatment time, s, Ecr ⫽ critical electric field strength, kV/cm, and k ⫽ a constant, kV/cm. The parameters Ecr, tcr, and k were functions of the microorganism chosen. The same model was extended to other organisms (Hulsheger et al., 1983) and parameters were established for E. coli K12, K. pneumoniae, P. aeruginosa, S. aureus, two strains of L. monocytogenes, and C. albicans. Using four static chambers – each with a different electrode configuration (plate– plate, needle–plate, wire–cylinder and rod–rod) – and exponential decay pulses, the survival of S. cerevisiae and B. natto, when suspended in 0.1 per cent and 0.3 per cent NaCl solutions, followed the Weibull distribution (Mizuno and Hori, 1988): S ⫽ Exp(⫺␣cnWi)

(11)

where S ⫽ survival ratio or fraction (0–1), ␣c ⫽ a constant and is a function of microbial species and Wi ⫽ input energy per pulse, J. Peleg (1995) reported that the microbial survival curve had a characteristic sigmoid shape and established a model based on Fermi’s equation: S% (Ef, n) ⫽ 100/[1 ⫹ exp(Ef ⫺ Ec(n))/ac(n)]

(12)

where Ec ⫽ critical electric field strength where the survival level is 50 per cent or the inflection point of S%(Ef), kV/cm, and ac ⫽ a parameter indicating the steepness of the survival curve around Ec, kV/cm. Ec(n) and ac(n) were described by a single exponential decay model: Ec(n) ⫽ Eco exp[⫺k1n]

(13)

ac(n) ⫽ ao exp[⫺k2n]

(14)

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where Eco and ao ⫽ constants, kV/cm and k1 and k2 ⫽ constants. At Ef ⬎⬎ Ec, Equation (7) reduces to the following: S% (Ef, n) ⫽ 100/[exp(Ef)/ac(n)]

(15)

Using results published by Castro et al. (1993), Peleg (1995) showed that the empirical data had a good fit (r2 ⫽ 0.973 to 0.999) with Equation (7). Experimental results of Ho et al. (1995) also supported this model. The model of Hulsheger et al. (1981) was extended to test the survival rate of E. coli, S. aureus and S. cerevisiae in a semisolid model food containing potato dextrose agar (Zhang et al., 1994c). They obtained 5–6 log cycles of reduction and determined that the inactivation rate followed first order kinetics, as shown in the following equations: S ⫽ exp[⫺(Ef ⫺ Ecr)/kE]

(16)

S ⫽ exp[⫺(n ⫺ ncr)/kn]

(17)

where ncr ⫽ critical number of pulses applied and kE and kn ⫽ rate constants indicating the additional Ef and n required to achieve one additional log cycle of microbial reduction, kV/cm. The two constants were functions of treatment temperature and microbial growth stage. Models to describe inactivation of E. coli (Alvarez et al., 2003c; Rodrigo et al., 2003a), Salmonella senftenberg (Raso et al., 2000) and Lactobacillus plantarum (Abram et al., 2003; Rodrigo et al., 2003b) by PEF have been published.

9.3 Process temperature The effect of temperature increase per process volume in the fluid medium, Tv (°C/m3) was outlined by: ⌬Tv ⫽ (tE 2f )/(HC ⫺ ␳)

(18)

where HC ⫽ heat capacity, J/°C.

10 Conclusions PEF has been investigated as a potential non-thermal technique for food preservation and demonstrated a moderate to significant microbicidal effect by discharging high voltage, short electric pulses through foods. The process parameters used for batch processing had a very wide range: DC voltage 2.5–43 kV; Ef 0.6–100 kV/cm; electrode distance 3–77 mm; pulse width 1 ␮s–10 ms; pulse frequency 0.2–50 Hz; number of applied pulses 1–120; and process volume 0.5 ml–1.6 l. Based on all the available studies, the microbial reduction rate could be classified as moderate (1–3 log cycle reduction in counts) to significant (⬎6 log cycles) and the efficacy of the treatment seemed to be a function of various process parameters, conditions and procedures.

Conclusions 129

The process of PEF pasteurization is complex in the number of variables involved, which can generally be grouped into three categories: system, medium and subject parameters. These parameters have been tested by several research groups. However, the varying conditions and equipment used by different groups means that not all results are in agreement and comparisons between experiments may not always be valid. In some instances, it is possible to identify key characteristics of PEF processing and to observe some generally agreed-upon trends in the three variable divisions. The PEF treatment was very effective in inactivating E. coli O157:H7 cells in apple cider. A ⬎5 log cfu/ml reduction in count was observed as the treatment temperature increased from 20 to 42°C with 10 electrical pulses at 80 kV/cm. PEF treatment, when combined with either cinnamon or nisin, could induce cell inactivation by 6–8 log cfu/ml. A 7.0 log cycle reduction of naturally occurring microorganisms in raw skimmed milk has been achieved through a combination of PEF treatment (80 kV/cm, 50 pulses), mild heat (52°C) and addition of the natural antimicrobial agents, nisin (3000 U/ml) and lysozyme (1000 ␮g/ml). The combination of PEF, mild heat and antimicrobials resulted in a much higher microbial inactivation than the sum of the individual reductions achieved from each treatment alone, indicating synergy. Varying the pH from 6.7 to 5.0 had no effect on microbial inactivation. PEF has the potential to reduce Salmonella counts in orange juices and the efficiency of the bactericidal effect is affected by the nature of the juice as well as the treatment conditions. Even though higher temperatures and pulse numbers are required to inactivate S. typhimurium in orange juice, PEF seems to be a promising method for inactivation of Salmonella and other microorganisms (e.g. yeast). For naturally occurring spoilage microorganisms in orange juice, optimal conditions consisting of 20 pulses of an electric field of 80 kV/cm, at pH 3.5 and a temperature of 44°C with 100 U nisin/ml resulted in over a 6 log cycle reduction in the microbial population. There was 97.5 per cent retention of vitamin C, along with a 92.7 per cent reduction in pectinmethylesterase activity. The microbial shelf-life of the orange juice was also improved and determined to be at least 28 days when stored at 4°C without aseptic packaging. Gas chromatography revealed no significant differences in aroma compounds before and after pulsing. For tomato juice, at an Ef of 80 kV/cm, 20 pulses, 50°C and in the presence of nisin (100 U/ml), there was about a 4.4 log cycle reduction in microbial counts. For red grape juice, a log reduction in microbial count of 6.2 was achieved at 51°C when 20 pulses, each of 80 kV/cm, were applied in combination with nisin addition. For white grape juice, the greatest reduction in count (4.4 log cfu/ml) was achieved when 20 pulses of 65 kV/cm were applied at 50°C in the presence of a nisin:lysozyme mix. For mango juice, in the presence of antimicrobials and mild heat (50°C), PEF at 20 and 50 pulses of 87 kV/cm reduced microbial count by 3.07 and 3.38 log cfu/ml respectively compared to control at 24°C. With the increase in temperature to 52°C, microbial count was reduced by 4.40 log cfu/ml. From the results published to date, it would appear that PEF is most effective when applied to stressed cells, especially if the stress imposed has an effect on cell membrane integrity.

130 Pulsed Electric Field Processing of Liquid Foods and Beverages

Acknowledgements Most of the material is taken from the reviews and research works of graduate students of the authors especially Shirley Ho, Matt Hodgins, Keith Smith and Alex Ross.

Nomenclature A A⬘ a ac aw B C C0 C1 C2 Cc d D Ec Eco Ecr Ef Emax G HC HPP h Imax k k1 k2 kE kn L L1 n ncr Pc PEF Q Qc R

area of the electrode surface, m2 constant cell radius, m steepness of the survival curve around Ec water activity constant capacitance, F electrolytic capacitor bank, F pulse capacitor, F high voltage capacitor, F charging capacitor, F distance between electrodes, m log cycle reductions in colony counts external field strength, V constant, kV/cm critical field strength, kV/cm or V/m field strength, V/m or kV/cm maximum field strength, kV/cm or V/m membrane shear elastic modulus, Pa heat capacity, J/°C high-pressure processing unstrained membrane thickness, m maximum peak current, A constant, kV/cm constant constant rate constant, kV/cm rate constant, kV/cm cell length, m inductor, H number of pulses critical number of pulses applied power per pulse, W pulsed electric field energy stored in a capacitor, J or J/ml energy per pulse, J resistance, ⍀

References 131

RxC r2 S S% SMUF t tcr TMP U V Vm Vc Vs W ␣o ␣c ⑀ ⑀o ⑀r ␴ ␥ ␳ ⌽ ␻ ␶ ␶p ⌬Tv

dielectric relaxation constant of the membrane, s/rad coefficient of determination survival ratio or fraction surviving organisms, % simulated milk ultrafiltrate treatment time, s critical treatment time, s transmembrane potential, V potential difference, V charging voltage, V induced potential for cells, V potential difference for membrane breakdown, V supply voltage, V input energy per pulse, J constant, kV/cm constant relative elastic permittivity permittivity of free space relative permittivity, i.e. dielectric constant conductivity of the food, S/m surface tension food resistivity, ⍀m cell orientation, rad angular frequency of the field, rad/s pulse duration or width, s pulse period, s temperature increase per process volume, °C/m3

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Wouters PC, Bos AP, Ueckert J (2001b) Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields. Applied and Environmental Microbiology, 67, 3092–3101. Wouters PC, Dutreux N, Smelt JPPM, Lelieveld HLM (1999) Effects of pulsed electric fields on inactivation kinetics of Listeria innocua. Applied and Environmental Microbiology, 65, 5364–5371. Yeom HW, McCann K, Streaker CB, Zhang QH (2002) Pulsed electric field processing of high acid liquid foods: A review. Advances in Food and Nutrition Research, 44, 1–32. Zhang Q, Chang FJ, Barbosa-Canovas GV, Swanson BG (1994c) Inactivation of microorganisms in a semisolid model food using high voltage pulsed electric fields. Food Science & Technology, 27, 538–543. Zhang Q, Monsalve-Gonzalez A, Barbosa-Canovas GV, Swanson BG (1994a) Inactivation of E. coli and S. cerevisiae by pulsed electric fields under controlled temperature conditions. Transactions of the American Society of Agricultural Engineers, 31 (2), 581–587. Zhang Q, Monsalve-Gonzalez A, Barbosa-Canovas GV, Swanson BG (1994b) Inactivation of S. cerevisiae in apple juice by square-wave and exponential-decay pulsed electric fields. Journal of Food Process Engineering, 17 (4), 469–478. Zhang Q, Barbosa-Canovas GV, Swanson BG (1995a) Engineering aspects of pulsed electric field pasteurization. Journal of Food Engineering, 25 (2), 261–281. Zhang Q, Qin BL, Barbosa-Canovas GV, Swanson BG (1995b) Inactivation of E. coli for food pasteurization by high-strength pulsed electric fields. Journal of Food Processing and Preservation, 19 (2), 103–118. Zimmerman U, Pilwat G, Riemann F (1974) Dielectric breakdown of cell membranes. Biophysics Journal, 14, 881–899.

Effect of High Intensity Electric Field Pulses on Solid Foods Magnus Gudmundsson and Hannes Hafsteinsson Matra, Technological Institute of Iceland Keldnaholt, Reykjavik, Iceland

There are few studies on the use of high intensity electric fields (PEF) on solid foods. The information available on the use of PEF treatment is mostly limited to the inactivation rate of microorganisms. Very little information is available regarding structural changes of cells in real foods during and after application of PEF treatment. However, it is quite clear from the few studies available that most solid foods do not tolerate low intensity electric fields between 100 and 1000 kV/m without damage to the microstructure and changes to the texture. Meat and fish products are particularly vulnerable to detrimental effects of such treatment. PEF treatment of solid foods for preservation is therefore unrealistic as most species of microorganisms are not affected by low intensity PEF treatment to any extent. It may be possible to use PEF treatment in combination with other methods, e.g. high pressure in minimal processing of foods, but further research is needed to evaluate its feasibility. However, the use of PEF treatment in combination with compression is a promising way to use this new method. The combination of PEF and compression can be used in juice production, to extract metabolites or other materials from organic materials. It can also be used for dehydration of cellular materials from either animal or plant origin. To improve the PEF process further, more knowledge is needed on how different foods react to external electric fields.

1 Introduction Many solid foods are of cellular organic origin like plant foods, fish or meat. As they are made up of cells, the effect of treatment with high intensity electric field pulses (PEF) can be explained by a theory of how an electric field affects the membrane of cells. The dielectric rupture theory explains the effect of a high electric field on cells (Zimmermann, 1986; Zimmermann et al., 1976). The applied external electric field induces an electric potential across the membrane, which causes charge separation in the cell membrane. When a pulsed electric field (PEF) is applied to cellular material above a critical level, it can lead to electric breakdown of the cell membrane. For Emerging technologies for food processing ISBN: 0-12-676757-2

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142 Effect of High Intensity Electric Field Pulses on Solid Foods

many different types of cells the critical level is about 1 V across the membrane and, when that level is exceeded, local structural changes and the formation of pores will be induced. The consequence is an increase in the permeability of the membrane. The permeability can either be reversible or irreversible and that depends on the intensity of the PEF treatment (Mertens and Knorr, 1992; Ho and Mittal, 1996). When a membrane ruptures there is an increase in conductivity because of the formation of an aqueous phase at the membrane interfaces (Angersbach et al., 2000). It is possible that a rapid rupture occurs in limited areas without affecting the entire cell membrane but that depends on the external electric field applied (Chernomordik et al., 1987; Freeman et al., 1994; Ho and Mittal, 1996; Angersbach et al., 2000). The electric breakdown is reversible if only a small fraction of the membrane is occupied with pores (Freeman et al., 1994; Ho and Mittal, 1996; Angersbach et al., 2000). The pores are thought to form in the lipid bilayer rather than in the protein channels (Angersbach et al., 2000). PEF treatment is a non-thermal food processing method as the rise in temperature is small during application in most cases. High intensity pulsed electric field treatment has, therefore, a potential as a minimal processing method (Knorr, 1995; Knorr and Angersbach, 1998; Barbosa-Cánovas et al., 1998). The main effort in previous research has been on the possible uses of PEF for food preservation. The emphasis has therefore been on inactivation of microorganisms in growth and stationary phases or as spores (Sale and Hamilton, 1967; Hülsheger and Nieman, 1980; Castro et al., 1993; Martín-Belloso et al., 1994; Qin et al., 1995; Wouters et al., 1999, 2001). However, food safety is not the only issue that concerns the consumer. Equally important are the texture and other sensory properties of the food. Changes in microstructure and texture can be expected as a consequence of the permeabilization of cell membranes which can lead to water release into the extracellular space or changes in water retention properties as well as damage to the cell membrane. It is also necessary to evaluate the effects of PEF on microstructure and texture of solid foods or foods containing solid particles. Traditional processes for meat, fish and other solid foods, e.g. frozen storage, drying, salting and canning, can affect the microstructure of the product severely when compared to fresh foods (Chu and Sterling, 1970; Dunajski, 1979; Bello et al., 1982; Fennema, 1990; Mackie, 1993; Edwards, 1999; Greaser and Pearson, 1999; Saurel, 2002). Possibly, the non-thermal PEF process has less effect on microstructure than traditional methods like freezing or processes based on heat. There has been very little research on the effect of PEF treatment on the microstructure of food (Barsotti and Cheftel, 1999; Fernandez-Diaz et al., 2000) and the only study available is on fish and meat products (Gudmundsson and Hafsteinsson, 2001). A few studies have been done on the use of PEF for electric permeabilization and cell membrane rupture of cellular plant material, e.g. to increase the yield of juices (Flaumenbaum, 1968; Knorr et al., 1994; Bazhal et al., 2001). The use of PEF in combination with compression on plant tissues offers ways to improve the expression and extraction of plant metabolites and make dehydration easier by affecting the mass and heat transfer of food products (Angersbach et al., 2000; AdeOmowaye et al., 2001, 2003; Bouzrara and Vorobiev, 2003).

Food safety 143

PEF treatment could also be used in combination with other processes such as high-pressure treatment in order to achieve a possible hurdle effect in preservation (Heinz and Knorr, 2000). To improve the PEF process further more knowledge is needed on the cell specific critical transmembrane voltage or the critical external field strength that specific biomaterial can tolerate before permeability occurs. The information available is mostly limited to the inactivation rate of microorganisms. Very little information is available regarding the kinetics of permeabilization and on reversible-irreversible structure changes of cells in real foods during and after application of PEF treatment.

2 Food safety PEF treatment can have a lethal effect on living cells, which has been explained by dielectric breakdown of the cell membrane (Sale and Hamilton, 1967; Zimmermann, 1986; Zimmermann et al., 1976). If the applied external electric field induces a transmembrane potential above a critical value, which usually is above 1 V, it will cause pore formation in the cell membrane that can be lethal to microorganisms. The irreversible changes occur to a cell when an external electric field between 100 and 1000 kV/m is used for more than 10–15 ms (Zimmermann et al., 1976). It has been shown that the rate of bacterial killing is related to the field strength, time and also to the number of pulses and the pulse width. The inactivation depends also on the type of microorganism, the microbial growth stage, the initial amount of microbes and the ionic concentration and conductivity of the suspension (Hülsheger et al., 1981; Wouters and Smelt, 1997). Bacteria in the growth phase are more sensitive to electric fields than stationary bacteria and spores are the most resistant (Sale and Hamilton, 1967; Marquez et al., 1997; Wouters et al., 1997, 2001). It has also been shown that larger microorganisms are more sensitive to PEF treatment than smaller ones (Sale and Hamilton, 1967; Hülsheger et al., 1983). Some factors seem to have a protective effect, e.g. cations, proteins and lipids (Hülsheger et al., 1981; Zhang et al., 1994; Grahl and Märkl, 1996; Martín-Belloso et al., 1997). As the bactericidal effect of PEF decreases with increased ionic strength (Hülsheger et al., 1981), it is more difficult to inactivate microorganisms in semi-solid or solid food materials than in dilute buffer solutions, as solid foods are rich in ions and other protective substances (Hülsheger et al., 1981; Zhang et al., 1994). Furthermore, as many foods are heterogeneous with areas of different electrical resistivity, the effects of PEF treatment can be varied, as some areas will be untreated and others over-treated in such material (Barsotti and Cheftel, 1999). The resistivity of different food materials can be very varied as products with high water and salt content can have resistivity as low as 0.4 ohms and oils and fats can have resistivity of more than 100 ohms and can act like electrical insulators (San Martín et al., 2003). The effect of PEF treatment on different types of bacteria in solid foods has not been specially studied, although the effect of PEF treatment on total bacteria count has

144 Effect of High Intensity Electric Field Pulses on Solid Foods

been reported on lumpfish roes, for which treatment of 1100 kV/m and seven pulses (2 ␮s in width) reduced the total bacteria count by one log cycle (Gudmundsson and Hafsteinsson, 2001). In meat and seafood the potential bacterial pathogens are many, e.g. Salmonella species, Escherichia coli, Campylobacter, Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Vibrio parhaemolyticus and others (Liston, 1990; Wekell et al., 1994; Lawrie, 1998). There are some studies that deal with the effect of PEF on some of these pathogens in solutions but not in solid foods. These studies show that relatively intensive PEF treatment from 2000 to 7000 kV/m is needed to reduce microorganisms from 2 to 9 log cycles in, e.g. E. coli, Staphylococcus aureus, Bacillus subtilis, Listeria and Salmonella species (Hamilton and Sale, 1967; Hülsheger et al., 1983; Dunn and Pearlman, 1987; Zhang et al., 1994; Ho et al., 1997; Marquez et al., 1997; Martín-Belloso et al., 1997; Calderón-Miranda et al., 1999; Heinz et al., 1999; Jeantet et al., 1999; Alvarez et al., 2000; Wouters et al., 2001). Solid foods like meat or fish would need even more intense PEF treatment to inactivate microorganisms than solutions because of protective substances in the products. The heterogeneity of many foods containing materials with areas of different electrical resistivity adds to the difficulty of using PEF treatment for preservation of solid foods.

3 Effects on food quality 3.1 Effects on proteins and enzyme activity Various proteins and enzymes have different sensitivity towards PEF treatment. Some enzymes are more sensitive and others are more resistant towards PEF treatment than many microorganisms. There are few studies that deal with the effect of PEF treatment on certain proteins or enzymes found in food. It has been shown that surface hydrophobicity of egg white proteins was not changed by PEF-treatment with an electric field of 2000–3500 kV/m and pulse number from 2 to 8 (Jeantet et al., 1999). Ovalbumin apparently did not denature when treated with PEF with an electric field of 2700–3300 kV/m and using from 50 to 400 pulses (Fernandez-Diaz et al., 2000). However, it was shown that there was partial unfolding of ovalbumin, thus exposing all four sulphydryl groups to the surface. Recently, it was shown that PEFtreatment consisting of 200 exponential decay pulses of up to 1.3 ␮s and up to 3000 kV/m did not lead to marked unfolding of -lactoglobulin nor aggregation (Barsotti et al., 2002). The change in sulphydryl groups reactivity of -lactoglobulin was reversible. Another study (Perez and Pilosof, 2004) showed that egg white proteins and -lactoglobulin were partially denatured when subjected to 1250 kV/m and 10 long length pulses (2 ms) and that the denaturation temperature of ovalbumin decreased by 4–5°C as measured by a differential scanning calorimeter. The same study (Perez and Pilosof, 2004) indicated that aggregates were formed that involved covalent bonds. A study of the effects of PEF treatment on seafood has reported that

Effects on food quality 145

212 170 116

76

53

1

2

3

4

5

6

7

8

Figure 6.1 Electrophoresis of cod proteins from PEF-treated cod muscle and untreated samples. Rows 1 and 8 are standard proteins with the highest molecular weight at the top. Samples 2–5 are cod proteins from PEF-treated cod muscle with electric pulses (1800, 1500, 1250 and 1060 kV/m respectively and 7 pulses) and 6 and 7 are untreated control samples.

treatment of cod muscle with electric fields between 1050 and 1800 kV/m and seven pulses (2
s width) did not affect the proteins (Gudmundsson and Hafsteinsson, 2001) as can be seen in Figure 6.1. According to the results from SDS-electrophoresis no changes were seen in molecular protein bands in PEF-treated cod muscle compared to untreated samples. Studies on enzyme activity show that many enzymes are unaffected even at electric fields above 3000 kV/m. These enzymes include amylases, lipase, NADH dehydrogenase, succinic hydrogenase and hexogenase (Hamilton and Sale, 1967). However, PEF treatment inactivates some enzymes like proteases from Pseudomonas fluorescens at 1500 kV/m and 98 pulses and plasmin at 3000 kV/m and 50 pulses (VegaMercado et al., 1995a, b). Papain was inactivated after treatment of 2000 kV/m and 400–500 pulses, however, the inactivation did not occur immediately but only after 24 h storage at 4°C (Yeom et al., 1999). The enzymes -amylase, a lipase and glucose oxidase were inactivated at high electric fields of 6400 and 8700 kV/m, whereas peroxidase and polyphenoloxidase were even more resistant (Ho et al., 1997).

146 Effect of High Intensity Electric Field Pulses on Solid Foods

3.2 Effects on texture and microstructure As mentioned previously, one can expect changes in water-holding properties, microstructure and texture of PEF-treated foods as a consequence of the permeabilization of the cell membrane. Lebovka and co-workers (2001) have put forward a hypothesis on how PEF treatment affects cellular tissues. They consider the effect as correlated percolation which is governed by two processes. The first one is resealing of cells and the other is moisture transfer inside cellular structures which is sensitive to repetition of PEF treatments. At low strength electric fields (20 kV/m) the electric breakdown is reversible as the resealing process is quick to repair the membranes immediately after the PEF treatment has been terminated. At moderate PEF treatment (50–200 kV/m and duration of pulses between 104 and 105 s) the integrity of cells rapidly decreases but, due to the resealing process, some of the cells lose their permeability but in others the pores may persist (Lebovka et al., 2001). High-intensity PEF treatment (1000–5000 kV/m and pulse duration 106 s) causes irreversible damage to the cell membrane (Barbosa-Cánovas et al., 1998). Long-term changes in conductivity after application of PEF treatment can also be related to osmotic flow and redistribution of moisture inside the sample (Lebovka et al., 2001). There are only a few studies on the effect of PEF treatment on the quality of foods, mainly on juices and other pumpable foods, which show that sensory properties are not affected (Knorr et al., 1994; Qin et al., 1995; Barbosa-Cánovas et al., 1996). Research on the effect of PEF treatment on texture and microstructure of meat and seafood or other solid foods is very limited (Gudmundsson and Hafsteinsson, 2001). The effect of PEF treatment of 136 kV/m and 60 pulses (width of pulse was 2
m) on the average cell size of salmon and chicken breast muscle is shown in Table 6.1. The PEF treatment reduced significantly the average cell size for both the salmon and chicken breast muscle. The salmon cells were reduced to about 34 per cent of the original cell size and the chicken cells were reduced to 61 per cent of the original cell size after the PEF treatment. Obviously the PEF treatment had a detrimental effect on both chicken breast and salmon muscle cells. The PEF treatment of 136 kV/m and 60 pulses also induced a gap or looseness in the salmon muscle on a macroscopic level but not in the chicken breast muscle. In Figure 6.2, microscopic pictures compare PEF-treated chicken breast muscle and salmon muscle and untreated samples. The PEF treatment of 136 kV/m induced a separation between the muscle cells where salmon cells were more separated than chicken muscle cells. Apparently there

Table 6.1 Average cell area (␮m2) of untreated and PEF-treated samples of chicken and salmon. Percentage of original cell size is in parenthesis Samples

Control (␮m2)

136 kV/m/60 pulses (␮m2)

Chicken Salmon

3600a (100) 13200a (100)

2190b (61) 4480b (34)

Different letters a, b; mean that treatments are significantly different at P  0.05 within each sample.

Effects on food quality 147

was a collagen leakage into the extracellular gap between the muscle cells as seen microscopically (Gudmundsson and Hafsteinsson, 2001). The probable explanation of these effects is pore formation in the cell membranes that causes leakage of cell fluids into the extracellular space. Fish like salmon contain low amounts of connective tissue (0.66 per cent) in the muscle (Dunajski, 1979; Eckoff et al., 1998). Chicken muscle contains about 2 per cent of connective tissue (Baily and Light, 1989), which could explain why chicken muscle withstands PEF treatment of 13 kV/m without a gap formation but not salmon muscle. It is also known for different bacteria species that increased cell size makes them more vulnerable to PEF treatment (Sale and Hamilton, 1967; Hülsheger et al., 1983). The muscle cells of salmon and chicken are usually between 50 and 100
m in diameter which is considerably larger than any bacteria which are between 0.3 and 2.0
m (Nester et al., 1983; Wong, 1989). The cell size of salmon muscle is greater than chicken breast muscle, which could also contribute to

50
m

50
m

(b)

(a) 200
m

(d) (c) Figure 6.2 Chicken and salmon muscle samples treated with PEF. Muscle proteins are stained orange and collagen is stained blue: (a) untreated chicken breast muscle, (b) chicken breast muscle treated with 136 kV/m and 40 pulses (each pulse is 2
s width), (c) untreated salmon sample, (d) salmon sample treated with 136 kV/m and 40 pulses.

148 Effect of High Intensity Electric Field Pulses on Solid Foods

the vulnerability of salmon muscle to PEF treatment. These two factors, cell size and collagen content, could explain why there is a difference in vulnerability to PEF treatment between meat and fish. However, neither fish nor meat can withstand even a mild PEF treatment without damage to the microstructure. The impact of PEF treatment on microstructure of fish or meat muscle cannot possibly be related to protein denaturation as the intensity of the electric field is too low. Fresh lumpfish roes treated with 1200 kV/m and 12 pulses (2
s pulse width) were intact after the treatment except for a very low percentage of damage to the roes. Firmness of PEF-treated roes, measured with a compression test in a texture analyser instrument, also showed that the PEF treatment only marginally affected the firmness of the roes (Gudmundsson and Hafsteinsson, 2001). For comparison another study on frozen and then thawed salmon roes showed that 46 per cent less energy is needed to rupture such roes than fresh roes (Craig and Powrie, 1988). The three-layer membrane of a roe probably gives it the strength to tolerate the PEF treatment. The roes could probably withstand more intense PEF treatment, which perhaps could be plausible for preservation of roes. However, the general conclusion is that solid foods do not tolerate intensive PEF treatment without damage to the tissue cells. The consequences of such damages are detrimental changes in texture and microstructure.

4 Use of PEF in combination with other methods Some combination processes have been tried to inactivate microorganisms effectively with less intense PEF treatment than is needed when the method is used alone. PEF treatment in the presence of nisin has been shown to have a synergistic effect on several microorganisms but the inactivation mechanism is not clear, probably the PEF treatment makes it easier for nisin to enter through the membrane and react inside the microorganisms (Calderón-Miranda et al., 1999; Dutreux et al., 2000; Pol et al., 2000). The combination of high pressure and PEF treatment has been tried on vegetative Bacillus subtilis. When pressure as high as 200 MPa was applied with PEF the inactivation effect increased considerably compared to that of the PEF treatment alone (Heinz and Knorr, 2000). PEF treatment has been used in combination with other methods for other purposes, mainly extraction and osmotic dehydration of cellular materials. For example PEF treatment was used in combination with compression on various foods such as carrots, apples, sugar beets, red bell peppers and coconuts (Flaumenbaum, 1968; Knorr et al., 1994; Rastogi et al., 1999, 2002; Ade-Omowaye et al., 2001; Bazhal et al., 2001; Eshtiagi and Knorr, 2002; Bouzrara and Vorobiev, 2003). Mechanical compression has various industrial applications for biological materials. These applications include extraction of fruit juices and vegetable oils, dewatering of plant materials and dehydration of organic waste and more. The amount of compounds released into the extracellular liquid depends on the degree of cell disintegration and the type

References 149

of biological material, which influences the efficiency of the compression process. To facilitate extraction from biological materials, PEF can be used to permeabilize the cell membrane which consequently makes the rupture of the defective cells easier with mechanical compression and without any increase in temperature. PEF treatment is most effective when the conductivity of the food material is as low as possible. The combined application of pressure and PEF gives optimum results and improves the quality of apple juice (Bazhal et al., 2001) and increases the yield of juice from sugar beets for example (Eshtiagi and Knorr, 2002). Furthermore, using PEF as a pre-treatment can enhance mass transfer and accelerate water loss during osmotic dehydration (Rastogi et al., 1999, 2002; Angersbach et al., 2000; Bazhal et al., 2001; Ade-Omowaye et al., 2003; Taiwo et al., 2002). Therefore, PEF treatment can be a good and useful non-thermal pre-treatment in extraction and dehydration applications.

5 Conclusions The use of PEF treatment for preservation of fish, meat or other solid food products does not seem realistic as application of low intensity electric field pulses has a detrimental effect on the microstructure. At the same time the low field voltage does not effectively reduce the growth of bacteria. Fish roes seem to be one of very few solid foods that can withstand PEF treatment of 1200 kV/m and 12 pulses without a visible effect on the microstructure or texture. A PEF treatment could possibly be a pretreatment for roes but further study is needed. Other uses of PEF treatment on solid food material are more realistic as PEF treatment seems to be effective as a pre-treatment for the extraction of valuable materials, mainly from plant materials and also for dehydration of organic material from plant and animal origin. In the fish and meat industry a PEF treatment could possibly be used either to dehydrate by-products or to extract some valuable items such as enzymes, fish oils or valuable metabolites. However, these applications have not been thoroughly investigated at present.

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Enzymatic Inactivation by Pulsed Electric Fields Olga Martín-Belloso and Pedro Elez-Martínez University of Lleida, Department of Food Technology, UTPV-CeRTA Lleida, Spain

A new food processing technology, pulsed electric fields (PEF), is currently being investigated due to its capability of inactivating undesirable microorganisms and enzymes with limited increase in food temperature. As a result, more stable foods with fewer changes in composition, physicochemical properties and sensory attributes can be obtained. Several studies exist on the effects of PEF on enzymes suspended in aqueous solutions and in real foods like fruit juices and milk, which are products of great importance to the food industry. The studied enzymes have been pectin methyl esterase (PME), polygalacturonase (PG), polyphenoloxidase (PPO), peroxidase (POD), lipoxygenase (LOX), alkaline phosphatase (ALP), protease, lipase, ␣-amylase, glucose oxidase, lysozyme, pepsin, lactate dehydrogenase and papain. Depending on the particular enzyme, the medium where it is suspended and the PEF treatment conditions, most enzymes are almost completely inactivated, while others show resistance to PEF processing. Electric field strength, treatment time, number of pulses, pulse width, field polarity, frequency and treatment temperature are PEF factors that have significant effects on enzyme inactivation. Among them, the factors that most influence enzyme inactivation are field strength and treatment time. Enzyme inactivation has been described as an exponential function of treatment time or field strength but empirical models such as Hülsheger’s and Fermi’s have also been successfully used. Although the mechanism of enzyme inactivation is still unclear, it is believed that PEF processing may affect the native structure of enzymes and therefore could promote changes in enzymatic activity.

1 Introduction Pulsed electric fields (PEF) is a non-thermal preservation method that uses high voltage to inactivate enzymes and to produce microbiologically safe foods with fresh-like flavour and taste without significant loss of nutrients (Jia et al., 1999; Yeom et al., 2000a; Bendicho et al., 2002b; Hodgins et al., 2002; Espachs-Barroso et al., 2003a). PEF technology is gaining popularity in processing foods as a means of avoiding the negative effects of thermal treatments (Giner et al., 2000). PEF treatment involves the Emerging technologies for food processing ISBN: 0-12-676757-2

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7

156 Enzymatic Inactivation by Pulsed Electric Fields

Relative activity (%)

250 200 150 100 50 0 0

5

10 15 20 Voltage supplied (kV)

Peroxidase Amylase Lysoenzyme Polyphenol oxidase

25

30

Alkaline phosphatase Lipase Glucose oxidase Pepsin

Figure 7.1 Changes in activity of enzymes due to electric pulse treatment using 0.3 cm electrode distance of 30 ␮s at 2 seconds of pulse period (adapted from Ho et al., 1997).

application of a short burst (␮s or ms) of high voltage (kV/cm) to foods placed between two electrodes (Qin et al., 1996). Most of the published works are focused on microorganism inactivation. In general, PEF is effective in destroying microorganisms (Hülsheger et al., 1981; Jayaram et al., 1992; Zhang et al., 1994; Qin et al., 1995; Pothakamury et al., 1995a, b; Martín et al., 1997; Martín-Belloso et al., 1997; Calderón-Miranda et al., 1999; Wouters et al., 1999; Raso et al., 2000; Bendicho et al., 2002a; Álvarez et al., 2003a, b; Espachs-Barroso, 2003a). This treatment is as effective as traditional pasteurization heat treatments (Yeom et al., 2000a). In comparison to the extensive research devoted to the destruction of microorganisms by PEF, there are few studies about the inactivation of enzymes by PEF. Being a matter of recent interest, there are several studies that exist about the effects of PEF on enzymes suspended in aqueous solutions and in real foods. These studies show some controversial results. Some enzymes are throughly inactivated, such as pectin methyl esterase (PME) (Giner et al., 2000; Yeom et al., 2002b), polyphenol oxidase (PPO) (Ho et al., 1997; Giner et al., 2001, 2002), papain (Yeom et al., 1999), glucose oxidase, lysoenzyme, amylase (Ho et al., 1997), plasmin (Vega-Mercado et al., 1995), lipase (Ho et al., 1997; Bendicho et al., 2002c), protease (Bendicho et al., 2003a) and alkaline phosphatase (Barbosa-Cánovas et al., 1998). However, some researchers have found that PEF treatment did not affect some enzymes (Ho et al., 1997; Van Loey et al., 2002) or even produced an increase of activity (Ho et al., 1997) (Figure 7.1, see Table 7.1). This chapter reviews the current information available about the effect of PEF on enzymes, including what is known on the inactivation mechanisms, the factors that influence the changes of activity, as well as the models that are currently used to describe the reduction of enzyme activity.

Mechanism of enzyme inactivation by PEF 157

2 Mechanism of enzyme inactivation by PEF The mechanism of enzyme inactivation by PEF is still unclear. It is known that enzymes are proteins whose catalytic activity relies on the native configuration of their active site and the conformation of the surrounding proteins. The amino acid group present in enzyme proteins creates highly asymmetric spatial distributions of charge that lead to strongly polar and charged regions in the molecular structure of proteins (Laberge, 1998). Because of a complex network of non-covalent (electrostatic forces, ion pairing, Van der Waals forces, hydrogen bonding and interaction hydrophobic effect) as well as covalent interactions (disulphide bonds), the structural stability and catalytic functions of enzymes are maintained (Wong, 1995). If interactions cause a shift, changes in enzyme activity may occur due to the effect on the three-dimensional molecular structure of enzymes. The behaviour of proteins under intense electric fields is not well known, but it seems that these fields may cause protein unfolding and denaturation, breakdown of covalent bonds and oxidation– reduction reactions, like those between sulphide groups and disulphide bonds, because of the charge separation (Barsotti and Cheftel, 1999). Moreover, it is known that electric fields influence the conformational state of proteins through charge, dipole, or induced dipole chemical reactions (Tsong and Astunian, 1986). Charged groups and structures are highly susceptible to various types of electric field perturbations and these changes cause modifications of its structure and consequently the loss of activity due to the difficulty of assembling the substrate with the active site (Tsong, 1990). Some authors (Vega-Mercado et al., 1995; Ho et al., 1997; Castro et al., 2001a) have suggested that conformational changes in enzyme structure may be responsible for the modifications in enzyme activity when treated by PEF. Yeom et al. (1999) also related the reduction of papain activity by PEF to the loss of ␣-helix structure. Giner et al. (2002) reported that the differences between the inactivation of PPO and of PME might be due to the relevant differences between the molecular structures of these enzymes. Moreover, they reported that PPO from different sources may show different sensitivity during PEF treatment, which may be due to the huge differences in molecular size and structures displayed among enzymes. Castro et al. (2001a) related the inactivation of alkaline phosphatase (ALP) to degradation of the enzyme secondary structure. In addition, it was found that PEF altered the entire globular configuration of ALP. Yang and Zhang (2003) also observed structural changes in PEF-treated pepsin, lysozyme and papain. Therefore, if PEF processing may affect the native structure of enzymes, which likewise promote changes in activity, PEF treatments could be used to prevent the undesirable effects of enzymes in foods (Barbosa-Cánovas et al., 1999). Application of PEF affect the forces involved in maintaining the three-dimensional structure of lipase, because denaturation of the enzyme was observed. However, the conformational changes in the molecule that lead to an activity inhibition were more evident using a batch PEF system than a continuous PEF system (Bendicho et al., 2002c).

158 Enzymatic Inactivation by Pulsed Electric Fields

3 Factors affecting enzyme inactivation by PEF The effect of PEF on enzymes depends on the PEF treatment conditions, the enzyme itself and the medium in which it is suspended. Factors such as electric field strength, treatment time or number of pulses, pulse width, pulse frequency, pulse polarity, treatment temperature, batch or continuous circulating processing, wave shape, characteristics of the enzyme and the media containing the enzyme have been reported to have significant effects on enzyme inactivation (Vega-Mercado et al., 1995; Giner et al., 2000, 2001; Bendicho et al., 2002c, 2003a; Yeom et al., 2002; Min et al., 2003b).

3.1 PEF processing factors The effectiveness of enzyme inactivation by PEF is higher when electric field strength and treatment time are increased (Vega-Mercado et al., 1995; Ho et al., 1997; Yeom et al., 1999, 2002; Giner et al., 2000, 2002, 2003; Bendicho et al., 2002c, 2003b; Min et al., 2003b) (see Figures 7.2, 7.3, 7.4; Table 7.1). Table 7.1 summarizes the effect of some process parameters on the inactivation of enzymes by PEF. Pulse width is an important PEF-processing factor which affects enzyme activity. If the number of pulses and electric field strength are kept constant, an increase in pulse width will lead to higher levels of enzyme inactivation. Giner et al. (2000) achieved 20 per cent of tomato PME inactivation when it was processed with pulses of 20 ␮s, while a 78 per cent inactivation was achieved with 40-␮s pulses, for the same electric field strength and number of applied pulses. Peach PPO was inactivated around 10 per cent when 80-␮s pulse widths were applied and less than 5 per cent when pulses were about 20 ␮s width, for identical electric field strength and treatment time (Giner et al., 2002). Seventy-two per cent of relative activity was achieved after 200 pulses of 40 ␮s at 5.18 kV/cm, whereas the same number of pulses and electric field strength but using 160 ␮s pulse width led to 41 per cent of relative activity (Giner et al., 2003). The higher the pulse width the greater levels of peroxidase (POD) inactivation, reaching 97 per cent maximum inactivation at 10 ␮s pulse width (35 kV/cm, 600 ␮s) (ElezMartínez et al., 2003b). A reduction in PME activity in orange juice and in a commercial pectic enzyme complex (CPC) was achieved by increasing pulse width (Elez-Martínez et al., 2003a; Espachs-Barroso et al., 2003b). Bendicho et al. (2003a, b) reported that no significant changes in protease inactivation were observed if total treatment time was considered. When enzyme activity was related to the number of pulses, there was a significant difference between the inactivation achieved using the same number of pulses with 4- or 7-␮s pulse widths. It should be noted that a 4-␮s pulse width process requires a higher number of pulses than a 7-␮s pulse width to accumulate similar treatment times. So, the latter might be considered more effective because similar inactivation was achieved with the application of less but longer pulses. Enzyme inactivation by PEF increases with a rise of pulse frequency (Bendicho et al., 2002c, 2003a; Elez-Martínez et al., 2003a, b). Bendicho et al. (2002c) reported less than a 5 per cent inactivation of lipase at 2 Hz, whereas a 13 per cent enzyme inactivation was achieved at 3.5 Hz when samples were subjected to 80 pulses of 37.3 kV/cm.

Factors affecting enzyme inactivation by PEF 159

For skimmed milk, whole milk and simulated milk ultrafiltrate (SMUF), it was observed that the higher the frequency the higher the protease inactivation achieved when similar treatments in terms of cumulated time and field strength were applied (Bendicho et al., 2003a, b). Vega-Mercado et al. (2001a) reported a reduction of up to 60 per cent for a protease using low field strengths (14–15 kV/cm) and frequencies of 1 and 2 Hz, whereas at lower frequencies (0.6 Hz) and higher field strengths (25 kV/cm), an enhancement of proteolytic activity was observed. Elez-Martínez et al. (2003a) and Espachs-Barroso et al. (2003b) observed that orange juice PME and CPC activities lowered as pulse frequency increased. Residual POD activity was reduced to 6.9 per cent when orange juice was treated at 450 Hz (35 kV/cm, 600 ␮s) and it was observed that higher inactivations were achieved when pulse frequency was increased (Elez-Martínez et al., 2003b). The pulse polarity is another factor reported to affect PEF effectiveness for inactivating enzymes. Different results have been reported depending on the enzyme. The application of pulses in mono- or bipolar mode did not significantly affect the extent of inhibition of tomato PME (Giner et al., 2000) (see Figure 7.2). When orange juice was treated with mono- or bipolar pulses, it was observed that bipolar pulses led to greater PME inactivation than monopolar pulses (Elez-Martínez et al., 2003a). However, the CPC inactivation was higher for the monopolar than for the bipolar treatments and the maximum enzymatic activity reduction was 80 per cent after a PEF treatment with monopolar pulses of 8 ␮s at a frequency of 400 Hz (Espachs-Barroso et al., 2003b). Giner et al. (2002) concluded that PEF treatments inactivated peach PPO more effectively when applied in bipolar than in monopolar mode. PEF applied in monopolar mode were more effective than bipolar mode in the inactivation of POD in orange juice working at 25 kV/cm, but higher rates of enzyme inactivation were achieved in bipolar mode when orange juice was processed at 35 kV/cm (Elez-Martínez et al., 2003b). Pulse wave shape seems to be a factor that influences the inactivation of enzymes by PEF. Yeom et al. (2002b) inactivated less than 10 per cent of orange PME after a treatment of 25 kV/cm for 184 ms working with square wave pulses. Nevertheless, a 93.8 per cent inhibition of tomato PME was achieved after a processing of 24 kV/cm for 8 ms with exponential decay pulses (Giner et al., 2000). So, the inactivation of PME was greater when the enzyme was subjected to exponential decay pulses. Batch-mode and continuous equipments are the most common systems for processing foods by PEF (see Table 7.1). The batch-mode PEF process inhibits lipase activity more effectively than the continuous mode (Bendicho et al., 2002c). Tomato PME activity was reduced by 93.8 per cent when treated at 24 kV/cm for 8 ms in batch-mode equipment (Giner et al., 2000) and residual orange juice PME activity was less than 10 per cent when the enzyme was subjected to 25 kV/cm for 184 ms in a continuous flow equipment (Yeom et al., 2002b). Therefore, batch-mode systems were more effective than continuous systems in inactivating PME. Temperature during PEF processing plays an important role on the enzyme inactivation (see Table 7.1). The higher the temperature during PEF processing the higher the enzyme inactivation (Vega-Mercado et al., 1995; Yeom et al., 2002; Min et al., 2003b). Increasing the temperature from 30°C to 61.9°C implies an increase of PME inactivation from 0 to 83.2 per cent (Yeom et al., 2002). There was approximately 47 per cent difference between residual activity values of lipoxygenase (LOX) obtained

160 Enzymatic Inactivation by Pulsed Electric Fields

after PEF treatment at 10°C (62.2 per cent) and 50°C (15.4 per cent) at 20 kV/cm for 60 ␮s (Min et al., 2003b). Sixty per cent plasmin inactivation was achieved in skimmed milk when the milk was processed by PEF with 50 pulses of 45 kV/cm at 10°C, but 90 per cent enzyme inactivation was achieved after a treatment of 50 pulses of 45 kV/cm at 15°C (Vega-Mercado et al., 1995). Van Loey et al. (2002) observed that higher rates of inactivation of alkaline phosphatase and lactoperoxidase could be achieved by increasing the process temperature than processing samples at room temperature. On the other hand, Castro et al. (2001b) reported that an increase in temperature did not affect the inactivation of alkaline phosphatase. A parameter that includes the electric field strength and treatment time is the energy density (Q). This parameter is useful to compare the effectiveness of PEF treatments with different electrical conditions or even treatments conducted with different PEF equipment. Q can be calculated using the following equations whether the pulses have exponential or squared waveform (Martín-Belloso et al., 2004): t

Q⫽∫ 0 t

Q⫽∫ 0

V0 ⭈ I dt 2⭈v

(1)

V0 ⭈ I dt v

(2)

where V0 is the peak voltage, I the intensity of the current, t the treatment time and v the volume of the treatment chamber. Many studies have demonstrated that the higher the input electric energy supplied to samples, the greater the reduction of enzyme activity obtained (Grahl and Märkl, 1996; Giner et al., 2000, 2001, 2002, 2003; Bendicho et al., 2002c, 2003a, b; ElezMartínez et al., 2003a). However, lactoperoxidase in raw milk could not be inactivated under PEF treatment, even after energy inputs of 500 kJ/kg (Van Loey et al., 2002).

3.2 Enzyme characteristics The enzymatic inactivation levels by PEF also depend on the enzyme concentration (Castro et al., 2001b) and the enzyme characteristics (Vega Mercado et al., 1995; Ho et al., 1997; Yeom et al., 1999, 2002; Giner et al., 2000, 2003; Bendicho et al., 2002c, 2003a; Min et al., 2003b) (see Table 7.1). Grahl and Märkl (1996) studied the inactivation of alkaline phospatase, peroxidase and lipase by PEF. They observed that for the same PEF treatment the most resistant enzyme was alkaline phosphatase followed by peroxidase and the most sensitive one was lipase. Moreover, to cause similar inhibition levels on PPO enzyme activity, pear PPO required stronger PEF treatment conditions than apple PPO (Giner et al., 2001).

3.3 Product parameters The influence of the electric conductivity of the medium on the inactivation of enzymes by PEF is not clear. Giner et al. (2001) reported that the higher the electric

Effects of PEF on enzymes 161

conductivity of the treatment medium, the less inactivation of PPO. However, for both PPO and POD, the medium conductivity did not influence the enzyme stability towards PEF processing (Van Loey et al., 2002). Van Loey et al. (2002) studied the influence of pH on the stability of enzymes subjected to PEF processing. Changes in pH did not influence the stability of PPO to PEF treatment, whereas lowering the pH to 4 decreased the stability of POD processed by PEF. Giner et al. (2001) observed an increase in PPO inactivation when the pH of the medium decreased. The influence of the composition of the medium on enzyme inactivation by PEF is not well defined. The content of fat in the medium influences the inactivation by PEF. Inactivation of alkaline phosphatase (ALP) by PEF depends on the amount of fat in milk. PEF processing was more effective when the milk fat content was lower (Castro et al., 2001b). However, Grahl and Märkl (1996), Ho et al. (1997) and Van Loey et al. (2002) did not observed any significant ALP reduction in either milk or aqueous solution. Milk composition affected the inactivation of a protease by PEF since higher inactivation levels were reached in skimmed than in whole milk (Bendicho et al., 2003a). Proteins also seem to influence the inactivation of enzymes by PEF. No significant influence of the protein content of the medium on the PEF sensitivity of enzymes (PPO, POD, LOX) was observed by Van Loey et al. (2002). Protease inactivation by PEF was more effective in a medium with proteins (Bendicho et al., 2002d). However, Vega-Mercado et al. (2001a) reported that casein has a protective effect on protease against PEF treatment. Sugar concentration of the medium also affects the inactivation of enzymes by PEF. Espachs-Barroso et al. (2002) reported that the activity of a commercial pectic enzyme formulation treated by PEF decreased when the concentration of the medium increased. The inactivation of enzymes by PEF was also influenced by the temperature of the treatment medium. Yeom et al. (2002b) observed greater values of PME inactivation as inlet temperature of the medium increased.

4 Effects of PEF on enzymes Enzymatic activity is altered by PEF, though, in general, enzyme inactivation by PEF requires stronger electrical conditions to achieve significant reductions than microbial inactivation (Ho et al., 1997). This fact is important because some enzymes are useful for the food industry, thus PEF would allow the destruction of microorganisms while maintaining the activity of some enzymes (Martín-Belloso et al., 2004). PEF processing affects enzyme activity. In most of the cases, high levels of inactivation have been achieved, but in some cases no effect or an increase in initial activity has been detected (Ho et al., 1997; Giner et al., 2000, 2002; Bendicho et al., 2002c, 2003a; Van Loey et al., 2002; Min et al., 2003b) (see Table 7.1 and Figure 7.1). Van Loey et al. (2002) found no depletion of enzyme activity due to PEF treatment. These authors attributed the significant reductions of enzyme activity to thermal effects. Therefore, in the following sections the effects of electric field processing conditions, systems and suspending media on enzyme inactivation by PEF are discussed.

162 Enzymatic Inactivation by Pulsed Electric Fields

Table 7.1 Effects of pulsed electric fields on enzymes Enzyme

Media

Treatment conditions*

Inactivation (%)

Reference

Pectin methyl esterase (tomato)

NaCl solution (8.8%) Distilled water

a

93.8 ⬍10

Giner et al. (2000) Van Loey et al. (2002)

Pectin methyl esterase

Orange juice Orange juice Orange juice Blended orange-carrot juice

b

35 kV/cm, 59 ␮s, 60.1°C 25 kV/cm, 250000 ␮s, 65°C b 35 kV/cm, 1500 ␮s, 45°C b 25 kV/cm, 340 ␮s, 63°C

88 90 80 79

Yeom et al. (2000b) Yeom et al. (2002) Elez-Martínez et al. (2003a) Rodrigo et al. (2001)

a a

⬍10 ⬍10

Van Loey et al. (2002) Van Loey et al. (2002)

24 kV/cm, 8000 ␮s, 15°C 30 kV/cm, 40000 ␮s

a

b

Pectin methyl esterase (orange peel)

Orange juice Distilled water and McIlvaine buffer (pH ⫽ 3.7)

35 kV/cm, 1000 ␮s 30 kV/cm, 40000 ␮s

Polygalacturonase

Commercial enzyme formulation

a

10.3 kV/cm, 32400 ␮s, 25°C

98

Giner et al. (2003)

Polyphenoloxidase (mushroom)

Buffer (pH ⫽ 6.5) 50 mM K-phosphate Distilled water

a

40 ⬍10

Ho et al. (1997) Van Loey et al. (2002)

Polyphenoloxidase (apple)

McIlvaine buffer (pH ⫽ 6.5) ⫹ 1 M NaCl ⫹ 5% PVPP McIlvaine buffer (pH ⫽ 3.8)

96.85 ⬍10

Giner et al. (2001) Van Loey et al. (2002)

80 kV/cm, 60 ␮s, 20°C 30 kV/cm, 40000 ␮s

a

a

24.6 kV/cm, 6000 ␮s, 15°C 31 kV/cm, 1000 ␮s

a

Polyphenoloxidase

Apple juice

a

31 kV/cm, 1000 ␮s

⬍10

Van Loey et al. (2002)

Polyphenoloxidase (pear)

McIlvaine buffer (pH ⫽ 6.5) ⫹ 1 M NaCl ⫹ 5% PVPP

a

22.3 kV/cm, 6000 ␮s, 15°C

62

Giner et al. (2001)

Polyphenoloxidase (peach)

McIlvaine buffer (pH ⫽ 4.5) ⫹ 1 M NaCl ⫹ 5% PVPP

a

24.3 kV/cm, 5000 ␮s, 25°C

70

Giner et al. (2002)

Peroxidase (soybean)

Buffer (pH ⫽ 6.0) 100 mM K-phosphate

a

75 kV/cm, 60 ␮s, 20°C

30

Ho et al. (1997)

Peroxidase (horseradish)

Distilled water and phosphate buffer (pH ⫽ 7)

a

⬍10

Van Loey et al. (2002)

Peroxidase

Milk Milk Orange juice

a

a

21.5 kV/cm, 20 pulses 19 kV/cm, 500 ␮s b 35 kV/cm, 1500 ␮s, 35°C

25 0 100

Grahl and Märkl (1996) Van Loey et al. (2002) Elez-Martínez et al. (2003b)

Lipoxygenase (soybean)

Distilled water

a

30 kV/cm, 40000 ␮s

⬍10

Van Loey et al. (2002)

Lipoxygenase (green peas)

Green pea juice

a

20 kV/cm, 400 ␮s

0

Van Loey et al. (2002)

Lipoxygenase Alkaline phosphatase (milk)

Tomato juice SMUFc Skim milk 2% fat milk Whole milk Milk 1.5% fat Milk 3.5% fat Raw milk

b

80 65 59 ⬍10

Min et al. (2003b) Castro et al. (2001b)

Alkaline phosphatase (bovine intestinal mucosa)

Buffer (pH ⫽ 9.8)

30 kV/cm, 40000 ␮s

35 kV/cm, 50 ␮s, 30°C 22 kV/cm, 51800 ␮s a 18.8 kV/cm, 28000 ␮s a 21.5 kV/cm, 780 ␮s

a

a

20 kV/cm, 400 ␮s

a

80 kV/cm, 60 ␮s, 20°C

Grahl and Märkl (1996)

0

Van Loey et al. (2002)

5

Ho et al. (1997)

(continued)

Effects of PEF on enzymes 163

Table 7.1 (Continued) Enzyme

Media

Treatment conditions*

Inactivation (%)

Reference

Protease (P. fluorescens)

Casein solution Skimmed milk

b

14–15 kV/cm, 196 ␮s 23.3–31.6 kV/cm, 64 ␮s b 14–15 kV/cm, 196 ␮s b 25 kV/cm, 32 ␮s

0 0 60 Activation

Vega-Mercado et al. (2001a)

Protease (B. subtilis)

Skimmed milk Whole milk SMUFc SMUFc Skimmed milk (SM)

b

35.5 kV/cm, 866 ␮s, 46°C 35.5 kV/cm, 866 ␮s, 46°C b 35.5 kV/cm, 866 ␮s, 40°C a 16.4–27.4 kV/cm, 320 ␮s b 26.1–37.3 kV/cm, 143 ␮s

81.1 57.1 62.7 0 SM: 0 SM: activationinactivationd

Bendicho et al. (2003a) Bendicho et al. (2003b) Bendicho et al. (2001d)

Lipase

Milk

a

60

Grahl and Märkl (1996)

Lipase (P. fluorescens)

SMUFc

a

b

27.4 kV/cm, 314.5 ␮s, 34°C 37.3 kV/cm, 136 ␮s, 35°C

62.1 13

Bendicho et al. (2002c)

Lipase (wheat germ)

Deionized water

a

87 kV/cm, 60 ␮s, 20°C

85

Ho et al. (1997)

␣-amylase (B. licheniformis)

Deionized water

a

90

Ho et al. (1997)

Glucose oxydase (A. niger)

Buffer (pH ⫽ 5.1) 50 mM Na-acetate

a

75

Ho et al. (1997)

Papain (papaya)

1 mM EDTA

b

50 kV/cm, 2000 ␮s, 35°C

90e

Yeom et al. (1999)

Lysozyme (egg white)

Buffer (pH ⫽ 6.2) 66 mM K-phosphate

a

13 kV/cm, 60 ␮s, 20°C

60

Ho et al. (1997)

Lactate dehydrogenase (beef heart)

Buffer (pH ⫽ 7.2) 20 mM K-phosphate

a

0

Barsotti et al. (2002)

Pectic enzymes

Commercial enzyme formulation

b

35 kV/cm, 1000 ␮s

98.4

Espachs-Barroso et al. (2002)

Pepsin (porcine stomach mucose)

Buffer (pH ⫽ 2.0) 10 mM HCl

a

40 kV/cm, 60 ␮s, 20°C

Activation (250%)

Ho et al. (1997)

b

b

21.5 kV/cm, 20 pulses

80 kV/cm, 60 ␮s, 20°C 63 kV/cm, 60 ␮s, 20°C

31.6 kV/cm, 192 ␮s, 30°C

*

Electric field strength, treatment time, treatment temperature. Batch mode PEF treatment. b Continuous PEF treatment. c SMUF: simulated milk ultrafiltrate. d Depending on the frequency of the treatment. e After 24 h storage at 4°C. a

4.1 Pectin methyl esterase (PME) The PME activity reduction by PEF in tomato extract is very high. When 400 pulses of 0.02 ms pulse width and 24 kV/cm of electric field strength were applied, the PME inactivation reached a maximum of 93.8 per cent with no significant differences

164 Enzymatic Inactivation by Pulsed Electric Fields

100

PW ⫽ 0.02 ms

Relative activity (%)

80 60

40 20 0

0

100

200

300

400

Number of pulses E ⫽ 5 kV/cm BF E ⫽ 20 kV/cm BF E ⫽ 6 kV/cm MF E ⫽ 24 kV/cm MF

E ⫽ 6 kV/cm BF E ⫽ 24 kV/cm BF E ⫽ 12 kV/cm MF

E ⫽ 12 kV/cm BF E ⫽ 5 kV/cm MF E ⫽ 20 kV/cm MF

Figure 7.2 Effect of the number of pulses on pectin methyl esterase extracted from tomato treated by PEF at several electric field intensities and field polarities. Plotted lines show the mono- and bipolar data adjusted to a first-order kinetic model. MF and BF data are for monopolar and bipolar pulses, respectively. Exponential decay electric pulses of 0.02-ms pulse width (Giner et al., 2000).

between applying mono- or bipolar pulses. These conditions included increasing the input energy to 43.2 GJ/m3 with the temperature less than 15°C during PEF treatment (Giner et al., 2000) (Figure 7.2). However, less than 10 per cent inactivation was attained on a commercial tomato PME dissolved in distilled water by applying a PEF process of 40 ms at 30 kV/cm (Van Loey et al., 2002). Yeom et al. (2000b) found a depletion of 88 per cent of PME activity when applying pulses at 35 kV/cm for 59 ms to orange juice. The inactivation was similar to the heat pasteurization of orange juice at 94.6°C for 30 s, which produced an activity depletion of 98 per cent (Yeom et al., 2000a). When the supplied electric field was lower than 35 kV/cm, the PME activity reduction was insignificant. If the treatment time was 49 ␮s, the activity reduction reached 40 per cent; while a shorter treatment time such as 39 ␮s did not cause any inactivation of PME at 35 kV/cm. The temperature of the orange juice increased to 60.1°C during the PEF treatment, but it was demonstrated that the increase of the temperature during the process was not the major cause for the reduction of PME activity (Yeom et al., 2000b). About 80 per cent of PME activity was inactivated when orange juice was treated for 1500 ␮s (4-␮s pulse width) at 35 kV/cm and 200 Hz in bipolar mode without exceeding 45°C and with an input energy of 8.269 GJ/m3 (Elez-Martínez et al., 2003a). The effects of electric field strength on PME activity in orange juice at a constant water bath temperature were studied using electric field strengths up to 35 kV/cm at 30°C (Yeom et al., 2002). An increase of electric field strength caused significant inactivation of PME with an increase in orange juice temperature. However, a thermal inactivation study showed that heating orange juice to the same temperature as that reached during PEF treatment was not as effective as PEF treatment in inactivating

Effects of PEF on enzymes 165

PME. Therefore, the main cause of enzymatic inactivation was PEF processing not the temperature reached during PEF treatment. The effects of electric field strength at different water bath temperatures were studied by Yeom et al. (2002) using electric field strengths up to 25 kV/cm and water bath temperatures of 10–50°C. Higher electric field strengths at higher water bath temperature were the most effective for PME inactivation. A combination of PEF treatment at 25 kV/cm and a water bath temperature of 50°C caused 90 per cent inactivation of PME. Moreover, 79 per cent PME inactivation was obtained at 25 kV/cm and 340 ␮s with a maximum temperature of 63°C in blended orange and carrot juice (Rodrigo et al., 2001). Rodrigo et al. (2001) also found a synergistic effect between PEF and thermal treatment. On the other hand, Van Loey et al. (2002) could not achieve more than 10 per cent inactivation of commercial PME from orange peel in distilled water and buffer solution with a treatment of 30 kV/cm and 40 ms. Moreover, PME in orange juice could not be inactivated more than 10 per cent with a treatment of 1 ms at 35 kV/cm. However, the combination of PEF processing (80 kV/cm and 20 pulses) with pH 3.5, at 44°C with 100 U nisin/ml resulted in a 92.7 per cent reduction in PME activity of orange juice (Hodgins et al., 2002).

4.2 Polygalacturonase (PG) PEF treatments significantly reduced the PG activity of a commercial enzyme preparation. Maximum inactivation of PG activity (98 per cent) required 32.4 ms PEF treatment at 10.28 kV/cm with bipolar pulses of 160 ␮s. The temperature was always below 25°C. When comparing the relative activity after an identical number of pulses at matching electric field intensities, the inactivation of the PG enzyme was found to be higher for pulses of 160 ␮s than for pulses of 40 ␮s. An energy density input of 22.56 GJ/m3 led to the maximum enzyme activity reduction (98 per cent), whereas 0.37 GJ/m3 produced minimal inactivation of PG (2 per cent) (Giner et al., 2003).

4.3 Polyphenoloxidase (PPO) Ho et al. (1997) achieved approximately 40 per cent inactivation of a commercial PPO from mushrooms suspended in a buffer solution with PEF treatment of up to 60 ␮s at 80 kV/cm with 2-␮s pulse width at 20°C. On the other hand, commercial mushroom PPO in distilled water was not inhibited more than 10 per cent after a PEF processing of 40 ms at 30 kV/cm (Van Loey et al., 2002). The relative activity of apple PPO decreased up to 3.15 per cent upon treatment with 24.6 kV/cm for 6 ms (31.2 GJ/m3) with 0.02 ms bipolar pulses maintaining the treatment temperature at 15°C (Giner et al., 2001) (Figure 7.3). However, treatments of 1000 pulses of 1 ␮s at 31 kV/cm and 1 Hz resulted in no more than 10 per cent inactivation of apple PPO (Van Loey et al., 2002). Van Loey et al. (2002) also found that PPO could be inactivated up to 32 per cent after 1000 pulses of 40 ␮s at 7 kV/cm by increasing the pulse frequency to 10 Hz, but the highest pulse frequency resulted in a

166 Enzymatic Inactivation by Pulsed Electric Fields

100

Relative activity (%)

80

60

40

20

0

0

1

2

3

4

5

6

7

8

Treatment time (ms) Figure 7.3 Effect of HIPEF applied in bipolar at 24 kV/cm on the polyphenol oxidase activity of pear (♦), apple (■) and peach (▲). Plotted lines show the data adjusted to a first-order kinetic model (adapted from Giner et al., 2001, 2002).

temperature increase up to 60°C within the treated sample and the observed PPO inactivation was attributed mainly to thermal effects. A reduction of 62 per cent of pear PPO activity was found after 6 ms of treatment at 22.3 kV/cm (27.9 GJ/m3) with 0.02 ms bipolar pulses without exceeding 15°C (Giner et al., 2001) (Figure 7.3). An activity reduction of peach PPO of up to 70 per cent was achieved in bipolar mode at 24.3 kV/cm electric field strength for a total PEF treatment of 5 ms using 0.02 ms pulse width. These conditions included an input energy density of 27.75 GJ/m3 and the temperature of the samples never exceeded 25°C during PEF treatment. Furthermore, pulses applied in bipolar mode tend to cause a larger depletion in peach PPO activity than in monopolar mode at a fixed input energy (Giner et al., 2002) (Figure 7.3).

4.4 Peroxidase (POD) Grahl and Märkl (1996) achieved a decrease of 25 per cent on POD activity when processing raw milk by 20 pulses at 21.5 kV/cm. These PEF conditions led to an input energy density nearly of 0.4 GJ/m3. However, Van Loey et al. (2002) did not find any effect on milk POD after exposing milk samples to PEF treatments of 500 ␮s at 19 kV/cm. A reduction of 30 per cent was attained when a POD from soybean suspended in a phosphate aqueous solution was treated with 60 ␮s at 75 kV/cm with pulses of 2 ␮s at 20°C (Ho et al., 1997).

Effects of PEF on enzymes 167

The effects of PEF on the activity of a commercial POD from horseradish diluted in distilled water were tested for processing samples at different field strengths and treatment times. Within the range of the experimental conditions, the activity of this POD did not decrease more than 10 per cent (Van Loey et al., 2002). However, when the pH of the media was reduced to 4, an inactivation of up to a 60 per cent was reported. A 100 per cent POD inactivation was achieved when orange juice was processed at 35 kV/cm for 1500 ␮s (4-␮s pulse width) and 200 Hz in bipolar mode without exceeding 35°C. Residual POD activity was reduced to 6.9 per cent when orange juice was treated at 450 Hz (35 kV/cm, 600 ␮s, bipolar pulses of 4 ␮s) and it was observed that higher inactivations were achieved when pulse frequency was increased. The higher the pulse width, the greater the levels of POD inactivation, with the maximum inactivation of 97.0 per cent at 10 ␮s of pulse width (35 kV/cm, 600 ␮s, 200 Hz, bipolar pulses) (Elez-Martínez et al., 2003b).

4.5 Lipoxygenase (LOX) A LOX from soybean suspended in distilled water could not be inactivated more than 10 per cent with a treatment of 30 kV/cm and a treatment time of 40 ms (Van Loey et al., 2002). LOX was not inactivated in pea juice after a PEF treatment of 400 pulses of 1 ␮s at a field strength of 20 kV/cm and frequency of 1 Hz (Van Loey et al., 2002). However, an 80 per cent reduction of LOX activity was observed when tomato juice LOX was exposed to PEF at 35 kV/cm for 50 or 60 ␮s (3-␮s pulse width) at 30°C (Min et al., 2003b). A maximum of 88.1 per cent inactivation of tomato juice LOX was observed with a PEF treatment at 30 kV/cm for 60 ␮s at 50°C. Moreover, a commercial scale PEF processing at 40 kV/cm, with a pulse duration time of 2 ␮s and a total treatment time of 57 ␮s at 53°C inactivated 54 per cent of the lipoxygenase in tomato juice (Min et al., 2003a).

4.6 Alkaline phosphatase (ALP) Castro et al. (2001b) studied the PEF inactivation of ALP in SMUF, non-fat milk, 2 per cent fat milk and whole milk. PEF treatment was able to reduce up to 65 per cent of ALP activity after 70 pulses of 740 ␮s at 22 kV/cm in SMUF and at 18.8 kV/cm in skimmed milk. When 2 per cent milk and whole milk were treated, ALP activity was reduced up to 59 per cent after a 70-pulse treatment at 18.8 kV/cm with pulses of 400 ␮s. However, Grahl and Märkl (1996) observed only a slight inactivation (about 5 per cent) after subjecting milk samples to PEF processes up to 0.4 GJ/m3 (21.5 kV/cm, 20 pulses) and Van Loey et al. (2002) did not observe any significant ALP reduction in PEF-processed milk after applying treatments of 200 pulses of 2 ␮s at 20 kV/cm. On the other hand, if the treatment temperature reached 70°C, ALP activity decreased up to 26 per cent (Van Loey et al., 2002). In addition, the activity of a commercial ALP from bovine intestinal mucosa suspended in a buffer solution decreased about 4.8 per cent

168 Enzymatic Inactivation by Pulsed Electric Fields

when it was treated at 80 kV/cm for 60 ␮s with pulses of 2 ␮s at 20°C (Ho et al., 1997).

4.7 Protease Inactivation of a dairy protease such as plasmin by PEF was evaluated in SMUF by applying a continuous treatment of up to 50 pulses of 2 ␮s at field strengths of 15, 30 and 45 kV/cm (Vega-Mercado et al., 1995). Plasmin activity decreased by 60 per cent after 50 pulses at field strengths of 30 or 45 kV/cm at 10°C, while a reduction of up to 90 per cent was achieved when the treatment temperature was 15°C. For microbial proteases, Vega-Mercado et al. (2001a) achieved up to 80 per cent inactivation of an extracellular protease from Pseudomonas fluorescens. The enzyme, suspended in tryptic soy broth enriched with a yeast extract, was subjected to 20 pulses of 18 kV/cm at 0.25 Hz, resulting in an energy density input of 0.01421 GJ/m3. On the other hand, inactivation levels changed significantly when the medium was skimmed milk. In this case, with energy inputs of 0.02075 GJ/m3 and 0.06963 GJ/m3, which were achieved by applying 32 pulses at 14.3 kV/cm (1 Hz) and 98 pulses at 15 kV/cm (2 Hz) respectively, decreases of up to 40 and 60 per cent of enzyme activity were achieved. When samples were subjected to higher field strengths (25 kV/cm), lower frequencies (0.6 Hz) showed an increase of the proteolytic activity. It is interesting to note that when the enzyme was suspended in casein-Tris buffer no significant inactivation of protease was obtained. This indicated that casein has a protective effect against PEF treatment on protease. The most severe evaluated treatment at 67 Hz (35.5 kV/cm for 866 ␮s) with monopolar pulses of 7 ␮s delivered 6.56 GJ/m3 to samples of skimmed milk with a commercial protease from Bacillus subtilis and the temperature never exceeded 46°C. This PEF treatment led to maximum levels of enzyme inactivation (37.9 per cent). In skimmed milk, inactivation levels varied from 37.9 to 81.1 per cent, depending on whether treatments of 35.5 kV/cm for 866 ␮s were performed at 67 or 111 Hz. In whole milk, both treatments led to 37.9 and 57.1 per cent inactivation, respectively (Bendicho et al., 2003a). Moreover, the maximum inactivation of a protease from Bacillus subtilis suspended in SMUF (62.7 per cent) was achieved after an 896-␮s treatment at 35.5 kV/cm and 111 Hz with monopolar pulses and a maximum temperature of 40°C. This treatment implied an energy density input of 6.787 GJ/m3 to the sample. When treatments were applied at a frequency of 67 Hz for the same electric field strength and treatment time, a reduction of only 48 per cent in enzyme activity was attained (Bendicho et al., 2003b). Bendicho et al. (2001d) studied the effect of PEF treatment using different devices on a protease from Bacillus subtilis suspended in SMUF or in milk. Protease activity did not show any inactivation after processing the samples of SMUF or milk with treatments of up to 80 pulses at 16.4–27.4 kV/cm (up to 0.5 GJ/m3) using batch mode PEF equipment. When treatments of similar input energy were applied with continuous flow equipment using treatments of up to 89 pulses at field strengths from 26.1 to 37.3 kV/cm, the effects of PEF on the enzyme activity depended on the treatment

Effects of PEF on enzymes 169

time, the field strength, the pulse repetition rate and the medium containing the enzyme. In SMUF, a maximum of 13 per cent inactivation was achieved by applying treatments of up to 0.5 GJ/m3 at 4 Hz, whereas at conditions of low frequency and low field strength no effects of PEF on enzyme activity were detected. In milk, results changed completely. In this case, most of the treatments at high frequencies (4 Hz) caused no changes on protease activity and on the contrary, a slight enhancement (10–15 per cent) of the proteolytic activity was observed after PEF treatments at low frequencies (2 Hz) (Bendicho et al., 2001d).

4.8 Lipase Grahl and Märkl (1996) observed about a 60 per cent reduction of lipase activity in raw milk after subjecting milk samples to an energy density input of nearly 0.4 GJ/m3 which was reached with 20 pulses of 21.5 kV/cm. Ho et al. (1997) achieved up to 85 per cent inactivation on a commercial lipase from wheat germ after exposure to PEF treatment of 60 ␮s at 87 kV/cm with 2-␮s pulses at 20°C. Another lipase whose behaviour has been studied after exposure to different PEF devices is a lipase from Pseudomonas fluorescens suspended in SMUF. Using batchmode PEF equipment, a 62.1 per cent maximum activity depletion was achieved after 80 pulses at 27.4 kV/cm (0.505 GJ/m3) at 34°C (Figure 7.4, where data for 16.4 kV/cm and 18.5 kV/cm are overlapped.). However, when PEF treatments were applied in a continuous flow mode, an inactivation rate of just 13 per cent was achieved, after applying 80 pulses at 37.3 kV/cm and 3.5 Hz (0.424 GJ/m3) at 35°C (Bendicho et al., 2002c). The batch-mode and continuous PEF equipments used to study the inactivation of lipase by PEF were the same as those used for protease (Bendicho et al., 2001d; Bendicho et al., 2002c). In the case of lipase, different from that for protease, the batch mode treatment showed more effectiveness than continuous mode, possibly due to the superiority of the voltage applied, the slowness of the treatment or the length of the pulse delay. Working in continuous PEF mode, it was observed that 2 Hz of frequency

Relative activity (%)

120 100 80 60 40 20 0 0

20

40 Number of pulses

60

80

Figure 7.4 Inhibition of lipase activity after several batch mode PEF treatments. Treatments were performed at 16.4 (♦), 18.5 (■), 22.7 (▲) and 27.4 kV/cm (●). The plotted lines correspond to the fit of experimental data to a first-order kinetic model (Bendicho et al., 2002c).

170 Enzymatic Inactivation by Pulsed Electric Fields

led to an inactivation of 6.9 per cent, whereas 13 per cent of enzyme inactivation was achieved when samples were processed at 3.5 Hz (Bendicho et al., 2002c).

4.9 Other enzymes The effect of PEF on other enzymes such as ␣-amylase, glucose oxidase, lysozyme, pepsin, lactate dehydrogenase and papain was studied by dissolving them in aqueous solutions. It was observed that some of them could be highly inactivated, whereas others showed little or no decrease in activity. Moreover, some others may suffer an increase on their initial activity after PEF processing (see Figure 7.1). For ␣-amylase and glucose oxidase, reductions of up to 90 per cent and 75 per cent were attained after PEF treatments of 60 ␮s at 80 kV/cm and 60 ␮s at 63 kV/cm, respectively with pulses of 2␮s at 20°C (Ho et al., 1997). On papain, the effect of PEF was evaluated immediately after the treatment as well as after storage. A PEF treatment of 50 kV/cm for 2 ms at 35°C had no significant effect on papain activity in a 1 mM EDTA solution. However, a significant activity depletion of up to 90 per cent was observed after 24 h of storage at 4°C (Yeom et al., 1999). The activity of lysozyme was reduced up to 60 per cent after a PEF process of 60 ␮s at 13 kV/cm with 2-␮s pulses at 20°C. On the other hand, applying the same treatment conditions but at a higher field strength (47 kV/cm) the enzyme activity decreased by only about 15 per cent (Ho et al., 1997). After a PEF process of 31.6 kV/cm for 192 ␮s (0.582 GJ/m3) and maintaining the treatment temperature below 30°C no significant effects were observed on the activity of lactate dehydrogenase (Barsotti et al., 2002). Pepsin is an enzyme that behaves in a completely opposite way to the other enzymes. After a PEF treatment of 60 ␮s at field strengths from 20 to 80 kV/cm, the activity of this enzyme was enhanced showing the maximum activation (250 per cent) when 40 kV/cm were applied (Ho et al., 1997). Espachs-Barroso et al. (2002, 2003b) studied the effect of PEF treatment on the inactivation of a commercial pectic enzyme complex. The maximum activity reduction was 98.4 per cent by applying 1 ms at 35 kV/cm with bipolar 4-␮s pulses and 200 Hz. A treatment of 35 kV/cm for 500 ␮s with monopolar pulses of 8 ␮s at a frequency of 400 Hz led to an enzymatic activity reduction of 80 per cent.

5 Modelling enzymatic inactivation by PEF Models describing the inactivation of enzymes are useful to establish appropriate PEF process conditions to obtain certain levels of inactivation. Knowing the inactivation levels obtained by PEF is necessary to achieve enzymatically stable products without over-processing. The intensity of the changes during PEF processing is affected by the treatment conditions, the composition of the medium and the specific characteristics of the enzyme (Vega-Mercado et al., 1995; Giner et al., 2000, 2001; Bendicho et al., 2002c, 2003a; Yeom et al., 2002; Min et al., 2003b). The factors that mainly influence

Modelling enzymatic inactivation by PEF 171

enzyme inactivation are field strength and treatment time. Enzyme inactivation has been described as an exponential function of treatment time or field strength (Bendicho et al., 2002c, 2003b; Giner et al., 2000, 2001, 2002, 2003; Min et al., 2003b), as detailed below: RA ⫽ RA0 ⭈ exp(⫺kE ⭈ t)

(3)

RA ⫽ RA0 ⭈ exp(⫺kt ⭈ E)

(4)

where RA is the relative activity, RA0 the initial relative activity (100 per cent), kE and kt are inactivation rate constants, t the treatment time and E the electric field strength. Moreover, Bendicho et al. (2001e, 2002c), Giner et al. (2000, 2001, 2002, 2003) and Min et al. (2003b) reported that kE and kt values could be fitted to linear, exponential or potential functions related to the field strength or the treatment time, respectively. Thus, exponential models related to both treatment time and field strength could be obtained to describe enzyme inactivation as given in the following equations. These models were used to describe the inactivation of vegetable (Giner et al., 2000, 2001, 2002, 2003) and microbial dairy enzymes (Bendicho et al., 2001e, 2002c). RA ⫽ RA0 ⭈ exp[⫺(k0 ⫺ k1 ⭈ E) ⭈ t]

(5)

RA ⫽ RA0 ⭈ exp[⫺(k2 ⭈ exp(k3 ⭈ E)) ⭈ t]

(6)

RA ⫽ RA0 ⭈ exp[⫺(k4 ⫺ k5 ⭈ t) ⭈ E]

(7)

RA ⫽ RA0 ⭈ exp[⫺(k6 ⭈ exp(k7 ⭈ t)) ⭈ E]

(8)

RA ⫽ RA0 ⭈ exp[⫺(k8 ⭈ tk9) ⭈ E]

(9)

where k0 to k9 are constants, t is the treatment time and E is the field strength. In all the cases, RA can also be related to the number of pulses (n) instead of the treatment time (t). In fact, both parameters are directly related by the pulse duration. The results of tomato PME activity reduction obtained by Giner et al. (2000) were adjusted properly to a first-order model for the treatment time and the electric field strength (Equations (3) and (4); see Figure 7.2). The inhibition constant related to the treatment time followed an exponential trend when increasing the electric field strength and the rate constant for the field strength increased potentially with the treatment time. Therefore, the inactivation of tomato PME could be described with exponential functions that contain field strength and treatment time as variables (Equations (6) and (9)) (Giner et al., 2000). The inactivation of PPO as a function of the treatment time also followed a firstorder kinetic (Equation (3)) for apple, pear and peach (Giner et al., 2001, 2002). Moreover, the first-order rate constants for all the three PPOs also followed an exponential model (see Figure 7.3). Thus, the inactivation of PPO could be described by Equation (6). As occurs in PPO, PEF inactivation of PG fitted well an exponential model (Equation (3)) where the calculated rate constants increased exponentially with electric

172 Enzymatic Inactivation by Pulsed Electric Fields

field strength. Therefore, the inactivation of PG as a function of field strength and treatment time could be well described by Equation (6) (Giner et al., 2003). Min et al. (2003b) reported that the first-order inactivation model (Equation (3)) is valid for describing the activity depletion of tomato juice lipoxygenase (LOX) by PEF. The rate constants showed an exponential dependence with field strength and the variations in LOX activity fitted Equation (6). A first-order kinetic (Equation (3)) properly matches lipase depletion suspended in SMUF with the number of applied pulses (see Figure 7.4). Moreover, the batch-mode rate constants increase exponentially when the applied electric field intensity goes up. Consequently, Equation (6), which provided the combined effect of the treatment time and electric field intensity to predict relative enzyme activity, could be obtained (Bendicho et al., 2002c) to describe the protease activity in SMUF which decreased exponentially with increase of treatment time and field strength (Bendicho et al., 2003b). Moreover, some empiric models such as Hülsheger’s (Hülsheger et al., 1983) and Fermi’s (Peleg, 1995) models, which were first proposed for predicting microbial inactivation, have also been used to describe the destruction of enzymes by PEF (Giner et al., 2000; Min et al., 2003b). Hülsheger’s model describes the decrease of enzyme activity (RA) as a function of both electric field strength (E) and treatment time (t) as described by  t ⫺ RA ⫽ 100 ⭈    t c 

(E⫺Ec ) k

(10)

where tc and Ec are the extrapolated critical t and E values for RA equal to 100 and k is an independent constant factor. Fermi’s model describes the level of residual relative enzyme activity (RA) as a function of the electric field intensity (E), given below RA ⫽

100  E ⫺ Eh   1 ⫹ exp   a 

(11)

where Eh is the critical level of electric field strength when RA is 50 per cent and a is a parameter indicating the steepness of the curve around Eh. The inactivation of tomato PME can be well described with the Hülsheger’s model (Equation (10)), with an Ec of 0.7 kV/cm and nc of 24 pulses (0.48 ms) and a k of 39 kV/cm (Giner et al., 2000). Furthermore, the Fermi’s model (Equation (11)) also described the inactivation of PME. The Eh values vary from 28 kV/cm for 50 pulses to 10.8 kV/cm for 400 pulses (Giner et al., 2000). The Hülsheger’s model was used to fit the experimental data obtained with the inactivation of tomato juice LOX by PEF (Min et al., 2003b) and it was observed that Ec, tc and k parameters varied with temperature. The Fermi’s kinetic model was fitted to the experimental data with good agreement at different levels of PEF treatment, with Eh parameter decreasing from 54.59 to 10.01 kV/cm as the PEF treatment time increased from 20 to 70 ␮s (Min et al., 2003b).

Modelling enzymatic inactivation by PEF 173

Bendicho et al. (2001e) studied the inactivation kinetics of a microbial lipase treated by PEF. They observed that Hülsheger’s model fitted the data with good accuracy and the resulting values of Ec, tc and k were 4.03 kV/cm, 31.08 ␮s and 64.31 kV/cm, respectively. Fermi’s model also described the inactivation of lipase by PEF with good agreement. Moreover, it was observed that a potential model related to the treatment time was the one that better described the evolution of both parameters Eh and a (Bendicho et al., 2001e). The following modified first-order fractional conversion model proposed by Levenspiel (1972) has also been used to describe the inactivation of enzymes by PEF (Espachs-Barroso et al., 2002). The inactivation of a commercial pectic enzyme formulation by PEF fitted well a fractional conversion model as a function of the total treatment time (Espachs-Barroso et al., 2002). RA ⫺ RA ⬁ ⫽ exp(⫺k1′ ⭈ P) RA0 ⫺ RA ⬁

(12)

where RA is the relative activity, RA0 the initial value of relative activity, RA⬁ is the stabilized value of relative activity, P the PEF processing parameter, and k⬘1 a firstorder rate constant. The electric energy density (Q) delivered through the treatment chamber is an important parameter in PEF technology and can be related to the relative residual activity (RA) of the enzyme through the following exponential decay relationship (Giner et al., 2000, 2001, 2002, 2003; Bendicho et al., 2002c, 2003a, b). RA ⫽ RA0 ⭈ exp(⫺k10 ⭈ Q)

(13)

where k10 is a first order constant. The first order constants (k10) for the PPOs of apple and pear were 0.091 and 0.027 m3/GJ, respectively (Giner et al., 2001) and k10 for peach PPO treated in mono- or bipolar mode were 0.019 or 0.039 m3/GJ (Giner et al., 2002). Therefore, less energy was needed to achieve similar activity reduction of PPO from apple than from pear and peach. Giner et al. (2000) found 0.068 m3/GJ for the k10 value in the inhibition of tomato PME by PEF, which is within the range for apple, pear and peach PPO. However, another vegetable enzyme such as PG needed much higher levels of energy to be inactivated because the k10 value was 0.196 m3/GJ (Giner et al., 2003) (Figure 7.5). Inactivation of dairy enzymes also followed exponential decay relationships with electric energy density input. Decrease of lipase activity in SMUF with input electric energy density can be adequately modeled by a first-order kinetic model in both batch and continuous mode, although the k-value obtained was much higher for the batchmode process (1.9 m3/GJ for the batch mode and 0.2 m3/GJ for the continuous mode) (Bendicho et al., 2002c). The continuous PEF treatment of a protease in SMUF led to a k-value of 0.07 m3/GJ (Bendicho et al., 2003b). So, the protease was more resistant to PEF than the lipase since the lower the k-values of the exponential model the higher the energy densities required to reach a large degree of inactivation. Bendicho et al. (2003a) reported that the decrease of relative protease activity in skimmed milk could be successfully related to the supplied energy density using a first-order kinetic model

174 Enzymatic Inactivation by Pulsed Electric Fields

PME tomato PPO apple

100

Relative activity (%)

PPO pear

80

PPO peach monopolar PPO peach bipolar

60 40 20 0 0

10

20

30

40

50

Energy density (GJ/m3) Figure 7.5 First-order model fits of the enzymatic residual activity subjected to different input energy densities supplied by PEF from 5 to 24 kV/cm (Espachs-Barroso et al., 2003).

(Equation (13)), leading to a k-value of 0.06 m3/GJ. Regarding the k-value of protease suspended in SMUF, it can be said that protease showed almost equal sensitivity towards PEF treatment when it is suspended in skimmed milk or in SMUF.

6 Enzyme inactivation by combining PEF with other hurdles Because of the resistance of several enzymes to PEF treatments, combined processes of PEF with other hurdles such as mild heat, acidification of the media or the use of some additives were studied (Table 7.2). The application of a mild heat treatment after the PEF process was investigated to enhance the inactivation of a protease from Bacillus subtilis and a lipase from Pseudomonas fluorescens; however, this combination did not provide great improvements on the inactivation of these enzymes over PEF alone (Bendicho et al., 2001a, 2001b). Bendicho et al. (2001a, 2001c) combined the acidification of a SMUF to pH ⫽ 5 (HCl) with the application of a batch PEF process of 60 pulses at 27.4 kV/cm and achieved approximately 30 per cent inactivation of a protease from B. subtilis and approximately 52 per cent inactivation of a lipase from P. fluorescens. Hodgins et al. (2002) studied the combination of PEF processing with reduction of pH, heat treatment and addition of nisin on the inactivation of PME in orange juice. A 92.7 per cent of PME inactivation was achieved when orange juice was processed at 80 kV/cm for 20 pulses, at pH 3.5 and a temperature of 44°C with 100 U nisin/ml. PEF treatment of SMUF samples containing EDTA enhanced the inactivation of the protease, whereas PEF inactivation of the protease was not a function of the calcium added (Vega-Mercado et al., 2001b).

Enzyme activity during storage of PEF processed foods 175

Table 7.2 Enzyme inactivation by combining pulsed electric fields with other hurdles Enzyme

Media

Combined treatment

Inactivation (%)

Reference

Protease (B. subtilis)

SMUFb

a

16.4–27.4 kV/cm, up to 60 pulses ⫹ Mild heat treatment (63°C, 5 min) a 16.4–27.4 kV/cm, up to 60 pulses ⫹ Acidification (pH ⫽ 5)

50

Bendicho et al. (2001a)

30

Bendicho et al. (2001a)

SMUFb

a

6.2 kV/cm, 20 pulses, 20°C ⫹15 mM Ca

30

Vega-Mercado et al. (2001b)

SMUFb

a 6.2 kV/cm, 20 pulses, 20°C ⫹20 mM EDTA

100

Vega-Mercado et al. (2001b)

SMUFb

a

35

Bendicho et al. (2001b)

52

Bendicho et al. (2001c)

SMUFb

Protease (P. fluorescens)

Lipase (P. fluorescens)

SMUFb

Pectin methyl esterase

Orange juice

16.4–27.4 kV/cm, up to 100 pulses ⫹ Mild heat treatment (63°C, 15 min) a 16.4–27.4 kV/cm, up to 100 pulses ⫹ Acidification (pH ⫽ 5) a

80 kV/cm, 20 pulses, 92.7 44°C ⫹ Acidification (pH ⫽ 3.5) ⫹ Nisin (100 U/ml)

Hodgins et al. (2002)

a

Batch mode PEF treatment. SMUF: simulated milk ultrafiltrate.

b

7 Enzyme activity during storage of PEF processed foods There is little information about the behaviour of the enzymes during the storage of foods processed by PEF. Yeom et al. (2000a) studied the effects of PEF on PME of orange juice and compared it with that of heat-pasteurized orange juice. PEF treatment of orange juice at 35 kV/cm for 59 ␮s decreased 88 per cent of PME activity and the inactivated PME was not restored at 4 and 22°C for 112 days. Heat pasteurization of orange juice at 94.6°C for 30 s inactivated 98 per cent PME activity and this inactivation was constant during 112 days of storage at 4 and 22°C (Figure 7.6). LOX activity was not detected in thermally (92°C for 90 s) processed tomato juice throughout the storage at 4°C for 112 days. Commercial scale PEF processing (57 ␮s at 40 kV/cm) inactivated 54 per cent of the LOX in tomato juice. The reduced LOX activity in PEF-processed juice decreased further during storage at 4°C. The LOX in tomato juice was irreversibly inactivated by thermal or PEF processing (Min et al., 2003a) (Figure 7.6).

176 Enzymatic Inactivation by Pulsed Electric Fields

Control PEF Heat

100 90 80 70 60 50 40 30 20 10

Relative LOX activity (%)

Relative PME activity (%)

110

Control PEF Heat

0

0 0 (a)

110 100 90 80 70 60 50 40 30 20 10

10 20 30 40 50 60 70 80 90 100 110 120 Storage time (days)

0 (b)

10 20 30 40 50 60 70 80 90 100 110 120 Storage time (days)

Figure 7.6 Effects of PEF and thermal processing on the pectin methyl esterase (PME) activity of orange juice (a) and the lipoxygenase (LOX) activity of tomato juice (b) during storage at 4°C (Yeom et al., 2000a; Min et al., 2003a).

8 Conclusions The feasibility of using PEF to inactivate enzymes has been positively demonstrated. However, to achieve a great extent of enzyme inactivation, higher energy levels than those required to destroy microorganisms are needed. The results of PEF inactivation of enzymes are encouraging in the exploration of an alternative non-thermal method of food preservation. Enzyme activity is affected by the particular enzyme, the media used for suspension and the PEF treatment conditions. Thus, most enzymes are almost completely inactivated while some show resistance to PEF processing. PEF can be employed as an effective hurdle when used in combination with other preservation factors such as pH and additives or as a complementary step with mild thermal processes. First-order kinetic models as a function of treatment time or field strength can be successfully used to describe enzyme inactivation. Moreover, the PEF inactivation could also be explained by empirical models such as those of Hülsheger and Fermi. Hence, the food industry is expressing an increasing interest in PEF processing as an alternative or complementary preservation technique to traditional thermal treatments. Further investigation of enzyme inactivation by PEF is required to better control the critical points involved, to achieve higher levels of enzyme inactivation, to learn more about the mechanism of enzyme inactivation and to enable the scale-up of the PEF technology for the food industry.

Nomenclature a ALP CPC E

steepness of the curve around Eh alkaline phosphatase commercial pectic enzyme complex electric field strength (kV/cm)

References 177

Ec Eh f I k k 1 k0, k1, k2, k3, k4, k5, k6, k7, k8, k9 k10 kE kt LOX n P PEF PG PME POD PPO Q RA RA RA0 SMUF t tc V V0

critical electric field strength critical level of electric field strength when RA is 50 per cent frequency (Hz) intensity of the current (A) independent constant factor first-order rate constant kinetic constants first-order constant first-order rate constant first-order rate constant lipoxygenase number of pulses PEF processing parameter pulsed electric fields polygalacturonase pectin methyl esterase peroxidase polyphenoloxidase energy density (J/m3) relative activity stabilized value of RA initial relative activity simulated milk ultrafiltrate treatment time (ms, ␮s) critical treatment time volume of treatment chamber (m3) peak voltage (V)

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Food Safety Aspects of Pulsed Electric Fields Olga Martín-Belloso and Pedro Elez-Martínez University of Lleida, Department of Food Technology, UTPV-CeRTA Lleida, Spain

Pulsed electric field food processing is a treatment with very short electric pulses (
s or ms) at high electric field intensities (kV/cm) and moderate temperatures. It is a non-thermal technology that has been studied in the last few years as an alternative to traditional thermal treatments. The effectiveness of PEF processing on the inactivation of microorganisms has been demonstrated while the appearance and quality of fresh foods are not altered. Microbial destruction by PEF depends on several factors related to process factors, microbial characteristics and food properties. Irreversible pore formation and destruction of the semipermeable barrier of the cell membrane cause microbial inactivation by PEF. This technology has the potential to provide minimally processed, safe, nutritious and fresh-like food products to consumers.

1 Introduction Food-borne diseases are caused by a wide range of agents with different degrees of severity ranging from mild indisposition to chronic or life-threatening illness (Käferstein et al., 1999). Food safety is not limited to microbiological aspects but also to chemical contamination and foreign bodies (Andrews et al., 2001). Biological contamination, i.e. bacteria, viruses and parasites, constitutes the major cause of food-borne diseases. Although a variety of bacteria has been implicated in food-borne disease, it is known that a few species cause the majority of health problems. The most important pathogenic bacteria are Clostridium botulinum, Bacillus cereus, Staphylococcus aureus, Salmonella, Campylobacter, Escherichia coli, Listeria monocytogenes, Vibrio cholerae and others like Vibrio parahemoliticus, Vibrio vulnificus, Aeromonas hydrophila and Yersinia enterocolitica (Van Schothorst, 1999). Moulds and yeasts are used sometimes intentionally in the production of some foods and drinks but they can also grow in food leading to its spoilage. In addition, strains of some moulds may produce toxins (Pitt and Hocking, 1989). Viruses and parasites that may be pathogenic for humans are transmitted through foods or water (Van Schothorst, 1999). Emerging technologies for food processing ISBN: 0-12-676757-2

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184 Food Safety Aspects of Pulsed Electric Fields

The presence of some toxic components must be controlled in order to assure food safety. Thus, considerable efforts have been undertaken to ensure the safety of food products against dangerous substances. Veterinary drug residues, pesticide residues, food additives, environmental chemicals and plant toxins are among the most important hazards for humans (Käferstein et al., 1999). Prevention and control of microbiological and chemical hazards could be achieved by the application of food-processing technologies that can prevent or reduce the use of chemicals in food (Shapton and Shapton, 1991). Processed foods may be safer than fresh foods due to the capability of preservation processes not only for removing, inhibiting, or killing pathogenic microorganisms, but also inactivating hazardous substances (Jones, 1992). The major concern of food preservation is the control of microbial growth to ensure product safety and to prevent spoilage, which makes the food undesirable (Aronsson and Rönner, 2001). Traditionally, the most popular preservation technologies for the reduction of microbial contamination of food, and pathogens in particular, have been the modification of the water activity and/or pH, heat treatments, the addition of chemical preservatives and the control of storage temperature of foods (IFT, 2001). Although traditional preservation technologies assure the safety of foods, the organoleptic, nutritional and physicochemical properties are extensively damaged (Jeyamkondan, 1999; Espachs-Barroso et al., 2003). Today’s consumers demand high quality foods that are both natural and fresh, while still microbially safe (Hoover, 1997), which is the reason why the food industry is exploring alternative processing methods to overcome negative effects during conventional food processing (Qin et al., 1996). Thus, other preservation technologies are emerging as alternatives for the extension of product shelf-life and reduction of pathogenic organisms, for safer and better quality products. Pulsed electric fields (PEF) is a non-thermal technology in which the food industry is increasingly interested (Giner et al., 2000). PEF are intended to be able to kill microorganisms in foods without significant loss of flavour, colour, taste and nutrients (Mertens and Knorr, 1992; Yeom et al., 2000a; Bendicho et al., 2002b; Hodgins et al., 2002). Pulsed electric field processing involves the application of short pulses (
s) of high voltage (kV/cm) to foods placed between two electrodes. Application of PEF is restricted to food products that can withstand high electric fields, have low electrical conductivity and do not contain or form bubbles. The particle size of the food is also a limitation (IFT, 2001). Research related to PEF and food safety has been focused on microbial safety. Nevertheless, not much information is available about the effect of PEF on hazardous substances. The effect of PEF on pathogenic and spoilage microorganisms has been studied in different media. In general, PEF process is effective in destroying microorganisms (Hülsheger et al., 1981; Dunn and Pearlman, 1987; Jayaram et al., 1992; Zhang et al., 1994a; Qin et al., 1995a, b; Pothakamury et al., 1995a, b; Martín et al., 1997; Martín-Belloso et al., 1997; Calderón-Miranda et al., 1999a, b; Wouters et al., 1999; Raso et al., 2000; Álvarez et al., 2003a, b) and is as effective as traditional pasteurization heat treatments in microbial destruction (Yeom et al., 2000a). However,

Microbiological safety of pulsed electric fields 185

there are contradictory results on the inactivation of spores by PEF (Marquez et al., 1997; Cserhalmi et al., 2002). The information about chemical safety and PEF is very limited and only a few studies about electrochemical reactions and electrode corrosion in PEF treatment chambers have been reported (Sale and Hamilton, 1967; Jacob et al., 1981; Morren et al., 2003). This chapter presents the current information available about the effect of PEF on microorganisms as well as on chemical reactions which can affect the safety of foods.

2 Microbiological safety of pulsed electric fields 2.1 Effect of PEF on microorganisms Most of the studies on the effects of PEF on microorganisms have been performed in buffer or model solutions (Jayaram et al., 1992; Qin et al., 1994; Pothakamury et al., 1996; Wouters et al., 1999; Dutrueux et al., 2000a; Pol et al., 2000; Rusell et al., 2000; Aronsson et al., 2001; Abram et al., 2003; Álvarez et al., 2003b). These studies are very useful but not conclusive, since inactivating microorganisms is more difficult in complex food materials than in dilute buffer solutions (Zhang et al., 1994a; Martín et al., 1997). The effects of PEF on microorganisms suspended in foods have been mainly studied in juices and milk (Tables 8.1 and 8.2). The destruction of microorganisms by PEF has been demonstrated in model solutions similar to milk ultrafiltrate (SMUF), in milk with different fat contents and in yogurt (Bendicho et al., 2002a). Raso et al. (1999) achieved 4 and 2 log reductions on the inactivation of Staphylococcus aureus and coagulase-negative Staphylococcus spp., respectively when these microorganisms were inoculated in raw milk, but no reduction of other microorganisms such as Corynebacterium spp. or Xanthomonas maltophilia was observed. Listeria monocytogenes suspended in milk was inactivated around 3 log reductions when inoculated milk was processed at 30 kV/cm for 600
s (Reina et al., 1998). Significant inactivation levels have been achieved in several microorganisms inoculated in SMUF. The effect of PEF treatments was evaluated on samples of SMUF inoculated with Lactobacillus delbrueckii and Bacillus subtilis, and reductions of 4 and 5 log cycles were achieved, respectively, after applying treatment of 50 and 40 pulses of 16 kV/cm (Pothakamury et al., 1995b). When PEF treatments was applied to samples of SMUF inoculated with Escherichia coli, reductions of 6 and 9 cycles were achieved, respectively, after applying 50 pulses of 60 kV/cm or 80 pulses of 70 kV/cm (Zhang et al., 1995; Qin et al., 1998). PEF-processed milk stored under refrigeration was found to have a microbial shelf-life of two weeks (Qin et al., 1995a). The effects of PEF treatments on juices have been extensively studied. McDonald et al. (2000) studied the inactivation of four microorganisms suspended in freshly squeezed orange juice (ascospores of Sacharomyces cerevisiae, Listeria innocua, Escherichia coli ATCC 26 and Leuconostoc mesenteroides). The results showed that a 5 to 6 log reduction of Escherichia coli and Listeria innocua was achieved by six 2
s pulses per unit of volume at 30 kV/cm; at least four 2
s pulses per unit of volume were

186 Food Safety Aspects of Pulsed Electric Fields

required at 50 kV/cm to reduce any of the inoculated bacterial species by 4 log or more. However, the counts of Sacharomyces cerevisiae were reduced by more than 4 log cycles by applying 5 pulses at an electric field strength of 7 kV/cm (Grahl and Märkl, 1996). The inactivation of Escherichia coli in apple juice at 34 kV/cm and a total treatment time of 166
s was 4.5 log cycles (Evrendilek et al., 2000). Applying two 2.5
s pulses at 50 kV/cm or ten pulses at 35 kV/cm to apple juice inoculated with Saccharomyces cerevisiae, more than 6 log reductions were achieved (Qin et al., 1995b). The destruction of moulds like Zygosacharomyces bailii in apple juice by a PEF treatment of 5
s at 32.3 kV/cm was 4.8 log for vegetative cells and 3.6 log in the case of their ascospores (Raso et al., 1998a). A PEF treatment at 35 kV/cm for 59
s kept the total plate count below 1 log cfu/ml after storage at 4, 22 and 37°C for 112 days (Yeom et al., 2000a). Qin et al. (1995a) showed a shelf-life extension of over 3 weeks in apple juice after a HIPEF treatment of ten 2.5
s pulses at 36 kV/cm.

2.2 Mechanism of microorganism inactivation by PEF The mechanism underlying the inactivation of microorganisms by PEF is not yet clearly understood. Knowledge of the microbial inactivation mechanism is essential in order to develop better equipment and define conditions for inactivating microorganisms in food products by PEF technology (Wouters et al., 2001a). Membrane damage is believed to be the direct cause of cell inactivation (Sale and Hamilton, 1967). Electric fields are hypothesized to electroporate cellular membranes (primary effect) and cause mechanical breakdown of cells (secondary effect) (Zimmermann et al., 1976). There are several theories to explain how pores are formed but it is still unclear whether it occurs in the lipid or the protein matrices (BarbosaCánovas et al., 1999). The most commonly accepted theory is that local instabilities in the membranes of the microorganisms are formed by electromechanical compression and electric field-induced tension, which causes pores to form in the membrane (electroporation) (Coster and Zimmermann, 1975; Tsong, 1990; Ho and Mittal, 1996; Weaver and Chizmadzhev, 1996; Barbosa-Cánovas et al., 1999). One of the major consequences of electroporation is a phenomenon called electropermeabilization, which is a dramatic increase in permeability (or conductivity) and, in some cases, mechanical rupture of the membrane (Wouters et al., 2001a). When an external electric field is applied to a cell, a transmembrane potential is induced and the potential difference across the membrane is proportional to the external electric field intensity (Sale and Hamilton, 1967). Mechanical instability of membranes only occurs when the applied electric field induces a certain critical membrane potential. When this potential reaches a critical value due to a strong electric field, the membrane breaks down (Kinosita and Tsong, 1977). Electropermeabilization has been demonstrated to be reversible or irreversible depending on the degree of membrane organizational changes due to PEF treatment (Weaver et al., 1988; Rols et al., 1990; Tsong, 1990). The resulting inactivation of microorganisms is considered to be related to both the electrical field strength and the total treatment time (Sale and Hamilton, 1967; Hülsheger et al., 1983). Some studies about the effect of PEF on microorganisms corroborated the theory that microbial death is due to the electromechanical compression and electroporation

Microbiological safety of pulsed electric fields 187

induced by PEF treatment (Jayaram et al., 1992; Harrison et al., 1997; Pothakamury et al., 1997; Shin and Pyun, 1999; Aronsson et al., 2001). Jayaram et al. (1992) and Shin and Pyun (1999) studied the effect of PEF on the ultrastructure of Lactobacillus brevis and Lactobacillus plantarum, respectively. They reported that the cell surface was rough after treatment with PEF when observed by electron microscopy. Moreover, the cell wall was broken, the cytoplasmatic contents were disorganized and cytoplasmatic contents were leaking out of the cell. When Pothakamury et al. (1997) treated cells of Staphylococcus aureus with 64-pulse treatments at different electric field strengths, they observed, by electron microscopy, that cells exhibited rough surfaces and that the ones treated under more severe conditions showed small holes in the membrane and leakage of cellular contents. Thus, the increase of microbial inactivation with the field strength is related to the increase of cell deterioration. Aronsson et al. (2001) studied, by means of electron microscopy, the effect of PEF on Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae. They found clear differences between PEF-treated and untreated E. coli and L. mesenteroides cells. An effect on microbial cell membranes by PEF treatment was deduced from the finding that the membrane was detached from the cell wall in approximately 10 per cent of L. innocua cells. However, they were unable to demonstrate consistent differences between samples of untreated S. cerevisiae cells and cells exposed to PEF treatment.

2.3 Factors affecting microbial inactivation by PEF There are several factors affecting the PEF inactivation of microorganisms. These factors can be grouped as process parameters, microbial characteristics and product parameters. 2.3.1 Process parameters

The main process parameters that affect microbial inactivation by PEF are electric field strength, treatment time or number of pulses, pulse width, pulse shape, pulse polarity and treatment temperature. Many researchers have reported that microbial inactivation is greater when electric field strength and treatment time are increased (Hülsheger et al., 1981; Zhang et al., 1994a, b; Qin et al., 1996, 1998; Martin et al., 1997; Evrendilek et al., 1999; Cserhalmi et al., 2002; Rodrigo et al., 2003; Abram et al., 2003; Elez-Martínez et al., 2003a, b) (Figure 8.1). Treatment time is the product of the applied number of pulses and the pulse width of each one. For treatments with similar field strength and number of pulses, inactivation of microorganisms increased with the increase in pulse width (Martín et al., 1997; Martín-Belloso et al., 1997; Sensoy et al., 1997; Wouters et al., 1999; Aronsson et al., 2001; Abram et al., 2003) (Figure 8.2). However, when microorganisms where subjected to the same treatment time and field strength, microbial inactivation decreased with pulse width (Jayaram et al., 1992; Elez-Martínez et al., 2003a, b). Therefore, short pulses might be considered more effective in destroying microorganisms because a large number of short pulses gave greater microbial inactivation levels than a few long pulses for the same treatment time.

188 Food Safety Aspects of Pulsed Electric Fields

Survival fraction

10.000

1.000 16 pulses 0.100

32 pulses 64 pulses

0.010

0.001 15

20

25

30

35

40

45

50

Field strength (kV/cm) Figure 8.1 Effect of the electric field strength and number of pulses on the inactivation of Escherichia coli in skimmed milk using pulsed electric fields (Martín et al., 1997).

9

0.002 ms, 20 pulses 0.002 ms, 40 pulses

Survival (Log cfu/ml)

8

0.004 ms, 20 pulses

7

0.004 ms, 40 pulses

6 5 4 3 2 1 0 0

25

30

35

E (kV/cm) Figure 8.2 Effect of pulse width on the inactivation of Escherichia coli in a nutritive treatment medium (Aronsson et al., 2001).

PEF may be applied in the form of exponentially decaying, square-wave and oscillatory pulses. Square wave pulses are more efficient than exponentially decaying pulses and oscillatory decay pulses are the least efficient (Qin et al., 1994; Zhang et al., 1994b; Pothakamury et al., 1996) (Figure 8.3). In terms of pulse polarization, bipolar pulses are more efficient than monopolar pulses on the destruction of microorganisms (Qin et al., 1994; Ho et al., 1995; Elez-Martínez, 2003a, b). PEF processing of foods may be carried out in batch mode or as a continuous process. In general, continuous processes reached higher microbial inactivation rates than batch modes (Martín et al., 1997). Microbial inactivation has also been related to the treatment temperature during PEF processing. An increase in PEF treatment temperature leads to higher effectiveness in

Microbiological safety of pulsed electric fields 189

10

Survival fraction

1 0.1 0.01 0.001 0.0001 0.00001

0

200

400

600

800

1000

Treatment time (
s) Figure 8.3 Survival fraction of Saccharomyces cerevisiae after a PEF treatment at 12 kV/cm with exponential decay pulses (◆) and square-wave pulses (▲) (Zhang et al., 1994b).

microbial inactivation (Jayaram et al., 1992; Zhang et al., 1995; Pothakamury et al., 1996; Vega-Mercado, 1996a; Reina et al., 1998; Shin and Pyun, 1999). 2.3.2 Microbial characteristics

The effectiveness of PEF in inactivating microorganisms changes with the type and the growth stage of microorganisms as well as the initial cell concentration. Many publications have demonstrated that yeasts are more sensitive to PEF treatment than vegetative bacteria and, within bacteria, Gram-negative bacteria are more susceptible to treatment (Sale and Hamilton, 1967; Castro et al., 1993; Zhang et al., 1994a; Wouters and Smelt, 1997; Qin et al., 1998; Aronsson et al., 2001). In general, bacterial spores are resistant to PEF treatment, but after germination they become PEF-sensitive (Marquez et al., 1997; Barbosa-Cánovas et al., 1998; Barsotti and Cheftel, 1999). Cells in the logarithmic phase were more sensitive to PEF treatment than those in the stationary phase, as was found by Hülsheger et al. (1983), Gásková et al. (1996), Pothakamury et al. (1996), Álvarez et al. (2000) and Rodrigo et al. (2003) (Figure 8.4). The microbial inactivation rate is also related to the initial microbial concentration. Several authors reported that microbial inactivation is not a function of the initial concentration (Zhang et al., 1995; Álvarez et al., 2000). However, Zhang et al. (1994b, c) reported that initial concentration of the microorganism was inversely correlated with its survival fraction after a PEF treatment. 2.3.3 Product parameters

The inactivation rates of microorganisms increase with decreasing conductivity of the treatment medium for Lactobacillus brevis, Escherichia coli, Saccharomyces cerevisiae, Salmonella dublin and Listeria innocua (Jayaram et al., 1992; Grahl and Märkl, 1996; Sensoy et al., 1997; Wouters et al., 1999). Álvarez et al. (2000) reported that conductivity did not influence the inactivation of Salmonella senftenberg and Gásková et al.

190 Food Safety Aspects of Pulsed Electric Fields

Survival fraction

1 0.1 2 pulses 4 pulses

0.01 Log

Lag

Stationary

0.001 0

1

2

3

4

5

6

Time of growth (h) Figure 8.4 Effects of growth stage on the inactivation of Escherichia coli in SMUF. Samples were subjected to a field strength of 36 kV/cm (Pothakamury et al., 1996).

(1996) found that the inactivation of Saccharomyces cerevisiae was inversely related to the medium conductivity. Therefore, the effect of conductivity on microbial destruction depends on the characteristics of the microorganisms. Evidence of a relationship between the inactivation rate and the ionic strength was verified by Hülsheger et al. (1981) and Vega-Mercado et al. (1996b) who found that decreasing the ionic strength of the medium increased the cell inactivation. The influence of the pH on microbial inactivation by PEF is still unclear. Several authors reported that the pH had no influence (Sale and Hamilton, 1967; Hülsheger et al., 1981). However, more recently, it has been demonstrated that pH plays an important role in microbial inactivation by PEF. Liu et al. (1997), Vega-Mercado et al. (1996b) and Wouters et al. (1999) found that the inactivation was greater at lower pH values for Escherichia coli and Listeria innocua, respectively. On the other hand, Jeantet et al. (1999) and Álvarez et al. (2000) reported that Salmonella enteritidis and Salmonella senftenberg, respectively, were more resistant at low values of pH. Aronsson and Rönner (2001) studied the influence of pH on the inactivation of Escherichia coli and Saccharomyces cerevisiae by pulsed electric fields. They reported that the viability of E. coli declines significantly when pH value drops, but the effect of pH on the PEF resistance of S. cerevisiae was unclear (Figure 8.5). Although the role of water activity in the survival of microorganisms subjected to PEF treatment has not been widely studied, it was found that lowering the water activity protects microorganisms from PEF inactivation (Min and Zhang, 2000; Aronsson and Rönner, 2001) (Figure 8.5). The inactivation of microorganisms by PEF is significantly affected by the temperature of the product at the beginning of the treatment. Increasing the inlet temperature of the product to moderate values significantly enhances the killing effect of PEF (Wouters et al., 1999; Aronsson and Rönner, 2001; Aronsson et al., 2001). Hülsheger et al. (1981) also reported the synergistic effect of the medium temperature with PEF treatment on the inactivation ratio. The influence of the composition of the medium on microbial inactivation by PEF is not well known. While some investigators have reported a protective effect of several food constituents such as xanthan gum (Ho et al., 1995), proteins (Martín et al., 1997) or fats (Grahl and Märkl, 1996), others did not observe differences between microbial

Microbiological safety of pulsed electric fields 191

Inactivation (Log reduction)

5

Water activity 1

0.97

0.94

4 3 2 1 0 4.0

5.0

6.0

7.0

pH Figure 8.5 Effects of pH and water activity on the inactivation of Saccharomyces cerevisiae in a nutritive treatment medium by PEF. PEF treatment conditions: 20 pulses of 4
s and 25 kV/cm (Aronsson and Rönner, 2001).

inactivation in buffers with different components and in a complex medium such as milk (Reina et al., 1998; Dutrueux et al., 2000a; Mañas et al., 2001). The presence of non-conductive large-sized particles or air bubbles markedly decreased the efficacy of processing by PEF on the inactivation of microorganisms (Mañas et al., 2001). However, Dutrueux et al. (2000a) observed that PEF processing does efficiently inactivate bacteria attached to polystyrene beads. Electric resistivity of juices changes with the pulp concentration (Arántegui et al., 1999a). So, as microbial inactivation is influenced by electric resistivity, microbial inactivation could be affected by the pulp concentration of juices.

2.4 Combination of PEF with other hurdles to inactivate microorganisms The microbial inactivation rates achieved by PEF can be improved by combining the electrical treatment with other processes. When moderate heating is applied before or after PEF treatment, higher values of inactivation are achieved. Increasing the initial or inlet temperature before PEF treatment resulted in a greater inactivation level (Wouters et al., 1999; Aronsson and Rönner, 2001; Aronsson et al., 2001; Smith et al., 2002). Fernández-Molina et al. (2000) increased the shelf-life of milk treated by PEF up to 30 days (stored under refrigeration) by applying a mild thermal treatment before the PEF process. Sobrino et al. (2001) observed that the inactivation achieved by PEF could be increased if moderate heating was applied after the PEF process. It has been reported that the combination of PEF with the addition of acetic or propionic acid in skimmed milk can significantly improve the inactivation of L. innocua, but not the inactivation of P. fluorescens (Fernández-Molina, 2001). Moreover, the addition of hydrochloric acid to raw skimmed milk had no effect on the microbial inactivation achieved by PEF treatment (Smith et al., 2002).

192 Food Safety Aspects of Pulsed Electric Fields

Log (C/Co)

⫺3

⫺2

⫺1

0 Nisin 10 (IU/ml)

Nisin 100 (IU/ml)

(a)

PEF

PEF-10 IU PEF-100 IU (nisin/ml) (nisin/ml)

Treatment ⫺4

Log (C/Co)

⫺3 ⫺2 ⫺1 0 Nisin 10 (IU/ml) (b)

Nisin 100 (IU/ml)

PEF

PEF-10 IU PEF-100 IU (nisin/ml) (nisin/ml)

Treatment

Figure 8.6 Effect of the addition of nisin after a PEF treatment of (a) 32 pulses at 30 kV/cm and (b) 32 pulses at 50 kV/cm on the inactivation of Listeria innocua (Calderón-Miranda et al., 1999b).

Antimicrobial agents have been used in order to increase the inactivation rate of PEF processing. Inactivation of E. coli O157:H7 by PEF treatment increased dramatically with the presence of benzoic or sorbic acid (Liu et al., 1997). Some studies reported the synergistic effect of PEF treatment and nisin on the inactivation of certain vegetative microorganisms (Calderón-Miranda, 1999a, b; Pol et al., 2000; Terebiznik et al., 2000; Hodgins et al., 2002; Smith et al., 2002) (Figure 8.6). The synergistic effect of nisin and PEF on the inactivation of Micrococcus luteus was achieved by adding nisin before or after PEF treatment (Dutrueux et al., 2000b). Pol et al. (2000) found that treatment of Bacillus cereus spores with nisin and PEF did not lead to direct inactivation of the spores. The use of PEF treatment in combination with the addition of lysozyme is an effective method for the pasteurization of raw skimmed milk (Smith et al., 2002) and orange juice (Hodgins et al., 2002). The application of high pressure treatments combined with PEF processing enhanced microbial inactivation (Wouters et al., 2001b). The inactivation of vegetative Bacillus subtilis by PEF was improved when the cells were exposed to a high pressure treatment of 200 MPa for 10 minutes (Heinz and Knorr, 2000). However, when Bacillus subtilis spores were subjected to a high pressure treatment of 150 MPa

Microbiological safety of pulsed electric fields 193

for 30 min at 40°C combined with a PEF treatment of 20 pulses at 60 kV/cm, no inactivation of Bacillus subtilis spores was observed (Pagan et al., 1998).

2.5 Modelling the inactivation of microorganisms by PEF Mathematical models that describe the inactivation kinetics of spoilage and pathogenic microorganisms facilitate establishing appropriate PEF process conditions. Microbial inactivation kinetics lead to knowledge of which are the most efficient PEF-processing conditions in order to achieve stable and safe products without over-processing. The numerous critical factors, the broad range of experimental conditions and the diversity of equipment available limit the comparison among results and the drawing of conclusions on kinetics of microbial inactivation (Wouters et al., 2001b). Therefore, it is very important to have a reliable model that accurately describes the rate of cell death and how that rate varies with different environmental conditions (Raso et al., 2000). Models describing microbial inactivation kinetics should be based on reliable experiments. In addition, models should be as simple as possible and based on the understanding of the physiological mechanism of inactivation. In order to study microbial inactivation kinetics, static chambers may be preferable because they avoid the complexity of continuous treatment and simplify the control of critical factors. Finally, the models should be validated in a continuous process and in real food systems (Wouters et al., 2001b). Commonly, when survival curves cover a few log cycles, microbial inactivation by PEF follows a linear inactivation and, if the inactivation is extended for more than 3–4 log cycles non-linear relationships are observed. Several authors have found a linear relationship between the survivor number logarithm and the time of treatment (Mizuno and Hori, 1988; Martín-Belloso et al., 1997; Martín et al., 1997; Sensoy et al., 1997; Reina et al., 1998; Heinz et al., 1999). Other authors have observed a doublelogarithmic relationship between log reductions and treatment time (Qin et al., 1995b; Grahl and Märkl, 1996; Pothakamury et al., 1996). Hülsheger et al. (1981) developed a model to describe microbial inactivation kinetics by PEF. They obtained a mathematical relationship that described the survival curves of different microorganisms assuming a logarithmic dependency between the survival fraction and the electric field strength and a double-logarithmic relation between the survival fraction and the treatment time. Thus, the survival fraction could be expressed according to the electric field strength and the treatment time as is shown below:  E⫺Ec    k 

 t ⫺ s(E, t) ⫽    t c 

(1)

where s is the survival fraction (%), E is the electric field strength, t is the treatment time, tc is the extrapolated critical treatment time value for s equal to 100, Ec is the extrapolated critical field strength value for s equal to 100, and k is the independent constant factor.

194 Food Safety Aspects of Pulsed Electric Fields

A mathematical model based on the Weibull’s distribution (Peleg and Cole, 1998) has also been used to describe the microbial inactivation by PEF as a function of treatment time (Rodrigo et al., 2001, 2003; Álvarez et al., 2003a, b):  t m Log10 s(t) ⫽ ⫺   b 

(2)

where Log10 s(t) is the logarithm of the survival fraction, t is the treatment time and b and m are the scale and shape parameters, respectively. The b parameter is related to the treatment strength and represents the time to reduce the microbial population to the first decimal logarithmic cycle. The m value accounts for upward concavity of a survival curve (m  1), a linear survival curve (m ⫽ 1) and downward concavity (m ⬎ 1) (Van Boekel, 2002). Peleg (1995) also proposed a model based on the Fermi equation that describes the sigmoid form of the survival curve when the number of surviving microorganisms is plotted against the electric field strength that reads as follows: s(E,n) ⫽

100  E ⫺ Vc (n)   1⫹ exp   a(n)   

(3)

where s is the percentage of surviving microorganisms, E is the electric field strength, Vc is a critical value of E where the survival level is 50 per cent and a is a parameter indicating the steepness of the survival curve around Vc. Both parameters, Vc and a, are exponentially related to the number of applied pulses. Some authors have successfully used this model for modelling the inactivation of microorganisms by PEF (Peleg, 1995; Sensoy et al., 1997; Rodrigo et al., 2001). The Log-logistic model described by Cole et al. (1993) was used to fit the survivor curves of Salmonella senftenberg (Raso et al., 2000). The Log-logistic model is justified by a distribution of resistance within the bacterial population. Moreover, concave upward survival curves corresponding to the microbial inactivation by PEF have been analysed with different mathematical modelling approaches by Álvarez et al. (2003a). One model is an extension of the exponential model that assumes that there are two populations of microorganisms which differ on sensitivity to PEF (Pruitt and Kamau, 1993). Another model proposed by Augustin et al. (1998) is based on a distribution of resistances within the bacterial population. The modelling of upward concave curves could also be done by a purely empirical equation (Peleg and Penchina, 2000).

2.6 Effect of PEF on pathogenic microorganisms The most important pathogenic bacteria concerned with food-borne diseases are Clostridium botulinum, Bacillus cereus, Staphylococcus aureus, Salmonella, Campylobacter, Escherichia coli, Listeria monocytogenes, Vibrio cholerae and others like Vibrio parahemoliticus, Vibrio vulnificus, Aeromonas hydrophila and Yersinia enterocolitica (Van Schothorst, 1999). Knowledge of the effect of PEF on pathogenic microorganisms is vital in order to obtain safe products processed by PEF (Table 8.1).

Microbiological safety of pulsed electric fields 195

Table 8.1 Effects of pulsed electric fields on pathogenic microorganisms Microorganism

Media

Treatment conditions*

Microbial reduction (log reductions)

Reference

Escherichia coli

Phosphate buffer (pH ⫽ 6.8)

b

5

Dutrueux et al. (2000a)

Escherichia coli

Citrate-phosphate McIlvaine buffer (pH ⫽ 7.0)

25 kV/cm, 700 pulses, 2 ␮s, 35°C

6

Álvarez et al. (2003a)

Escherichia coli

Nutritive treatment medium (pH ⫽ 7.0)

b b

1.0 2.1

Aronsson and Rönner (2001)

Nutritive treatment medium (pH ⫽ 5)

3.8 6.2

Aronsson et al. (2001)

b

Escherichia coli O157:H7

0.1% NaCl

b

2.9

Unal et al. (2002)

Escherichia coli

SMUF c

a

36 kV/cm, 16 pulses, 2 ␮s, 7°C 36 kV/cm, 8 pulses, 2 ␮s, 20°C a 36 kV/cm, 8 pulses, 2 ␮s, 33°C

2–3 2.5 2.5

Zhang et al. (1995)

a

4 5

Pothakamury et al. (1996)

Escherichia coli

41 kV/cm, 63 pulses, 2.5
s, 37°C

30 kV/cm, 20 pulses, 4 ␮s, 10°C 30 kV/cm, 20 pulses, 4 ␮s, 30°C

b

35 kV/cm, 20 pulses, 2 ␮s, 30°C 35 kV/cm, 20 pulses, 4 ␮s, 30°C

20 kV/cm, 48.5 pulses, 3 ␮s, 35°C

a

Escherichia coli

SMUF c

36 kV/cm, 64 pulses, 7°C 36 kV/cm, 64 pulses, 20°C

a

Escherichia coli

Skimmed milk

a

Escherichia coli

Milk (1.5% fat)

a

Escherichia coli O157:H7

Apple juice

45 kV/cm, 64 pulses, 1.8–6 ␮s, 15°C

3

Martín et al. (1997)

4

Grahl and Märkl (1996)

b

5.0

Evrendilek et al. (1999)

23 kV/cm, 20 pulses, 45–50°C 30 kV/cm, 43 pulses, 4 ␮s, 25°C

Escherichia coli

Pea soup

b

6.5

Vega-Mercado et al. (1996a)

Listeria monocytogenes

Distilled water

b

4

Rusell et al. (2000)

Listeria monocytogenes

0.1% NaCl

b

2.1

Unal et al. (2002)

Listeria innocua

Nutritive treatment medium (pH ⫽ 5)

b

1.0 8.1

Aronsson et al. (2001)

b

Skimmed milk

b

3.9

Dutrueux et al. (2000a)

Listeria innocua

Skimmed milk

b

2.4

Calderón-Miranda et al. (1999b)

Listeria innocua

Liquid whole egg

b

3.5

Calderón-Miranda et al. (1999a)

Listeria monocytogenes

Whole milk

b

30 kV/cm, 400 pulses, 1.5 ␮s, 25°C 30 kV/cm, 400 pulses, 1.5 ␮s, 50°C

2.5 4

Reina et al. (1998)

b

Salmonella typhimurium

Distilled water

a

20 kV/cm, 10000 pulses, 50 ␮s, 40°C

6

Rusell et al. (2000)

Salmonella serovar typhimurium

Citrate-phosphate McIlvaine buffer (pH ⫽ 7.0)

28 kV/cm, 700 pulses, 2 ␮s, 35°C

7

Álvarez et al. (2003b)

Salmonella serovar enteritidis

Citrate-phosphate McIlvaine buffer (pH ⫽ 7.0)

25 kV/cm, 700 pulses, 2 ␮s, 35°C

6

Álvarez et al. (2003b)

Listeria innocua

33 kV/cm, 30 pulses, 2 ␮s, 55°C

20 kV/cm, 10000 pulses, 50 ␮s, 40°C

20 kV/cm, 48.5 pulses, 3 ␮s, 35°C

35 kV/cm, 40 pulses, 2 ␮s, 30°C 35 kV/cm, 40 pulses, 4 ␮s, 30°C

41 kV/cm, 63 pulses, 2.5 ␮s, 37°C

50 kV/cm, 32 pulses, 2 ␮s, 34°C

50 kV/cm, 32 pulses, 2 ␮s, 36°C

(Continued)

196 Food Safety Aspects of Pulsed Electric Fields

Table 8.1 (Continued) Microorganism

Media

Treatment conditions*

Microbial reduction (log reductions)

Reference

Salmonella senftenberg

Citrate-phosphate McIlvaine buffer (pH ⫽ 7.0)

28 kV/cm, 625 pulses, 2
s, 35°C

6.5

Raso et al. (2000)

Salmonella dublin

Skimmed milk

b

25 kV/cm, 100 pulses, 1 ␮s, 20°C 25 kV/cm, 100 pulses, 1 ␮s, 50°C

2.7 4

Sensoy et al. (1997)

b

35 kV/cm, 8 pulses, 30°C

3.5

Jeantet et al. (1999)

1.25

Pol et al. (2000)

Salmonella enteritidis

Egg white

a

Bacillus cereus

Phosphate buffer solution (pH ⫽ 7.0)

a

16.7 kV/cm, 50 pulses, 2 ␮s, 30°C

Bacillus cereus

0.15% NaCl

b

1.3

Cserhalmi et al. (2002)

Bacillus cereus spores

0.15% NaCl

b

25 kV/cm, 8.3 pulses, 2 ␮s, 30°C

0.4

Cserhalmi et al. (2002)

Bacillus cereus spores Bacillus subtilis spores

0.15% NaCl

a

50 kV/cm, 50 pulses, 2 ␮s, 25°C

>5

Marquez et al. (1997)

0.15% NaCl

a

3.4

Marquez et al. (1997)

Bacillus subtilis

SMUFc

a

5

Qin et al. (1994)

Bacillus subtilis

Pea soup

b

33 kV/cm, 30 pulses, 2 ␮s, 55°C

5.3

Vega-Mercado et al. (1996a)

Staphylococcus aureus

Phosphate buffer solution (pH ⫽ 7.0)

a

20 kV/cm, 30 pulses, 36 ␮s

3

Hülsheger et al. (1983)

Staphylococcus aureus

SMUF c

a

6

Zhang et al. (1994a)

Staphylococcus aureus

SMUF c

a

16 kV/cm, 50 pulses, 200–300 ␮s, 30°C

4–5

Pothakamury et al. (1995a)

Pseudomonas aeruginosa

Phosphate buffer solution (pH ⫽ 7.0)

a

3.5

Hülsheger et al. (1983)

Pseudomonas fluorescens

0.1% Peptone solution a10 kV/cm, 10 pulses, 2 ␮s, 20°C

6

Ho et al. (1995)

Yersinia enterocolitica

0.1 M NaCl solution (pH ⫽ 7.2)

a

7

Lubicki and Jayaram (1997)

Clostridium tyrobutyricum endospores

Milk (1.5% fat)

a

0.3

Grahl and Märkl (1996)

25 kV/cm, 8.3 pulses, 2 ␮s, 30°C

50 kV/cm, 30 pulses, 2 ␮s, 25°C 16 kV/cm, 40 pulses, 180 ␮s, 30°C

40 kV/cm, 64 pulses, 3 ␮s, 15°C

20 kV/cm, 30 pulses, 36 ␮s

75 kV, 250 pulses 22.4 kV/cm, 30 pulses

*

Electric field strength, number of pulses, pulse width, treatment temperature. Batch mode PEF treatment. b Continuous mode PEF treatment. c SMUF: simulated milk ultrafiltrate. a

2.6.1 Escherichia coli

The majority of the studies have been carried out on the inactivation of E. coli suspended in phosphate buffer solutions by PEF. Around 5 log reductions were achieved by Dutrueux et al. (2000a) when 63 pulses of 41 kV/cm were applied to E. coli suspended in a phosphate buffer solution. Similar results were reported by Mañas et al.

Microbiological safety of pulsed electric fields 197

(2001) when E. coli was processed with pulses of 1.7
s at 33 kV/cm for 300
s. More than 6 log reductions were observed by Álvarez et al. (2003a) in a citrate phosphate McIlvaine buffer of pH 7.0 inoculated with E. coli after a PEF treatment of 1400
s at 25 kV/cm with pulses of 2
s and 1 Hz of pulse frequency. Increasing the inlet temperature from 10 to 30°C significantly enhances the killing effect of PEF for E. coli suspended in a nutritive treatment medium (Aronsson and Rönner, 2001). E. coli inactivation increased from 3.8 log reductions to 6.2 log reductions when the pulse width was increased from 2 to 4
s for 35 kV/cm and 20 pulses (Aronsson et al., 2001). However, only 2.9 log reductions in E. coli O157:H7 were achieved by Unal et al. (2002) when the microorganism, suspended in a 0.1 per cent NaCl solution, was treated with pulses of 3
s at 20 kV/cm for 145.6
s. Compared to other studies, the inactivation reported by Unal et al. (2002) was lower because of the lesser values of pulse width, electric field strength and treatment time. PEF technology inactivates 4–5 log cycles of E. coli inoculated in simulated milk ultrafiltrate (SMUF) with 50–60 pulses at 16 kV/cm and width of 200–300
s (Pothakamury et al., 1995a). Increasing the temperature of the inoculated SMUF from 7 to 20°C promoted microbial inactivation by PEF treatment (Pothakamury et al., 1996). These results agreed with those obtained by Zhang et al. (1995). Vega-Mercado et al. (1996b) studied the effect of pH and ionic strength of SMUF on the E. coli destruction by PEF. They reported that the inactivation was higher at pH 5.69 than at pH 6.82 when samples were processed at 20–55 kV/cm up to 8 pulses. They also obtained a difference of 2.5 log cycles in the inactivation level between 0.168 M and 0.028 M solutions when up to 32 pulses of 40 kV/cm were applied to the samples. PEF treatments at 45 kV/cm, 64 pulses and 15°C led to a nearly 3 log cycle reduction of E. coli suspended in skimmed milk (Martín et al., 1997). In the same study it was observed that microbial inactivation increased when higher values of pulse width were applied to the sample. The application of 63 pulses at an intensity of 41 kV/cm and at 37°C induced a reduction of 4 log units for E. coli suspended in skimmed milk (Dutrueux et al., 2000a). Grahl and Märkl (1996) reported about 4 log reductions of E. coli in milk (1.5 per cent fat) when 20 pulses of 23 kV/cm were applied. Some studies have been conducted in order to study the effect of fat content in milk on the inactivation of E. coli by PEF and the conclusions were that the simpler the medium composition the higher the inactivation (Grahl and Märkl, 1996; Martín et al., 1997). Evrendilek et al. (1999) observed a 5.0 log cycle reduction of E. coli O157:H7 and a 5.4 log cycle reduction of E. coli 8739, both suspended in apple juice by applying a PEF treatment of 172
s at 30 kV/cm. Moreover, the inactivation of E. coli O157:H7 suspended in apple juice at 34 kV/cm and a total treatment time of 166
s in a benchscale PEF system was 4.5 log cycles (Evrendilek et al., 2000). McDonald et al. (2000) tested the survivability of E. coli suspended in orange juice using a pilot plant PEF system. The results showed that 5–6 log reductions were achieved by six 2-
s pulses per unit of volume at 30 kV/cm. At least four 2-
s pulses per unit of volume were required at 50 kV/cm to inactivate E. coli by 4 logs or more. Because the outlet temperature was near 60°C, the inactivation was mainly attributed to thermal effects. Therefore, a higher electric field does not always produce greater levels of microbial inactivation.

198 Food Safety Aspects of Pulsed Electric Fields

A reduction of 6.5 logs in E. coli suspended in pea soup was obtained at 33 kV/cm, 4.3 Hz and 30 pulses. When inoculated pea soup was treated with 30 pulses at 25 kV/cm and 4.3 Hz, only about 1 log reduction was achieved (Vega-Mercado et al., 1996a). E. coli inactivation was greater when electric field strength and treatment time were increased in all the studies mentioned above. Some researchers have studied the combination of PEF treatment and other factors on the inactivation of E. coli. A synergistic killing effect between PEF and organic acids was observed. The inactivation of E. coli O157:H7 by PEF and benzoic or sorbic acid was investigated by Liu et al. (1997). When the cell suspension of E. coli was treated with 5 pulses at 12.5 kV/cm in the presence of benzoic or sorbic acid, the counts decreased by 5.6 and 4.2 logs, respectively. The effectiveness of combining PEF treatment and nisin on the inactivation of E. coli was studied by Terebiznik et al. (2000). They reported that a 4 log cycle reduction may be accomplished with around 1000 IU/ml (7.15
M) of nisin and 3 pulses of 11.25 kV/cm or 500 IU/ml for 5 pulses of 11.25 kV/cm. 2.6.2 Listeria

In recent years, Listeria has become a major concern for the food industry due to an apparent increase in the incidence of listeriosis (McLaughin, 1987). Listeria monocytogenes suspended in distilled water was inactivated more than 4 log reductions when it was treated at 20 kV/cm for 10000 pulses (Rusell et al., 2000). In phosphate buffer at pH 4.0 and 0.5 S/m at 40°C, a 30 kV/cm PEF treatment at an inlet temperature of 40°C resulted in more than 6.3 log inactivation of Listeria innocua at 49.5°C (Wouters et al., 1999). In the same study, a 3.9 log reduction was achieved with a pulse width of 2
s, whereas a 5.2 log reduction was observed with pulse widths of 3 and 3.9
s. Moreover, a synergistic effect between temperature and PEF inactivation was also observed. After 63 pulses of 2.5
s at 41 kV/cm the reduction observed in L. innocua suspended in a phosphate buffer amounted approximately 3.5 log units (Dutrueux et al., 2000a). Aronsson et al. (2001) studied the effect of PEF on the inactivation of L. innocua suspended in a nutritive treatment medium and they observed a maximum inactivation of 8 logs when PEF treatment was 35 kV/cm with 40 pulses of 4
s. A significant difference in inactivation was observed for different levels of electrical field strength only when a pulse duration of 4
s was used. For example, at 30 kV/cm and 20 pulses, when using 2
s, there was 8.1 log survival; when pulses were extended to 4
s there were 7.0 log of survivals. Electric field intensity of 20 kV/cm and treatment time of 145.6
s inactivated 2.1 log of L. monocytogenes suspended in a 0.1 per cent NaCl (Unal et al., 2002). The inactivation by PEF of L. monocytogenes in milk was studied at different electric field strengths, treatment times, treatment temperatures and fat contents (Reina et al., 1998). The degree of inactivation depended on applied electric field strength, treatment time and treatment temperature, but not on the fat content in milk. The maximum inactivation achieved in inoculated whole milk was around 4 log reductions when processed at 30 kV/cm for 600
s with pulses of 1.5
s at 1700 Hz. A PEF treatment of 63 pulses at 41 kV/cm led to 3.9 log reductions in L. innocua suspended in skimmed milk (Dutrueux et al., 2000a). The highest inactivation of L. innocua suspended in skimmed milk obtained with PEF was 2.4 logs for an electric field strength of 50 kV/cm and 32 pulses (Calderón-Miranda et al., 1999b).

Microbiological safety of pulsed electric fields 199

When liquid whole egg was inoculated with L. innocua and processed at 5 kV/cm and 32 pulses, the reduction on the microbial population was 3.5 logs (CalderónMiranda et al., 1999a). At 30 kV/cm and 2-
s pulse width, a 5 to 6 log reduction of L. innocua suspended in orange juice was achieved by 6 pulses per volume and at 50 kV/cm, at least 4 pulses per volume were required to reduce L. innocua by 4 logs or more (McDonald et al., 2000). Calderón-Miranda et al. (1999a, b) reported the effect of PEF combined with nisin on the inactivation of L. innocua suspended in skimmed milk and in liquid whole egg. The obtained inactivation of L. innocua suspended in skimmed milk for an electric field intensity of 50 kV/cm and 32 pulses followed by exposure of the microorganism to nisin was 3.4 and 3.8 log units for 10 and 100 IU nisin/ml, respectively. When L. innocua was suspended in liquid whole egg, the inactivation increased from 4.1 to 5.5 logs when the concentration of nisin was increased from 10 to 100 IU/ml for a PEF treatment of 32 pulses at 50 kV/cm. In both skimmed milk and liquid whole egg, a synergistic effect on the inactivation of L. innocua as a result of the two preservation factors was observed when the electric field intensity, number of pulses and nisin concentration increased. 2.6.3 Salmonella

Salmonella is a well-known food-borne pathogen that frequently causes food poisoning outbreaks (Wheeler et al., 1999). After a PEF treatment of 10 000 pulses of 50
s at 20 kV/cm, 6-log cycle reductions in the viable cell counts of S. typhimurium inoculated in distilled water were reported by Rusell et al. (2000). About 7 log reductions in Salmonella serovar typhimurium suspended in citrate-phosphate McIlvaine buffer were achieved when samples were processed for 1400
s at 28 kV/cm (2-
s pulse width and 1 Hz) without exceeding 35°C (Álvarez et al., 2003b). Therefore, more time is needed to inactivate S. typhimurium when the electric field strength is diminished. Álvarez et al. (2003b) also observed about 6 log reductions in Salmonella serovar enteritidis inoculated in citrate-phosphate McIlvaine buffer after a treatment of 1400
s at 25 kV/cm and temperature below 35°C. A treatment of 28 kV/cm for 1250
s (2-
s pulse width and 1 Hz) led to the inactivation of about 6.5 log of S. senftenberg in a McIlvaine buffer (Raso et al., 2000). Álvarez et al. (2000) and Raso et al. (2000) studied the effect of different factors of PEF processing on the inactivation of S. senftenberg. They reported that after the same treatment time, inactivation of S. senftenberg depended neither on pulse width (1–15
s) nor frequency of treatment (1–5 Hz). They also observed that conductivity did not influence S. senftenberg inactivation at the same electric field strength. Microbial inactivation was not a function of the initial cell concentration and the inactivation was higher at neutral than acidic pH. Sensoy et al. (1997) studied the effect of PEF processing on the inactivation of S. dublin suspended in skimmed milk. About 4 log reductions were achieved when inoculated skimmed milk was processed at 35 kV/cm for 163.9
s with pulses of 1
s and 2000 Hz. Increasing treatment temperature from 30 to 50°C, produced an increase in microbial inactivation from 1 to 2 log reductions (25 kV/cm, 100
s). PEF was applied to destroy Salmonella enteritidis in diaultrafiltered egg white by Jeantet et al. (1999). They reported about 3.5 log reductions when egg white was processed at 35 kV/cm with 8 pulses. These authors also studied the effect of some

200 Food Safety Aspects of Pulsed Electric Fields

parameters such as electric field strength, pulse frequency, pulse number, temperature, pH and inoculum size on microbial inactivation. They concluded that the electric field strength was the dominant factor and pulse number, temperature and pH had also significant positive effects but to a lesser extent. 2.6.4 Bacillus

The destruction of Bacillus spp. is an important goal for food processors because of their capability of producing food spoilage and inducing food-borne diseases (Choudhery and Mikolajcik, 1980; Hassan and Nabbut, 1995; Langeveld et al., 1996). About 1.25 log reductions on vegetative cells of B. cereus in a phosphate buffer solution were achieved after a PEF treatment of 16.7 kV/cm for 100
s with 2-
s pulse width (Pol et al., 2000). Maximum reduction of only 1.3 log cycles was achieved in vegetative cells of B. cereus suspended in 0.15 per cent NaCl at 25 kV/cm using 8.3 pulses of 2
s. An electric field strength of 25 kV/cm and 8.3 pulses was practically ineffective on B. cereus spores suspended in 0.15 per cent NaCl (Cserhalmi et al., 2002). The B. cereus spores were resistant to the application of PEF as has been reported by Grahl et al. (1992). These results are the opposite to the observation of Marquez et al. (1997), who obtained a reduction greater than five log cycles in the number of B. cereus spores using 50 pulses of 50 kV/cm electric field intensity. The contradictory results could be explained by the different electric field intensity. When spores of B. subtilis suspended in 0.15 per cent NaCl solution were treated with 30 pulses at 50 kV/cm, 3.4 log reductions were achieved (Marquez et al., 1997). A PEF treatment of 16 kV/cm and 40 pulses of 180
s in bipolar mode led to more than 5 log reductions in B. subtilis suspended in skimmed milk ultrafiltrate (Qin et al., 1994). Moreover, compared with monopolar pulses, bipolar PEF provided more efficient inactivation of B. subtilis. Vega-Mercado et al. (1996a) reported the inactivation by PEF of B. subtilis in pea soup and observed that 5.3 log reductions were achieved when samples were subjected to 33 kV/cm at 4.3 Hz and 30 pulses. These authors also reported that the inactivation of B. subtilis suspended in pea soup increased with increases in intensity of the electric field, number of pulses and pulsing rate. A synergistic effect of PEF and nisin on the inactivation of B. cereus was reported by Pol et al. (2000). More than 3.5 log reductions were achieved in vegetative cells of B. cereus inoculated in a phosphate buffer solution after the combination of PEF treatment (16.7 kV/cm, 50 pulses each of 2-
s duration) and nisin (0.06
g/ml). 2.6.5 Other pathogenic microorganisms

Other microorganisms such as Staphylococcus aureus, Pseudonomas, Yersinia and Clostridium are food-poisoning microorganisms which must be taken into consideration for adequate effectiveness of PEF processing. More than three log reductions were achieved in Staphylococcus aureus suspended in a phosphate buffer solution (pH ⫽ 7.0) when samples were processed with 30 pulses of 20 kV/cm (Hülsheger et al., 1983). Some studies have been published about the effect of PEF processing on the inactivation of S. aureus in simulated milk ultrafiltrate (SMUF). Six log cycles of reduction in S. aureus could be achieved with 64 pulses of

Microbiological safety of pulsed electric fields 201

3
s duration at 40 kV/cm and 15°C (Zhang et al., 1994a). S. aureus was inactivated by about 4–5 log cycles with a field strength of 16 kV/cm and 50 pulses (Pothakamury et al., 1995a). Therefore, higher field intensity and/or longer treatment time increased inactivation of S. aureus (Pothakamury et al., 1997; Qin et al., 1998). After a PEF treatment of 30 pulses at 20 kV/cm, about 3.5 log reductions were achieved in Pseudomonas aeruginosa suspended in a phosphate buffer solution (Hülsheger et al., 1983). A pulse electric field strength of 10 kV/cm at 2-second intervals for 10 pulses was more than enough to achieve a P. fluorescens reduction of more than 6 log cycles when microorganism was suspended in 0.1 per cent peptone solution (Ho et al., 1995). In the same study, Ho et al. (1995) reported that the critical field strength required for cell lysis was reduced by inducing additional stress to the cell membrane by bipolar pulses in sodium chloride solutions or the osmotic pressure in sucrose solutions. The critical electric field strength required for cell lysis could also be higher by the formation of a protective layer for the cells, as in the case of xanthan gum solutions. Grahl and Märkl (1996) observed that the inactivation of P. fluorescens suspended both in solution of sodium-alginate and UHT milk (1.5 per cent fat) was greater when electric field strength and treatment time were increased. About 7 log reductions were achieved in Yersinia enterocolitica inoculated in a NaCl solution (pH ⫽ 7.2) after a treatment of 250 pulses at 75 kV and 1 Hz of frequency. Moreover, the lethal effect on the bacteria strongly depended on the peak voltage and the number of pulses applied (Lubicki and Jayaram, 1997). Grahl and Märkl (1996) studied the effect of PEF on the inactivation of endospores of Clostridium tyrobutyricum. They observed that the extent of inactivation of endospores of C. tyrobutyricum by PEF was negligible.

2.7 Effect of PEF on spoilage microorganisms The main objective of any food preservation process is to inactivate pathogenic or spoilage microorganisms or slow down their development under controlled conditions (Rodrigo et al., 2001). Therefore, one of the major concerns of food preservation by PEF technology is the study of the effect of PEF on the inactivation of spoilage microorganisms (Table 8.2). Most of the reported information has been focused on microorganisms which cause the spoilage of food such as juices and milk. 2.7.1 Lactobacillus

Lactobacillus is a major putrefactive bacterium, especially in acidic products (Shin and Pyun, 1997). Jayaram et al. (1992) studied the effects of PEF treatments on L. brevis suspended in phosphate buffer solution and observed that L. brevis survivability decreased with field strength and treatment time. They achieved a maximum inactivation near to 9 log reductions by exposing the medium to a field strength of 25 kV/cm for 10 ms with monopolar pulses, but temperature increased to 60°C. When L. plantarum suspended in a phosphate buffer solution was treated at 80 kV/cm for 1000 ␮s (300 Hz of frequency) and 50°C, around 5.5 log reductions were achieved, but if the processing temperature was 30°C, around 2.5 log reductions were obtained (Shin and Pyun, 1999). Therefore, an increase in PEF treatment temperature led to higher

202 Food Safety Aspects of Pulsed Electric Fields

Table 8.2 Effects of pulsed electric fields on spoilage microorganisms Microorganism

Media

Treatment conditions*

Microbial reduction (log reductions)

Reference

Lactobacillus brevis

Phosphate buffer solution (pH ⫽ 7.1)

a

9

Jayaram et al. (1992)

Lactobacillus plantarum

Phosphate buffer solution (pH ⫽ 7.0)

a

2.5 5.5

Shin and Pyun (1999)

Lactobacillus plantarum

Phosphate buffer solution (pH ⫽ 4.5)

b

4.4

Abram et al. (2003)

Lactobacillus plantarum

0.6% peptone water

b

3.5

Rodrigo et al. (2003)

Lactobacillus leichmannii

0.1% NaCl

b

1.6

Unal et al. (2002)

Lactobacillus brevis

Orange juice

b

5.8

Elez-Martínez et al. (2003a)

Lactobacillus plantarum

Orange-carrot juice

b

35.8 kV/cm, 46.3 ␮s

2.5

Rodrigo et al. (2001)

Lactobacillus brevis

Peach juice

a

32.5 kV/cm, 16 pulses, 4 ␮s, 20°C

2.7

Arántegui et al. (2000)

Saccharomyces cerevisiae

Nutritive treatment medium (pH ⫽ 5)

b

30 kV/cm, 40 pulses, 2 ␮s, 30°C 30 kV/cm, 40 pulses, 4 ␮s, 30°C

4.5 6.4

Aronsson et al. (2001)

Saccharomyces cerevisiae

Apple juice

a

25 kV/cm, 30 pulses, 25°C

3.8

Zhang et al. (1994c)

Saccharomyces cerevisiae

Apple juice

a

5.5

Zhang et al. (1994a)

Saccharomyces cerevisiae

Apple juice

b

6

Qin et al. (1995b)

Saccharomyces cerevisiae

Apple juice

b

20 kV/cm, 10.4 pulses, 2 ␮s, 30°C

4

Cserhalmi et al. (2002)

Saccharomyces cerevisiae

Orange juice

a

7 kV/cm, 5 pulses, 50°C

4.9

Grahl and Märkl (1996)

Saccharomyces cerevisiae

Orange juice

b

5.1

Elez-Martínez et al. (2003b)

Zygosaccharomyces bailii

Apple juice Orange juice Grape juice Pineapple juice Cranberry juice

b

32.3 kV/cm, 2 pulses, 2.5 ␮s, 19°C 34.3 kV/cm, 2 pulses, 2.0 ␮s, 20°C b 35.0 kV/cm, 2 pulses, 2.3 ␮s, 20°C b 33.0 kV/cm, 2 pulses, 2.2 ␮s, 20°C b 36.5 kV/cm, 2 pulses, 3.3 ␮s, 22°C

4.8 4.7 5.0 4.3 4.6

Raso et al. (1998a)

Zygosaccharomyces bailii ascospores

Apple juice Orange juice Grape juice Pineapple juice Cranberry juice

b

32.3 kV/cm, 2 pulses, 2.5 ␮s, 19°C 34.3 kV/cm, 2 pulses, 2.0 ␮s, 20°C b 35.0 kV/cm, 2 pulses, 2.3 ␮s, 20°C b 33.0 kV/cm, 2 pulses, 2.2 ␮s, 20°C b 36.5 kV/cm, 2 pulses, 3.3 ␮s, 22°C

3.6 3.8 3.5 3.4 4.2

Raso et al. (1998a)

Byssochlamys fulva conidiospores

Tomato juice Apple juice Orange juice Pineapple juice Grape juice Cranberry juice

b

30 kV/cm, 15 pulses, 2 ␮s, 23°C 32.3 kV/cm, 15 pulses, 2.5 ␮s, 23°C b 34.3 kV/cm, 15 pulses, 2.0 ␮s, 20°C b 33.0 kV/cm, 15 pulses, 2.2 ␮s, 20°C b 35.0 kV/cm, 8 pulses, 2.3 ␮s, 20°C b 36.5 kV/cm, 2 pulses, 3.3 ␮s, 22°C

3.8 5.1 5 5.5 4.9 5.9

Raso et al. (1998b)

*

25 kV/cm, 217.4 pulses, 46
s, 60°C

80 kV/cm, 1000 pulses, 1 ␮s, 30°C 80 kV/cm, 1000 pulses, 1 ␮s, 50°C

a

25 kV/cm, 9.6 pulses, 5 ␮s, 37°C 25 kV/cm, 64 pulses, 2.5 ␮s, 35°C 20 kV/cm, 48.5 pulses, 3 ␮s, 35°C 35 kV/cm, 250 pulses, 4 ␮s, 40°C

b

40 kV/cm, 65 pulses, 3 ␮s, 15°C 35 kV/cm, 10 pulses, 2.5 ␮s, 34°C

35 kV/cm, 250 pulses, 4 ␮s, 39°C

b

b

b

Electric field strength, number of pulses, pulse width, treatment temperature. Batch mode PEF treatment. b Continuous mode PEF treatment. a

Microbiological safety of pulsed electric fields 203

levels of L. plantarum inactivation. Abram et al. (2003) achieved 4.4 log reductions when L. plantarum suspended in a sodium phosphate buffer was treated at 25 kV/cm for 48
s with pulses of 5
s and 400 Hz at 37°C. The differences between inactivation could be attributed to the differences in treatment conditions and equipment. As Shin and Pyun (1999) reported, Abram et al. (2003) observed the same pattern on the evolution of microbial inactivation with PEF processing temperature. Grahl and Märkl (1996) reported that the death of L. brevis suspended in sodium alginate processed by HIPEF was greater when electric field strength and treatment time were increased. Other microorganisms belonging to the same genus (L. plantarum, L. leichmannii) suspended in different media (phosphate buffer solution, peptone water, NaCl solution) showed similar patterns when subjected to HIPEF-processing: the higher the field strength and the treatment time, the higher the microbial inactivation reached (Shin and Pyun, 1999; Unal et al., 2002; Rodrigo et al., 2003). The inactivation of L. plantarum in orange-carrot juice reached a maximum of 2.5 log reductions when treated at 35.8 kV/cm for 46.3
s (Rodrigo et al., 2001). ElezMartínez et al. (2003a) reported the effect of PEF on the inactivation of L. brevis suspended in orange juice. A maximum inactivation of 5.8 log reductions was achieved when L. brevis was treated for 1000
s (4
s-pulse width) at 35 kV/cm and 200 Hz in bipolar mode without exceeding 40°C. The effectiveness of PEF treatments depended on the electrical conditions such as electric field strength, treatment time, pulse frequency, width and polarity. The higher the electric field strength and the treatment time, the greater the L. brevis destruction. Moreover, L. brevis inactivation increased when the pulse frequency and the pulse width decreased. The inactivation of L. brevis in peach juice was studied by Arántegui et al. (2000). The study of the effects of PEF on L. brevis was performed by applying up to 16 pulses of 4
s width and an electric field strength between 20 and 32.5 kV/cm. All the experiments were carried out at temperatures lower than 20°C. The destruction of L. brevis in peach juice and in peach purée was 2 to 2.7 log reductions. The inactivation of L. brevis suspended in UHT milk (1.5 per cent fat) was greater when the electric field strength and treatment time were increased (Grahl and Märkl, 1996). 2.7.2 Saccharomyces

Although Saccharomyces are the yeasts most used in the production of foods and drinks, they can also grow in food leading to its spoilage. When S. cerevisiae was inoculated in a nutritive treatment medium and treated for 80
s at 30 kV/cm with pulses of 4
s at 30°C, about 6 log reductions were reached (Aronsson et al., 2001). These authors also observed that raising the pulse duration from 2 to 4
s at 25 kV/cm and 20 applied pulses resulted in a reduction in viable counts from 5.1 to 1.4 log. Grahl and Märkl (1996) reported that the inactivation of S. cerevisiae suspended in a sodium alginate solution was greater when the electric field strength and treatment time were increased. Some studies on the effect of PEF on S. cerevisiae suspended in apple juice have been found in the literature. Zhang et al. (1994c) achieved 3.8 log reductions in S. cerevisiae when apple juice was processed with 30 pulses of 25 kV/cm. In another study by Zhang et al. (1994a), a treatment at 40 kV/cm for 195
s at 15°C led to about 5.5 log reductions in S. cerevisiae. Qin et al. (1995b) obtained 6 log reductions in

204 Food Safety Aspects of Pulsed Electric Fields

S. cerevisiae inoculated in apple juice after a treatment of 35 kV/cm for 25
s at 34°C. Using 10.4 pulses of 2-
s pulse width (20.8
s total treatment time) and 20 kV/cm at 30°C, the treatment resulted in approximately 4 log cycles reduction in S. cerevisiae (Cserhalmi et al., 2002). The survival fraction of S. cerevisiae in apple juice treated by square-wave PEF was significantly smaller than that corresponding to a treatment by exponential decay pulses with equivalent electrical energy input (Zhang et al., 1994b). PEF processing produced less than 1 log reduction of ascospores of S. cerevisiae suspended in orange juice until temperatures greater than 60°C were reached, by applying three 2-
s pulses at 50 kV/cm (McDonald et al., 2000). However, the presence of S. cerevisiae in orange juice was reduced by more than 4.9 log cycles when 5 pulses at 7 kV/cm and 50°C were applied (Grahl and Märkl, 1996). Elez-Martínez et al. (2003b) studied the effect of PEF on the inactivation of S. cerevisiae suspended in orange juice. They reported a maximum inactivation of 5.1 log reductions after the exposure of S. cerevisiae to 1000
s (4
s-pulse width) at 35 kV/cm and 200 Hz in bipolar mode at 39°C and reported that electric pulses applied in bipolar mode were more effective than in monopolar mode in destroying S. cerevisiae. A study on inactivation of S. cerevisiae suspended in a model solution of peach juice by PEF was carried out by applying 0.02and 0.04-ms pulses at electrical field strength of 5, 5.6 and 6.2 kV/cm. The obtained destruction was 3 log reductions after 20 pulses at temperatures below 20°C during PEF treatment (Arántegui et al., 1999b). As was observed in juices, the inactivation of S. cerevisiae suspended in UHT milk (1.5 per cent fat) was greater when the electric field strength and treatment time were increased (Grahl and Märkl, 1996). 2.7.3 Other spoilage microorganisms

The low pH of some foods enables the growth of yeast, moulds and a few groups of aciduric bacteria (Splittstoesser, 1987). Zygosaccharomyces bailii inoculated in orange juice suffered a reduction of 4.7 logs for vegetative cells and 3.8 logs for the ascospores, after a treatment of 34.3 kV/cm for 4
s (Raso et al., 1998a). PEF treatment did not affect the ascospores of Byssochlamys nivea and Neosartorya fischer. Nevertheless, 1.5 log reductions after a treatment of 4
s at 34.3 kV/cm were achieved in conidiospores of Byssochlamys fulva suspended in orange juice (Raso et al., 1998b). The destruction of Z. bailii in apple juice by a PEF treatment of 5
s at 32.3 kV/cm was 4.8 logs for vegetative cells and 3.6 logs in the case of their ascospores when they are inoculated in apple juice (Raso et al., 1998a). In contrast, PEF treatment did not affect the ascospores of B. nivea and N. fischer; but the conidiospores of B. fulva were reduced by 1.5 logs by applying 2 pulses of 2.5
s at 32.3 kV/cm (Raso et al., 1998b). Raso et al. (1998a) studied the effect of PEF on the inactivation of Z. bailii suspended in grape, pineapple and cranberry juices. The resistance of vegetative cells of Z. bailii was similar to their ascospores. For a treatment of 4.4
s at 33 kV/cm in pineapple juice, reductions of 4.3 log of vegetative cells and 3.4 log of ascospores were obtained. After 4.6
s at 35 kV/cm, a destruction of 5 log for vegetative cells and 3.5 log for ascospores was found in grape juice. An inactivation of 4.6 log for vegetative cells and 4.2 log for ascospores was observed after a treatment of 6.6
s at 36.5 kV/cm in cranberry juice.

Microbiological safety of pulsed electric fields 205

Raso et al. (1998b) observed that ascospores of B. nivea and N. fischer were resistant to PEF treatment of 40 pulses at 51 kV/cm. A hierarchy of effectiveness was found for conidiospores of B. fulva in various juices. This hierarchy was cranberry, grape, pineapple, orange, apple and tomato juice. The inactivation in cranberry juice was almost 6 log cycles after 2 pulses of 6.6
s width at 36.5 kV/cm. However, in tomato juice, the population of conidiospores of B. fulva decreased less than 1 log cycle after a treatment of 4
s at 30 kV/cm.

2.8 Shelf-life of foods processed by PEF Information about the shelf-life of foods processed by PEF is limited. Table 8.3 shows the shelf-life of different products such as apple juice, raw skimmed milk, beaten eggs and green pea soup treated at different PEF processing conditions and different temperatures of storage. The shelf-life of refrigerated foods processed by PEF ranged from 10 days for green pea soup to 21 days for apple juice. The effects of PEF on the shelf-life of orange juice have been extensively studied. Qiu et al. (1998) applied pulses with three different shapes: square, exponential decay and underdamped RLC waveform. Square pulses were the most effective shape, with up to 1 year of shelf-life under refrigerated conditions and a reduction of more than 4 log cycles for yeasts and aerobic plate counts in orange juice after a treatment of 60
s at 35 kV/cm. It has been demonstrated that 480
s of PEF at 30 kV/cm was effective in reducing the total plate count of orange juice and, therefore, in extending its shelf-life, but it was not as effective as the heat process of 90°C for 1 minute (Jia et al., 1999). However, a PEF treatment at 35 kV/cm for 59
s produced a large destruction of total plate counts and mould and yeast counts (7 logs of cfu/ml) (Yeom et al., 2000b). Therefore, PEF treatment at 35 kV/cm and 59
s as well as a heat pasteurization at 94.6°C for 30 seconds kept the number of microorganisms in the orange juice at 1 log cfu/ml at 4, 22 and 37°C for 112 days (Yeom et al., 2000a; Ayhan et al., 2002). Optimal conditions consisting of 20 pulses of an electric field of 80 kV/cm at pH 3.5 and a temperature of 44°C with 100 U nisin/ml resulted in over a 6-log cycle reduction in the microbial population. The microbial shelf-life of the orange juice was also improved and determined to be at least 28 days when stored at 4°C without aseptic packaging Table 8.3 Shelf-life of food products processed by pulsed electric fields (Qin et al., 1995a) Food

Apple juice from concentrate

Fresh apple juice

Raw skimmed milk

Beaten eggs

Green pea

Peak electric field (kV/cm) Pulse duration (
s) Pulse number Initial temperature (°C) Maximum treatment temperature (°C) Storage temperature (°C) Shelf-life (days)

50 2 10 8.5  1.5 45  5

50 2 16 8.5  1.5 45  5

40 2 20 10.0  1.5 50  4

35 2 10 8.5  1.5 45  5

35 2 32 22.0  2.0 53  2

22–25 28

4–6 21

4–6 14

4–6 28

4–6 10

206 Food Safety Aspects of Pulsed Electric Fields

(Hodgins et al., 2002). Min et al. (2003a) studied the shelf-life of orange juice processed with commercial-scale pulsed electric field equipment compared with thermal treatment. After thermal processing (90°C for 90 s) or PEF processing (40 kV/cm, pulse duration of 2.6 ms and total treatment time of 97 ms), the total aerobic plate count and the yeast and mould count were always less than 10 cfu/ml. Both thermally processed and PEF processed orange juices maintained the microbial counts at less than 1 log at 4°C for 112 days. When apple juice was treated with ten 2.5
s pulses at 36 kV/cm, over 3 weeks of shelf-life extension were observed at both 4 and 25°C storage (Qin et al., 1995b). Moreover, the shelf-life of fresh apple juice was extended by a few days after a PEF treatment, in comparison to untreated juice (Cserhalmi and Czukor, 1999). The shelflife of PEF-processed reconstituted apple juice at 35 kV/cm and 94
s was more than 67 days at 4°C, approximately 67 days at 22°C and 14 days at 37°C (Evrendilek et al., 2000). Likewise, the shelf-life of apple juice from concentrate stored at 22–25°C was over 4 weeks after applying 10 pulses of 2
s at 50 kV/cm (Qin et al., 1995a). The shelf-life of apple cider treated at 35 kV/cm for 94
s followed by a heat treatment of 60°C for 30 s was over 67 days at 4°C, whereas the samples processed only by PEF had a shorter shelf-life (Evrendilek et al., 2000). Cranberry juice treated with PEF at 32 kV/cm stored at 4, 22 and 37°C contained less than 100 cfu/ml of bacteria, yeast and mould after 5 weeks, but the controls reached more than 1 000 000 cfu/ml after 2 weeks. In addition, treatment with only PEF at 32 kV/cm for 47
s, or combined with heat at 60°C for 32 seconds, extended the shelf-life of the cranberry juice samples stored at 22 and 37°C (Jin et al., 1998). No significant differences in the means of the total aerobic bacteria count and total yeast and mould counts of cranberry juice were observed when juice was treated by PEF or PEF combined with heat (Evrendilek et al., 2001). Min et al. (2003b) studied the shelf-life of tomato juice processed by PEF compared to thermal treatment. Tomato juice was thermally processed at 92°C for 90 s or PEF processed at 40 kV/cm for 57
s and stored at 4°C. The total aerobic plate count and the yeast and mould count were evaluated through 112 days of storage. Both thermally and PEF processed juices showed microbial shelf-life of 112 days at 4°C. The shelf-life of raw skimmed milk stored at 4–6°C was about 14 days when milk was processed by PEF at 40 kV/cm with 20 pulses of 2
s (Qin et al., 1995a). FernándezMolina et al. (2000) increased the shelf-life of milk treated by PEF up to 30 days (stored refrigerated) by applying a mild thermal treatment (73°C for 6 s) before the PEF process (50 kV/cm for 60
s).

3 Chemical safety and PEF Little information exists about PEF related to chemical safety and only a few studies about electrochemical reactions and electrode corrosion in PEF treatment chambers have been reported. Although the problems of electrochemical reactions and corrosion are well known in other application fields, very little attention has been paid to the occurrence of these problems related to food processing with PEF treatment systems.

Conclusions 207

One of the advantages of the PEF technique in comparison with the application of DC-current seems that it did not lead to the contamination of the food with residual products caused by electrolysis and food disintegration. However, contamination could also occur in PEF food treatment systems. Large currents have to pass the electrode– liquid interface in a PEF treatment chamber. These currents can cause electrochemical reactions at the electrode–solution interface, which may have consequences in three fields, namely food quality, electrode fouling and electrode corrosion (life time) (Morren et al., 2003). With respect to food quality the electrode reactions can cause changes to the chemical structure of the liquids in the vicinity of the electrode surfaces, produce toxic chemicals (mostly H2O2) and introduce small particles of electrode material in the liquid (Jayaram, 2000). Reaction products that are formed can react with other compounds in the liquid. The problem of electrochemical reactions in PEF food treatment systems has been reported by several researchers whose studies were focused on the production of toxic components during PEF processing (Sale and Hamilton, 1967; Hülsheger and Niemann, 1980; Lubicki and Jayaram, 1997; Jayaram, 2000). Jayaram (2000) reported that toxic compounds can be formed in PEF processing. He observed that hydrochloric and hypochlorous acid can be formed as a secondary reaction of chlorine with water. Hülsheger and Niemann (1980) reported that solutions containing chloride compounds led to the undesired electrolytic production of chlorine, causing the treated solutions to be toxic even after pulse treatment has been completed. Therefore, they advise to use solutions that do not contain chlorides, but phosphate or sulphate, in which no toxic chemicals were observed (Hülsheger et al., 1981). Another toxic action may arise from the reaction of oxygen or direct anodal treatment of cell proteins. This also has to be considered for solutions free of chlorides and leaves the medium without remaining toxicity (Hülsheger and Niemann, 1980). A very low amount of electrolysis product was generated during the PEF treatment of S. cerevisiae suspended in a 0.9 per cent NaCl solution at 35 kV/cm (Jacob et al., 1981). In order to limit the electrochemical reactions and their effects on the quality of food treated by PEF, it is necessary for the pulse duration be short. It is possible to reduce the formation of electrochemical reactions by using steep front short duration pulses (5–10 ns rise time and 20–30 ns duration); however, it has not been demonstrated that all electrode phenomena due to discharges in the fluid are fully eliminated (Lubicki and Jayaram, 1997). Electrolysis does not take place when PEF were applied in bipolar mode (Bruhn et al., 1997). Corrosion of the electrodes can be limited by applying very short pulses. Cumulative build-up of charge must be avoided. This can be done either by removing the charge after each pulse or by applying bipolar pulses. It could be seen that below a certain critical frequency (and above a certain pulse width) different elements of the stainless steel electrodes appeared in the liquid (Morren et al., 2003).

4 Conclusions PEF treatment is a non-thermal preservation technique which is capable of guaranteeing food safety. PEF processing has good prospects for being used by the food industry, since

208 Food Safety Aspects of Pulsed Electric Fields

promising results have been obtained on the inactivation of undesirable microorganisms when processing different foodstuffs. Moreover, chemical safety problems in PEFtreated foods seem to be limited. The use of this technology in combination with other non-thermal technologies or mild heating would increase the microbial destruction. Further investigation is needed to achieve higher levels of microbial inactivation, to learn more about the chemical reactions during processing and to achieve more precise control of all the critical points involved. Finally, to introduce this technique successfully to the food industry, systems of higher rates of production with identical or improved effectiveness must be designed.

Nomenclature a b C C0 E Ec EC f k M m n PEF s SMUF T t tc Vc

steepness of survival curve around Vc scale parameter (
s) microorganism concentration initial microorganism concentration electric field strength (kV/cm) critical field strength (kV/cm) electric conductivity (S/m) frequency (Hz) independent constant factor molarity (M) shape parameter number of pulses pulsed electric fields survival fraction simulated milk ultrafiltrate temperature (°C) treatment time (
s) critical treatment time (
s) critical value of E where survival level is 50 per cent (kV/cm)

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Developments in Osmotic Dehydration N K Rastogi1, K S M S Raghavarao1 and K Niranjan2 1

Department of Food Engineering, Central Food Technological Research Institute, Mysore, India 2 School of Food Biosciences, The University of Reading, Reading, Berkshire, UK

Due to energy and quality related advantages, the technique of osmotic dehydration is gaining popularity as a complementary processing step in the chain of integrated food processing. Generally, osmotic dehydration is inherently slow and there is a need to find ways of increasing mass transfer rates without adversely affecting quality. Various methods, such as the application of high hydrostatic pressure, high electrical field pulses, gamma irradiation, ultrasound, vacuum and centrifugal force have been attempted. Equations to estimate the diffusion coefficient in various foods having different sizes and geometry are discussed. The use of osmotic pretreatment in various food processing operations such as drying, freezing, rehydration, frying, jam manufacturing is reviewed. Certain limitations, which restrict its wider industrial use, are also discussed.

1 Introduction Osmotic dehydration is an operation used for the partial removal of water from plant tissues by immersion in a hypertonic (osmotic) solution. Water removal is based on the natural and non-destructive phenomenon of osmosis across cell membranes. The driving force for the diffusion of water from the tissue into the solution is provided by the higher osmotic pressure of the hypertonic solution. The diffusion of water is accompanied by the simultaneous counter diffusion of solutes from the osmotic solution into the tissue. Since the cell membrane responsible for osmotic transport is not perfectly selective, solutes present in the cells (organic acids, reducing sugars, minerals, flavours and pigment compounds) can also be leached into the osmotic solution (Dixon and Jen, 1977; Lerici et al., 1985; Giangiacomo et al., 1987), which affect the organoleptic and nutritional characteristics of the product. The rate of diffusion of water from any material made up of such tissues depends upon factors such as temperature and concentration of the osmotic solution, the size and geometry of the material, the Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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222 Developments in Osmotic Dehydration

solution-to-material mass ratio and, to a certain level, agitation of the solution. A number of publications have described the influence of these variables on mass transfer rates during osmotic dehydration (Raoult-Wack et al., 1992; Torreggiani, 1993; RaoultWack, 1994; Rastogi and Raghavarao, 1994, 1995, 1997a, b, 2004a; Rastogi et al., 1997, 2002). It is worth noting that these variables can only be varied over a narrow range, outside of which they adversely affect quality even though they enhance mass transfer rates. Hence, there is a need to identify methods that increase the mass transfer rates but which do not affect the quality significantly. Mass transfer during osmotic treatment occurs through semipermeable cell membranes of biological materials, which offers the dominant resistance to the mass transfer. The state of the cell membrane can change from partial to total permeability and this can lead to significant changes in the tissue architecture. During osmotic removal of water from foods, the dehydration front moves from the food surface that is in contact with the osmotic solution to the centre and the associated osmotic stress results in cell disintegration. The most likely cause of cell damage can be attributed to the reduction in size caused by water loss during osmotic treatment, resulting in the loss of contact between the outer cell membrane and the cell wall (Rastogi et al., 2000a). The difference in the respective chemical potentials of water and solute in a solid–liquid (solution) system results in fluxes of several components of material and solution. The water outflow and solute inflow mainly occur in the first 2–3 hours of immersion. After that the water content between food and osmotic solution gradually decreases, until eventually the system reaches a state of dynamic equilibrium of molecule transfer (Shi and LeMaguer, 2002). Osmotic dehydration is used as a pre-treatment to many processes to improve nutritional, sensorial and functional properties of food without changing its integrity (Torreggiani, 1993). It generally precedes processes such as freezing (Ponting, 1973), freeze drying (Hawkes and Flink, 1978), vacuum drying (Dixon and Jen, 1977) or air drying (Nanjundaswamy et al., 1978). It also increases the sugar to acid ratio and improves the texture and stability of pigments during dehydration as well as storage (Raoult-Wack, 1994). It is effective even at ambient temperatures, so heat damage to texture, colour and flavour can be minimized (Torreggiani, 1993). The osmotic process may provide the possibility of modifying the functional properties of food materials, improving the overall quality of the final product and giving potential energy savings (Shi and LeMaguer, 2002). The other major application is to reduce the water activity of many food materials so that microbial growth will be inhibited. Since most foods contain large amounts of water, they are cost intensive to ship, pack and store (Rao, 1977; Biswal and Le Maguer, 1989). Osmotic dehydration is acknowledged to be an energy-efficient method of partial dehydration, since there is no need for a phase change (Bolin et al., 1983). In order to make osmotic dehydration more attractive in economic terms, the osmotic solution needs to be reconcentrated by some means, either by evaporation or by adding fresh osmotic reagent. It can be an efficient complementary, if not an alternative, processing step to thermal dehydration in the overall chain of integrated food processing.

Mechanism of osmotic dehydration 223

Active research in the area of osmotic dehydration of fruit and vegetables is continuing all over the world. Research on osmotic dehydration of foods was pioneered by Ponting and co-workers (Ponting et al., 1966). Many review articles have already been published (Le Maguer, 1988; Raoult-Wack et al., 1992; Torreggiani, 1993; Raoult-Wack, 1994; Rastogi et al., 2002; Shi and LeMaguer, 2002) dealing with various parameters, such as mechanism of osmotic dehydration, effect of operating variables on osmotic dehydration, modelling of water loss and solid gain, etc. Since osmotic dehydration is an inherently slow process, several researchers have tried to increase the rates of osmotic mass transfer. This chapter deals with the mechanism of mass transfer during osmotic dehydration, methods developed to enhance mass transfer rates and determine the corresponding values of diffusion coefficients and its applications and limitations.

2 Mechanism of osmotic dehydration In the early reports of the mechanism, it was proposed that during osmotic dehydration a superficial layer, 2–3 mm deep, forms in the product that has a major effect on mass transfer, favouring water loss, while limiting solute deposition and reducing the loss of water-soluble solutes (Raoult-Wack et al., 1991; Marcotte and Le Maguer, 1992). Mass transfer during osmotic treatment of biological materials occurs through the semipermeable cell membranes that offer the dominant resistance to mass transfer. Diffusion of water takes place through the semipermeable membranes of fruit and vegetable tissues. The changes occurring in the state of the cell membranes were not considered in most of the earlier studies, although the diffusion coefficients for water have been generally considered to be constant throughout the process. Since cell membrane properties change due to osmotic stress, the models previously reported are not very appropriate. The state of the cell membrane can change from partial to total permeability, leading to significant changes in the rate of mass (water) transfer across it. The difference between the mechanisms of osmotic water removal from homogeneous materials and from cellular biological materials, proposed by Rastogi et al. (2000b), can be explained with help of a schematic diagram (Figure 9.1). In homogeneous material (Figure 9.1a), it is generally assumed that a constant rate diffusion (with diffusion coefficient D) occurs under the influence of a uniform moisture gradient. However, this does not appear to be true, especially after the initial stages of the process are over and the physical structure of the material starts to change. In the mechanism for cellular biological materials (Rastogi et al., 2000b), it is proposed that the dehydration front (represented by ⌬x) moves towards the centre of the material. This results in cell membrane disintegration in the dehydrated region and the water is transported across three different regions (each with its own characteristic properties): diffusion of water from the core of the material to the dehydration front; diffusion of water across the front; and diffusion of water through the osmotically dehydrated layers into the surrounding medium. At first, water diffuses from the outer layer of the sample to the osmotic medium, thereby increasing the osmotic pressure at the surface. As the osmotic pressure reaches a

224 Developments in Osmotic Dehydration

D 1

M/Mo

0 0

1

Relative distance

(a) D1

D2

D3

1 Liquid ZP

M/Mo

0

0 0

(b)

t⬎0

⌬x Relative distance

1

Figure 9.1 Mechanism of osmotic dehydration of (a) non-biological/homogeneous material and (b) biological material. Zp and M/Mo are the cell disintegration index and relative moisture content. D is the diffusion coefficient of water from homogeneous material during osmotic dehydration. D1, D2 and D3 are the diffusion coefficients of water from the core of the material to the dehydration front, across the front and through the osmotically treated material into the osmotic solution, respectively. ⌬x is the thickness of moving dehydration front (from Rastogi et al., 2002).

critical value, the cell membranes shrink and rupture. This results in a steep reduction in the proportion of intact cells which is reflected in an increase in the cell permeabilization index (Zp). The condition of the cell or the degree of disintegration can be examined by Zp. This is measured by electrophysical measurement based on electrical impedance analysis (Knorr and Angersbach, 1998). In other words, Zp is an integral parameter which indicates the relative reduction in the proportion of intact cells. The diffusion coefficient of water released through these ruptured and shrunk cells into the osmotic solution at any point of time is denoted by D3. As osmotic dehydration proceeds, the dehydration front moves into the product. In this front, the cells are in the process of disintegration and hence the rate of mass transfer increases sharply. At this juncture a relatively large amount of water diffuses out with a diffusion coefficient D2 (D2 ⬎⬎ D3). As the cells in the core of material are intact, the diffusion coefficient of water from this core (D1) is much lower than D2 and D3. The representative profiles for the cell disintegration index (Zp) and relative moisture content (M/Mo) values are also shown in Figure 9.1 (Rastogi et al., 2002).

Mechanism of osmotic dehydration 225

Diffused moisture ratio (Mr)

1.0 0.8 0.6 0.4 0.2 0.0 0

0.2

(a)

0.4

0.6

0.8

1.0

Relative distance (x/l)

Diffused solid ratio (Sr)

1.0 0.8 0.6 0.4 0.2 0.0 0 (b)

0.2

0.4

0.6

0.8

1.0

Relative distance (x/l)

Figure 9.2 (a) Moisture and (b) solid concentration profiles developed during osmotic dehydration. (Slice thickness 1.0 cm). Osmotic solution concentration and temperature 50°B and 25°C, respectively. (Osmotic dehydration time ● ⫽ 1 h; ■ ⫽ 2 h; ◆ ⫽ 3 h; ▲ ⫽ 4 h; O ⫽ 5 h) (from Rastogi and Raghavarao, 2004b).

Rastogi and Raghavarao (2004b) further explained the mechanism of mass transfer during osmotic dehydration by determining the moisture and solute diffusion coefficients during osmotic dehydration based on their respective concentration profiles (Figure 9.2). The average moisture and solute diffusion coefficients [(Dew)av and (Des)av] were estimated using the following equation (Crank, 1975): l l1 l l l ⫹ 2 ⫹ 3 ⫹⋯⫹ n ⫽ (Dew )n (Dew )av (Dew )1 (Dew )2 (Dew )3

(1)

l l1 l l l ⫹ 2 ⫹ 3 ⫹⋯⫹ n ⫽ (Des )1 (Des )2 (Des )3 (Des )n (Des )av

(2)

(Dew)i and (Des)i are the moisture and solute diffusion coefficients for respective length scales li. Shi and LeMaguer (2002) proposed a similar mechanism indicating mass transfer and tissue shrinkage spread occurring concurrently from the surface to the centre of the material with increasing immersion time. The solute taken up from the osmotic solution is accumulated in the extracellular spaces.

226 Developments in Osmotic Dehydration

Fernandez et al. (2004) classified the mass transfer behaviour of different plant materials during osmotic dehydration. The ratio of bulk flow (water loss) transport to diffusion (solid gain) transport at the interface at the initial time (␾) was used to divide mass transfer behaviours into three different categories such as fast, average and slow. The materials for which ␾ ⬎ 1 lead to more dehydration than impregnation and are referred as materials that have fast mass transfer behaviour. The materials for which ␾ ⬍ 1 lead to more impregnation than dehydration and are referred as materials that have slow mass transfer behaviour. The materials for which the ␾ values are close to 1 are referred to as materials having average mass transfer behaviour. Ferrando and Spiess (2002) described mass transfer from isolated carrot protoplasts during osmotic treatment using confocal scanning laser microscopy. The ratio of cellular volume before and after the osmotic treatment with different concentrations of sucrose solutions was monitored. The transmembrane water flux, coefficient for water membrane permeability and effective water diffusivity during osmotic treatment were estimated.

3 Effect of process parameters on mass transfer The effect of the concentration and temperature of the osmotic solution has been studied in considerable detail and it has been shown that the rate of osmotic dehydration increases with an increase in both parameters. The rate of dehydration also increases as the level of agitation is increased up to a certain extent. Agitation is indeed one of the key factors and an adequate level of agitation ensures reduction or elimination of liquid-side mass transfer resistance and constant driving force. At low solid to solution ratio, as the dehydration progresses, the osmotic solution becomes increasingly dilute and the driving force for further release of water drops. It is therefore necessary to have a solid-to-solution mass ratio of around 1:20 in order to ensure a more or less uniform driving force. It is reported that the increase in fruit-tosugar syrup ratio (from 1:1 to 1:4.5) resulted in increased weight loss during osmotic dehydration of banana (Bongirwar and Srinivasan, 1977). If the solid size is greater, dehydration will occur more slowly because the length of the diffusion path is longer. Hence, smaller pieces dehydrate more rapidly. The shape of the solid material is another important factor. In order to evaluate the effect of size and shape on mass transfer during osmotic dehydration, Lerici et al. (1985) cut apples into stick, slice, cube and ring shapes and then subjected these pieces to osmotic dehydration. The weight loss and solid gain increased in proportion to the ratio of the surface area to the characteristic length (Lerici et al., 1985). To summarize, the parameters influencing mass transfer are concentration and temperature of the osmotic solution, agitation, solid-to-osmotic solution ratio, solid structure (porosity, etc.), shape and size (which determine surface area and resistance for mass transfer in the form of the thickness) and nature and molecular weight of the osmotic solute and pressure (high pressure, ambient or vacuum).

Determination of moisture and solid diffusion coefficients 227

The selection of these process parameters also depends on the application. For instance, candying needs high solid gain, which is favoured by the low molecular weight of the osmotic solute and low concentration of the solution. On the other hand, dehydration requires high water loss, which is favoured by a high molecular weight solute. Thus it is necessary to strike the right balance between these process parameters so that the relative rates of the two main mass transfers suit the application at hand. The shape and size of the food being osmotically dehydrated varies considerably from application to application. The common shapes are slab, cylinder, cube and sphere. The models used to estimate diffusion coefficients based on the geometry are presented in the following section.

4 Determination of moisture and solid diffusion coefficients There are several methods reported in the literature for the estimation of diffusion coefficients for moisture and solute transport during osmotic dehydration, all based on the solution of Fick’s second law as applied to the situation under consideration, which can be written for diffused moisture (Mr) and solid ratio (Sr) considering appropriate assumptions and boundary conditions (Crank, 1975; Rastogi et al., 2002).

4.1 Infinite flat plate For a well-agitated unlimited volume of osmotic solution (m ⫺ m⬁ ) 8 Mr ⫽ t ⫽ 2 (mo ⫺ m⬁ ) ␲

2    1  2  1   ∑ (2n ⫹ 1)2 exp ⫺n ⫹ 2  ␲ Fow    n⫽0   ⬁

(3)

and Sr ⫽

(s t ⫺ s ⬁ ) 8 ⫽ (so ⫺ s ⬁ ) ␲2

2    1 ⫺n ⫹ 1  ␲2F  exp ∑   os  2 2     n⫽0 (2n ⫹ 1)   ⬁

(4)

For a well-agitated limited volume of osmotic solution Mr ⫽

⬁ (mt ⫺ m⬁ ) 2␣(1⫹ ␣) exp ⫺qn2 ⭈ Fow  ⫽∑ (mo ⫺ m⬁ ) n⫽1 1⫹ ␣ ⫹ ␣2qn2

(5)

⬁ (s t ⫺ s ⬁ ) 2␣(1⫹ ␣) exp ⫺qn2 ⭈ Fos  ⫽∑ (so ⫺ s ⬁ ) n⫽1 1⫹ ␣ ⫹ ␣2qn2

(6)

Sr ⫽

228 Developments in Osmotic Dehydration

For small values of t 1

 D t 2 (mt ⫺ m⬁ ) ⫽ 1⫺ 4  e2  Mr ⫽ (mo ⫺ m⬁ )  ␲l 

(7)

4.2 Rectangular parallelepiped Mr ⫽

⬁   1 (mt ⫺ m⬁ ) 1  1 ⫽ ∑ Cn3 exp ⫺Dew tqn2  2 ⫹ 2 ⫹ 2    a c  (mo ⫺ m⬁ ) n⫽1 b 

(8)

⬁   1 (s t ⫺ s ⬁ ) 1  1 ⫽ ∑ Cn3 exp ⫺Des tqn2  2 ⫹ 2 ⫹ 2    a c  (so ⫺ s ⬁ ) n⫽1 b 

(9)

and Sr ⫽

For Det(1/a2 ⫹ 1/b2 ⫹ 1/c2) (Fourier number) ⬎0.1, only the first term is significant M   1 1 1 ⫺ln  3r  ⫽ q12Dew t  2 ⫹ 2 ⫹ 2   a  C1  b c 

(10)

S   1 1 1 ⫺ln  r3  ⫽ q12Des t  2 ⫹ 2 ⫹ 2     C1  b c  a

(11)

and

The values of Dew and Des can be determined from the slopes of the lines obtained by plotting ⫺ln(Mr /C13 ) and ⫺ln(Sr /C31 ), each against t (Rastogi and Niranjan, 1998).

4.3 Infinite cylinder ⬁ Mt 4 exp ⫺Fo(a␣n )2  ⫽ 1⫺ ∑ 2 M⬁ n⫽1 (a ␣n )

(12)

where a␣n’s are the roots of the equation Jo(a␣n ) ⫽ 0 and Fourier number of diffusion (Fo) is defined as Det/a2 (Rastogi et al., 1997).

4.4 Finite cylinder Mr ⫽

  q2  2  ⬁ ⬁  (mt ⫺ m⬁ )  pn q2  ⫺D t qcn  C C ⫽ ∑ CpnCcn exp ⫺Dew t  2 ⫹ cn exp ⫽  ∑   ew A2   l r 2  n⫽1 pn cn (mo ⫺ m⬁ ) n⫽1  

(13)

Methods to increase the rate of mass transfer 229

and Sr ⫽

  q2  2  ⬁ ⬁  (s t ⫺ s ⬁ )  pn q2  ⫺D t qcn  C C ⫽ ∑ CpnCcn exp ⫺Des t  2 ⫹ cn ⫽ exp  ∑   pn c n es   l r 2  n⫽1 (so ⫺ s ⬁ ) n⫽1 A2    (14)

where A is defined as 2  2 1 1   r   qpn   ⫽ 2 1⫹      A2 r   l   qcn    

For an infinite cylinder, l ⬎⬎ r and the above relation reduces to A ⫽ r (Rastogi et al., 1999). For Fourier number (Det/A2) ⬎ 0.1  M    q2 2   r  ⫽ D t  p1 ⫹ qc1  ⫺ln    Cp1Cc1   ew  l2 r 2   

(15)

 S    q2 2   r  ⫽ D t  p1 ⫹ qc1  ⫺ln     Cp1Cc1   es  l2 r 2   

(16)

and

The values of Fow and Fos can be obtained as a function of Mr and Sr from Equations (15) and (16). These values can then be plotted against the corresponding values of t and the Dew and Des values were estimated from the slopes of these plots. Fickian unsteady state diffusion models presented above have so far been deemed to be the most appropriate for the estimation of diffusion coefficients. Depending on the shape of the food material, a suitable model can be selected for the estimation of water as well as solute diffusion coefficients.

5 Methods to increase the rate of mass transfer Since the rate of mass transfer during osmotic dehydration is generally low, a number of techniques have been attempted to improve it. These techniques include subjecting the food to ultra high hydrostatic pressure (Rastogi and Niranjan, 1998), high intensity electrical field pulses (Rastogi et al., 1999) or gamma irradiation (Rastogi, 2004; Rastogi and Raghavarao, 2004c) prior to osmotic dehydration and applying ultrasound (Simal et al., 1998), partial vacuum (Fito, 1994; Rastogi and Raghavarao, 1996) or centrifugal force (Azuara et al., 1996) during or prior to osmotic treatment.

230 Developments in Osmotic Dehydration

5.1 Application of high hydrostatic pressure The application of high pressure changes the cell wall structure and the tissue architecture, making the cells more permeable (Farr, 1990; Dornenburg and Knorr, 1993; Eshtiaghi et al., 1994; Rastogi et al., 1994). In the case of pineapple this led to an increase in mass transfer rates (Rastogi and Niranjan, 1998). The diffusion coefficients were found to increase fourfold for water and twofold for sugar in the pressure range varying between 100 and 700 MPa. Compression and decompression taking place during the high pressure pre-treatment itself cause the removal of a significant amount of water in the case of pineapple and this is attributed to cell wall breakage (Rastogi and Niranjan, 1998). Differential interference contrast microscopic examination showed evidence of the extent of cell wall break-up with applied pressure. The synergistic effect of cell permeabilization due to high pressure and osmotic stress as the dehydration proceeds was also demonstrated in the case of potato (Rastogi et al., 2000a, c). The moisture content was reduced and the solid content as well as the cell permeabilization index (Zp) increased in samples treated at 400 MPa. The distributions of relative moisture (M/Mo) and solid (S/So) content as well as the cell permeabilization index (Zp) for control and pressure treated samples are shown in Figure 9.3 (Rastogi et al., 2000a). Sopanangkul et al. (2002) determined the diffusion coefficient of sucrose infusion in potato cylinders at various pressures (200–600 MPa) and temperatures (20, 40 and 60°C). Application of pressure opened up the tissue structure and facilitated diffusion. However, higher pressures (above 400 Mpa) also induced starch gelatinization and hindered diffusion. The values of the diffusion coefficient were dependent on cell permeabilization and starch gelatinization. In this study (Sopanangkul et al., 2002), the maximum value of the diffusion coefficient observed represented an eightfold increase over the values at ambient pressure. Thus application of appropriate levels of pressure (100–400 MPa) can be used to accelerate mass transfer during ingredient infusion into foods.

5.2 Application of high electric field pulse pre-treatment High intensity pulsed electrical field (HIPEF) treatment resulted in an increase in the permeability of plant cells (Geulen et al., 1994; Knorr et al., 1994; Knorr and Angersbach, 1998; Rastogi, 2003) due to induced cell damage, which resulted in tissue softening. This in turn resulted in the loss of turgor pressure leading to a reduction in compressive strength or product softening, which in turn led to increased mass transfer rates during osmotic dehydration. High intensity electrical field pulse (0.22–1.60 kV/cm) pre-treatment was shown for the first time to accelerate osmotic dehydration (Figure 9.4). In the case of carrot, applied energy (0.04–2.25 kJ/kg) increased the cell disintegration index (Zp) in the range 0.09–0.84 with 1°C rise in the product temperature (Rastogi et al., 1999). Angersbach et al. (1997) showed an increase in permeability of potato tissue by HIPEF treatment, which resulted in improved mass transfer during fluidized bed drying. Ade-Omowaye et al. (2002b) studied the effects of HIPEF and elevated osmotic solution temperature on mass transfer and nutrient composition of bell peppers during

Methods to increase the rate of mass transfer 231

1.0 Relative moisture content

Relative moisture content

1.0 0.9 0.8 0.7

1h 2h 3h 4h 5h

0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 (a)

Relative distance (l/lo)

1.8 1.6 1.4 1.2

1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Relative distance (l/lo)

(e)

2.0 1.8

1h 2h 3h 4h 5h

1.6 1.4 1.2

(d)

Relative distance (l/lo) 1.0

1h 2h 3h 4h 5h

0.8 0.6 Zp

Zp

0.6

2.2

1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

1.0 0.8

Relative distance (l/lo) 2.4

1h 2h 3h 4h 5h

(c)

0.7

1h 2h 3h 4h 5h

(b)

Relative solid content

Relative solid content

2.0

0.8

0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

2.4 2.2

0.9

0.4

0.4

0.2

0.2

0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Relative distance (l/lo)

(f)

1h 2h 3h 4h 5h

Relative distance (l/lo)

Figure 9.3 Distribution of (a, b) relative moisture and (c, d) solid content as well as (e, f) cell disintegration index with respect to distance from the centre of the potato sample (thickness 10 mm) during osmotic dehydration of control and pressure pre-treated at 400 MPa for 10 min (from Rastogi et al., 2000b).

osmotic drying. The water loss and solids gain during osmotic dehydration increased with increasing solution temperature and/or the application of HIPEF. The combination of HIPEF with osmotic dehydration resulted in a product with higher vitamin C and carotenoid content compared to the samples subjected to osmotic dehydration alone at elevated temperatures. Hence, combined HIPEF and osmotic dehydration

232 Developments in Osmotic Dehydration

7.5 Control 0.22 kV/cm 0.64 kV/cm 1.09 kV/cm 1.60 kV/cm

Moisture content (kg/kg)

7.0 6.5 6.0 5.5 5.0 4.5 4.0 0.0

1.0

(a)

2.0

3.0

4.0

5.0

6.0

Immersion time (h) 4.5

Solid content (kg/kg)

4.0 3.5 3.0 2.5

Control 0.22 kV/cm 0.64 kV/cm 1.09 kV/cm 1.60 kV/cm

2.0 1.5 1.0 0.0

(b)

1.0

2.0

3.0

4.0

5.0

6.0

Immersion time (h)

Figure 9.4 Variation of (a) moisture and (b) solid content with time during osmotic dehydration of high intensity electrical field pulse pre-treated carrot sample (from Rastogi et al., 1999).

treatment could be used as a pre-processing step prior to air drying to enhance the quality of the dried product. Ade-Omowaye et al. (2003a, b) related the extent of membrane permeabilization due to HIPEF to the rate of diffusion during osmotic drying of red bell pepper. It was shown that initiation of pore development and pore growth in the cell membrane after HIPEF was time dependent. Both the water loss and solids gain increased due to HIPEF treatment. Taiwo et al. (2003) showed increased cell membrane permeabilization with increasing field strength and pulse numbers, which facilitated water loss during osmotic dehydration. The application of HIPEF resulted in the development of brown colour. However, sample brightness was reported to increase with increase in dehydration time and it decreased with increasing pulse number. The vitamin C content of dried samples also reduced at higher field strengths and longer osmotic dehydration duration.

Methods to increase the rate of mass transfer 233

Conventional wisdom indicates that it is necessary to have the cell membrane intact for achieving high mass transfer rates. However, our present work on high pressure (HP) pre-treatment (Rastogi and Niranjan, 1998) and high electric field pulse (HELP) pretreatment (Rastogi et al., 1999) and other recent reports (Raoult-Wack, 1994) clearly indicate that this is not the case. This fact gains importance since it is inevitable to work with food items whose cell/tissue structure is disintegrated during ripening or processing (as discussed above) or freezing (storage).

5.3 Application of ultrasound during osmotic dehydration In a solid medium, sound waves cause a series of rapid and successive compressions and rarefactions, with rates depending on the frequency. This mechanism is of great relevance to the drying and dewatering of foods. The mechanical and physical effects of sound can be used to enhance many processes where diffusion takes place (Florous and Liang, 1994). Acoustic streaming will favourably affect the thickness of the boundary layer which exists between stirred fluid and solid. Cavitation, a phenomenon caused by sonication, results in the formation of bubbles in the liquid, which can explosively collapse and generate localized pressure fluctuations. This effect increases diffusion during osmotic processes and accelerates degassing of the tissue (Florous and Liang, 1994). Diffusion across the boundary between the suspended solid and liquid is substantially accelerated in an ultrasonic field. Pressure and frequency are the two main factors to take into consideration. No increase in diffusion rates was reported when intensity was maximal due to violent cavitation that produces extreme turbulence or vapour locks at the interface. Lenart and Auslander (1980) showed that solute diffusion rates increase with higher acoustic intensities once the threshold value is crossed. Sajas and Gorbatow (1978) reported slight improvement in the diffusion rates at higher frequencies. The mechanism underpinning the effect of ultrasonic frequency on diffusion has not been elucidated. Simal et al. (1998) reported the applicability of sonication to osmotic dehydration of porous fruit such as apple cubes and showed that the rates of mass transfer increased when ultrasound was applied. Figure 9.5 shows that ultrasound affects mass transport, increasing both the water loss and solute gain. Ultrasonic osmotic dehydration can be carried out at a lower solution temperature to obtain higher water loss and solute gain rates while preserving the natural flavour, colour and heat-sensitive nutritive components.

5.4 Application of gamma-irradiation in osmotic dehydration Gamma-irradiation pre-treatment has been reported to change the interior tissue structure (Wang and Chao, 2002), thereby increasing the permeability of plant cells and mass transfer during osmotic dehydration (Rastogi and Raghavarao, 2004c). Doses of gamma-irradiation applied in the range of 3.0–12.0 kGy resulted in a decrease in hardness of potato, thereby resulting in an increase in effective water as well as solute diffusion coefficients, which was attributed to an increase in cell wall permeability facilitating the transport of water and solute (Rastogi, 2004).

600

150

500

130

400

110

300

90

200

70

100

50

0

Osmotic pressure (atm)

Osmotic pressure (atm)

234 Developments in Osmotic Dehydration

30 0

2

4

6

8

10

Immersion time (h) Figure 9.5 Variation of osmotic pressure of coconut with immersion time. (■) ⫽ atmosphere, ( ) ⫽ vacuum (coconut) (Sucrose concentration 40°B, temperature 50°C), (●) ⫽ atmosphere, (O) ⫽ vacuum (apple) (sucrose concentration 65°B, temperature 50°C, based on the data reported by Fito, 1994). Arrows point to respective axes (from Rastogi and Raghavarao, 1996).

5.5 Application of vacuum during osmotic dehydration Mass transfer during osmotic dehydration under vacuum has been reported to be quicker than under ambient pressure (Fito, 1994; Rastogi and Raghavarao, 1996). Fito and co-workers have explained this on the basis of pressure gradient and capillary flow. In some fruits, such as apple, the presence of intercellular voids is characteristic of the parenchymal tissue (Fito, 1994; Fito and Pasteur, 1994). The pore volume represents about 20 per cent of the total volume of the apple. These pores are occupied by gas that can be removed by the application of low pressure as in vacuum osmotic dehydration. The reduction in pressure causes the expansion and escape of gas occluded in the pores. When the pressure is restored, the pores can be occupied by osmotic solution, increasing the available mass transfer surface area. Rastogi and Raghavarao (1996) have explained the effect of vacuum application during osmotic dehydration on the basis of the diffusional osmotic transport parameter, mass transfer coefficient and interfacial area. The vacuum applied only affects the rate at which equilibrium is achieved and not the equilibrium moisture content as such. The overall mass transfer under vacuum was higher than at atmospheric conditions due to increased interfacial area resulting from increased pore filling by osmotic solution and increased capillary action. It is evident from Figure 9.5 that the vacuum affects only the rate at which the equilibrium is attained and not the equilibrium osmotic pressure (Rastogi and Raghavarao, 1996).

5.6 Application of centrifugal force during osmotic dehydration The effect of centrifugal force during osmotic dehydration was studied by Azuara et al. (1996). The samples (apple and potato) were placed in stainless steel tubes,

Applications of osmotic dehydration 235

mounted on a centrifuge, which contained a mixed osmotic solution of sucrose and salt. During the osmotic dehydration, the centrifuge speed was set at 64 g. It was observed that centrifugation enhanced mass transfer (water loss) by 15 per cent while considerably retarding the solid uptake (by about 80 per cent). Further work is needed to investigate the effect of variables such as rotational speed, temperature and concentration of osmotic solution, type of solute(s) or their mix and the size as well as the shape of the food.

6 Applications of osmotic dehydration 6.1 Osmotic dehydration and air drying Osmotic dehydration as a pre-treatment to an air drying process can be used to improve nutritional, sensorial and functional properties of food without changing the integrity of the foods (Nanjundaswamy et al., 1978; Torreggiani, 1993; Torreggiani and Bertolo, 2002). Osmotic dehydration increases the sugar to acid ratio and improves the texture and stability of pigments during dehydration and storage of foods (Raoult-Wack, 1994). It is effective around ambient temperatures, so heat damage to texture, colour and flavour can be minimized. Drying rates of osmotically dehydrated pineapple samples were reported to decrease significantly due to a higher initial solid content and/or action of solute on water sorption behaviour, with samples dehydrated in concentrated sucrose solution resulting in a reduction of the drying rate. Effective diffusivity of moisture transport during air drying decreased with increasing solid gain during osmotic dehydration (Rahman and Lamb, 1991). Ade-Omowaye et al. (2002a) reported that osmotic dehydration of red paprika renders a product with better sensory quality. The effect of osmotic dehydration prior to air dying on the dehydration rate has been widely investigated. Many researchers observed that osmotic dehydration pre-treatment improved the quality of dehydrated products and decreased the time necessary for drying (Kim and Toledo, 1987). However, in the case of the air drying of potato slices at 65°C, immersion in 51 per cent glucose solution resulted in a decrease in the effective moisture diffusion coefficients (Islam and Flink, 1982). According to Uddin and Hawlader (1990), similar water diffusion coefficients were observed for fresh pineapples as well as for pineapples osmotically dehydrated with sucrose. Alvarez et al. (1995) found no effect of glucose-osmotic impregnation of strawberries on the diffusion coefficient of water during air drying. Flink (1980) also found similar air dehydration rates for carrot slices with and without osmotic pre-treatment. Valencia-Rodriguez et al. (2003) reported that there was no improvement in the quality of air-dried seaweed (Porphyra columbina Montagne) by involving osmotic drying as a pre-treatment. Krokida et al. (2000) studied the effects of osmotic drying prior to air drying on the viscoelastic behaviour of apples and bananas using compression and relaxation tests. Osmotically dried samples exhibited a viscous rather than an elastic behaviour, indicating that infusion of sugars causes plasticity of the fruit structure. The

236 Developments in Osmotic Dehydration

effects of osmotic dehydration on subsequent air drying of peas resulted in reduced air drying time, good sensory quality and higher rehydration ratios of osmotically-treated samples as compared to untreated samples (Ertekin and Cakaloz, 1996; Bhuvaneswari et al., 1999). Osmotic dehydration as a pre-treatment in the case of cherry tomatoes resulted in a higher retention of vitamin C. Osmotic dehydration led to collapsed cell structure following water loss and the dried product showed cell shrinkage and dense tissue. The quality of the dried cherry tomatoes treated by osmosis was reported to be superior compared to control samples (Kyung et al., 1999). Ade-Omowaye et al. (2003b) studied the effects of pre-treatments of high intensity pulsed electric fields (HIPEF) applied alone or in combination with osmotic drying on air drying kinetics of fresh red bell pepper. The application of HIPEF prior to air drying significantly enhanced the initial drying rate, whereas osmotic dehydration pretreatment resulted in a decrease in drying rates. It has been demonstrated that combined HIPEF and osmotic dehydration has the potential for enhancing mass transfer rates and the preserving quality of dried red peppers. The combined use of microwave and convective drying has been reported to enhance the drying rate as well as to improve the final product quality. Prothon et al. (2001) studied the combined effects of osmotic dehydration and microwave-assisted air drying of apple pieces on texture, microstructure and rehydration properties of the fruit pieces. Osmotic dehydration pre-treatment resulted in reduced drying rate and effective moisture diffusivity and increased cell wall thickness in apple pieces, increased firmness of rehydrated apple pieces and reduced rehydration capacity. The quality of the final product was reported to improve by osmotic dehydration prior to drying. Torringa et al. (2001) studied the added value of an osmotic drying step of mushrooms prior to combined microwave and hot air drying. Microwave heating profiles of osmotically treated mushrooms showed more even heating, while drying curves demonstrated a shorter drying time. Shrinkage in products was slightly reduced by osmotic pre-treatment and the final porosity increased. In addition, rehydration properties were improved compared to non-osmotically dried samples and those dried by hot air treatment alone. Venkatachalapathy and Raghavan (1999) studied the microwave drying of osmotically dehydrated blueberries and reported an improved drying rate and maintained product quality compared to the air-dried berries. Microwave drying was more rapid than convective drying, while quality parameters of microwave-dried berries were comparable to those of freeze-dried berries.

6.2 Osmotic dehydration and freezing The use of osmotic dehydration for partial concentration of food as a pre-freezing treatment (osmodehydrofreezing) has been shown to decrease enzymatic browning (Convay et al., 1983) and to reduce structural collapse and drip during thawing (Forni et al., 1990). During freezing/thawing of plant cells, it is thought that membranes, particularly the plasma membrane, play a key role in the changes that occur within the tissue; the plasma membrane is considered the primary site of freezing injury (Partmann, 1975; Steponkus, 1984; Salisbury and Ross, 1985; Webb et al., 1994). Osmotic removal of water results in a reduction in subsequent ice formation.

Applications of osmotic dehydration 237

Tregunnol and Goff (1996) indicated that the addition of sugars due to osmotic dehydration increased firmness of thawed/rehydrated apple tissue and much of the damage to the fruit membranes was done during pre-freezing treatment. Talens et al. (2002, 2003) studied the effects of osmotic drying and frozen storage at ⫺18°C for 1 month on volatile compounds present in kiwi fruit and strawberries. Fruits subjected to osmotic drying contained higher levels of some ethyl esters than fresh fruits and significantly lower levels of some of the compounds that contribute to the characteristic fresh and green aroma of kiwi fruit. Concentrations of all volatile compounds were greatly reduced by frozen storage resulting in very small differences between untreated and osmotically dried fruits. The sensory properties of osmotic dried and frozen papaya were investigated over a range of concentration (40–65°Brix), temperature (20–40°C) and immersion time (30–90 min). Fast freezing was found to be the best process for preserving fruit texture. The optimum osmotic dehydration conditions (65°Brix at 20°C for 60 min) resulted in higher overall acceptability of the product (Moyano et al., 2002). Osmodehydrofreezing (osmotic dehydration followed by air drying and freezing) has been proposed as a suitable process for the production of reduced moisture fruits and vegetables devoid of preservatives with a natural flavour, colour, texture and functional properties suitable for different applications (Torreggiani et al., 1995; Forni et al., 1997). Incorporation of different sugars (maltose, sucrose or sorbitol) into the product affected their low-temperature phase transitions and the percentage distribution of the sugars. Maltose had the greatest protective effect on colour stability during frozen storage. Biswal et al. (1991) reported osmotic dehydration as an intermediate step in freezing of vegetable tissue. Movement of salt and water was modelled for water loss from the product and salt uptake by the product. Green beans contacted with 10 per cent NaCl-water solution at 20°C for 30 min were processed further by freezing in an air blast freezer. Evaluation of colour, hardness, texture, taste and overall acceptability indicated that the product had acceptable sensory properties. A comparison of dehydrofrozen products with their frozen counterparts revealed better quality characteristics of the former in the case of pineapples and very similar quality characteristics in the case of strawberries (Tomasicchio et al., 1986).

6.3 Osmotic dehydration and frying Osmotic dehydration before frying partially dehydrates materials through the elimination of a large portion of the water present in the food. The effects of osmotic dehydration on oil absorption, moisture loss phenomena and changes in colour as well as structure of French fries during subsequent deep fat frying have been reported (Krokida et al., 2001a, b). The moisture and oil content of fried potato samples immersed in osmotic solutions containing either sucrose (40 per cent w/w), NaCl (20 per cent w/w), maltodextrine-12 (20 per cent w/w) or maltodextrine-21 (20 per cent w/w) at 40°C for 3 h is shown in Figure 9.6. The osmotic pre-treatment reduced moisture loss and oil uptake during frying. Darkening of colour occurred during osmotic drying, which produced more intense coloration in the fried products. The most acceptable colour was produced in samples subjected to osmotic drying in NaCl solution. The porosity of

238 Developments in Osmotic Dehydration

4 Moisture content (kg/kg db)

Types of pre-treatment Central Sugar solution NaCl solution Maltodextrine-12 solution Maltodextrine-21 solution Calculated

3

2

1

0 0 (a)

10

5

15

Frying time (min)

Oil content (kg/kg db)

0.3

0.2

0.1

0 0 (b)

5

10

15

Frying time (min)

Figure 9.6 Effect of osmotic pre-treatment and frying time on oil and moisture contents of French fries (from Krokida et al., 2001a).

samples increased with osmotic pre-treatments with all osmotic solutes with the exception of those treated with the sucrose solution. It is concluded that osmotic drying can be effective as a pre-treatment for producing high quality low fat French fries. The viscoelastic behaviour of French fries during deep fat frying after osmotic dehydration was examined under uniaxial compression tests. Debnath et al. (2003) investigated the mass transfer kinetics of moisture outflow and oil uptake during deep fat frying of osmotically dehydrated potato slices (1.2 mm). The osmotic dehydration treatment using sodium chloride solution prior to frying of potato slices resulted in a reduced oil content of the fried product.

6.4 Osmotic dehydration and rehydration Osmotic pre-treatment with sucrose and sodium chloride solution resulted in minimization of shrinkage and improved rehydration of dried vegetables. Lewicki et al. (1998) reported that soaking in water as well as dipping in starch solution improved

Applications of osmotic dehydration 239

the rehydration characteristics of dehydrated onion. Osmotic dewatering of onion before drying decreased its ability to absorb water but increased the loss of soluble solids during rehydration and improved the retention of constitutive dry matter. Neumann (1972) reported better rehydration characteristics of celery with the incorporation of polyhydroxy compounds such as sucrose and glycerol. Pre-treatment with a low concentration mixed osmotic solution of salt (3 per cent) and sucrose (6 per cent) caused a considerable increase in rehydration and reduced shrinkage due to the fact that solutes in this concentration range provide sufficient structural and mechanical strength to withstand the shock during hot air drying and prevent cell rupture due to shrinkage and improve water uptake during rehydration (Jayaraman et al., 1990). It may be attributed to the fact that the solute (salt and sugar) concentration was such that at this level the osmotic pressure difference between the plant tissue and rehydrating solution was not significant, which in turn offered protection against cell damage (Lewicki, 1998a, b). Reduced water absorption and solute retention during rehydration of samples pretreated with a higher concentration (61.5°B) of osmotic solution was reported by Lewicki et al. (1998). Similar results for other systems were also reported by Lenart (1991) and Lenart et al. (1992). An increase in osmotic solution concentration from 20 to 60°B resulted in a decrease in moisture absorption and solid retention during rehydration compared to the control samples. This may be attributed to the osmotic dewatering when samples are pre-treated with the osmotic solution of higher concentration (20–60°B). Osmotic dewatering affects the rehydration properties of dried material because of cell permeabilization due to osmotic stress and thus on rehydration, these cells cannot absorb as much as water as the controls (Lewicki, 1998b). At the same time, solute loss during rehydration also increases, possibly due to the structural changes induced by the osmotic pre-treatment and interaction of the osmoactive substance with the cell components. Rastogi et al. (2004) reported the effect of water (0°B), as well as osmotic pretreatment (5–60°B), on the rehydration kinetics of carrot slices compared with control samples (Figure 9.7). The treatment up to 10°B resulted in an increase in moisture diffusion and decrease in solid diffusion. It may be due to the lower concentration of sucrose, which imparts structure and mechanical strength to the tissue, as well as maintaining the general protein (present in plant cell membranes) structure in the dry state and hence protecting the membrane shows its functionality upon rehydration. Maximum water diffusion and lowest solids diffusion was observed for samples treated with water (0°B). The samples pre-treated with more than 20°B could not absorb as much water in comparison with the control, because of cell permeabilization due to osmotic stress. At the same time, solute loss during rehydration increased, possibly due to structural changes induced by osmotic pre-treatment and interaction of the osmoactive substance with the cell components. Rehydration kinetics of high-pressure pretreated (100, 300 and 500 MPa for 10 minutes) and osmotically dehydrated pineapple cubes (20 20 10 mm) were studied at different temperatures (5, 25 and 35°C) and compared with ordinary osmotically dehydrated samples. The effective diffusion coefficients for water and solute were determined, assuming the rehydration process to be governed by Fickian diffusion (Rastogi

240 Developments in Osmotic Dehydration

Moisture content (kg/kg)

4.5

3.0 0°B 5°B 10°B Control 20°B 40°B 60°B

1.5

0.0 0.0

1.0

2.0

3.0

4.0

5.0

6.0

Rehydration time (h)

(a)

Solid content (kg/kg)

1.0

0.8

0.6

0.4

0.2 0.0 (b)

0°B 5°B 10°B Control 20°B 40°B 60°B

1.0

2.0

3.0

4.0

5.0

6.0

Rehydration time (h)

Figure 9.7 (a) Variation in moisture and (b) solid content with time during rehydration of carrot sample immersed in different concentrations of sucrose solution (from Rastogi et al., 2004).

et al., 2000d). The diffusion coefficients for water absorption into the tissue as well as for solute diffusion out of the tissue were found to be lower in the samples subjected to high-pressure treatment. Furthermore, the diffusion coefficients decreased with increase in pressure. The observed decrease in the water diffusion coefficient was attributed to the permeabilization of cell membranes which reduced rehydration capacity. High-pressure pre-treatment causes compacting of the cellular structure and release of cellular components which leads to the formation of a gel-network with divalent ions binding to de-esterified pectin (Eshtiaghi et al., 1994; Basak and Ramaswamy, 1998; Rastogi et al., 2000d), which in turn reduces the solid diffusion coefficient.

6.5 Osmotic dehydration and jam manufacture Jams are made from fruit and sugar mixed in proportions so that the final product contains a minimum fruit content of 30 per cent and strength of minimum 45°B. Traditional manufacturing methods require concentration by heat treatment, which causes quality

Management of osmotic solution 241

changes that adversely affect sensory and nutritional properties, the latter being related mainly to ascorbic acid losses. An alternative to thermal concentration is to incorporate previously dehydrated fruit, avoiding thermal damage (Shi et al., 1996). Osmotic dehydration with sugar solutions has been described as a suitable method for the concentration of fruit. The production of kiwi and orange jam by using osmotically dehydrated fruits mixed with osmotic solution, without thermal treatment, has been studied. The physical (colour and mechanical properties) and physicochemical properties (aw, Brix, moisture content, pH, acidity) of jams and marmalades prepared by formulation of osmodehydrated fruit and osmotic solution, avoiding thermal treatments, were comparable or even better than those of commercial products prepared by traditional methods (García-Martínez et al., 2002).

7 Limitations of osmotic dehydration There are still some limitations in scaling up osmotic dehydration technology. The high viscosity of the osmotic solution and low density difference between the solid and the solution (⌬␳ ⫽ ␳s ⫺ ␳sol) are the main drawbacks. An increase in viscosity increases resistance to mass transfer as the diffusion coefficient is inversely proportional to the system viscosity (Einstein or Wilke-Chang equation, Wilke, 1949; Wilke and Chang, 1955). The labile nature of foods will also not permit an increase in agitation levels beyond a point to overcome this viscosity effect. The low density difference makes the product float, requiring an additional mechanical means to keep it immersed in the solution. The major constraint for the industrial adoption of osmotic dehydration is the cost of osmotic solution that necessitates a proper means for its recycling. During osmotic dehydration, the solution becomes diluted and acquires flavour as well as colour, the extent of which depends upon the food. Care should be taken to minimize these so that product quality is not affected by recycling the osmotic solution. The osmotic solution has to be concentrated in order to be recycled which can be achieved through concentration by evaporation and/or by the addition of solute. The processing steps involved in recycling of spent osmotic solution still remain proprietary in the form of patents. Normally, the reconcentration of the spent solution is done by evaporation and/or addition of solute. Conventional membrane processes such as reverse osmosis have limitations after reaching a point (say 30°B) due to the very high osmotic pressure at this concentration. Again an osmotic membrane process looks promising since it does not involve phase changes, unlike evaporation.

8 Management of osmotic solution During osmotic dehydration, a large amount of water is transferred from the food to the osmotic solution which results in a dilution of the osmotic solution, an increase of the

242 Developments in Osmotic Dehydration

solution mass as well as a decrease in the dewatering potential. The other components such as aromas, pigments, acids, pectin, vitamins, salts, lipids, acids, proteins, pulp fragments and protein are leached into the solution, which leads to chemical, physicochemical and sensory changes in the solution after utilization. The recycling of the syrup results in browning/darkening after several uses due to oxidative enzymatic browning as well as non-enzymatic browning. In order to make osmotic dehydration a feasible operation in food processing, concentration of spent osmotic solution involving evaporation, addition of solute, membrane concentration or cryo-concentration is necessary. The most popular technique for the concentration of osmotic solution is evaporation. The main problem during heat treatment is due to non-enzymatic browning such as caramelization and Maillard reaction since some amino acids or proteins have been extracted from the food. Restoring solution concentration by the addition of dry solute or concentrated solution can save energy by avoiding the evaporation step. However, this method again poses problems during reconcentration of effectively used osmotic solution. Osmotic solution can be reused for several cycles after coarse filtration, concentration and colour removal by adsorbents such as charcoal (active carbon) or polyvinyl polypyrrolidone (PVPP). Membranes are also used for treatment of the osmotic solution. The process involving membranes has to take into account fouling at the membrane surface and the difficulty of working with solutions of higher concentrations due to their high viscosity as well as osmotic pressure. Hence, microfiltration and ultrafiltration may be more useful techniques for the clarification of used osmotic solution. With advent of new techniques such as direct osmosis or osmotic membrane distillation, it may be possible to concentrate the osmotic solution at a much lower cost. The large-scale industrial development of the osmotic dehydration process is limited due to problems encountered in the handling of used osmotic solution. Furthermore, the economic feasibility of any process involving osmotic dehydration as a pre-processing step depends upon the cost of the osmotic agent as well as disposal of the osmotic solution and making the process economically viable by involving several uses, such as syrups for fruit canning, mixing with fruit juices, use as a carbonated fruit-flavoured beverage, use as a covering solution in canning as well as for the production of natural flavourings.

9 Conclusions Osmotic dehydration can be considered as the most eligible energy-saving method for the partial removal of water from foods, if and even as a method of preservation in case of candy preparation. It can affect marked reduction in the moisture content of the foods before they are subjected to further processing steps such as drying, freezing, frying etc. Pre-treatment with osmotic solution having concentrations lower than the natural cell concentration can improve the rehydration characteristics. Osmotic dehydration can also be used for the natural concentration of fruits, which helps in obtaining better characteristics of food prepared from them, such as jam. Application of various pre-treatments to osmotic dehydration such as high hydrostatic pressure, high

References 243

electrical field pulses, gamma irradiation, ultrasound, vacuum and centrifugal force, can overcome the long existing issues related to the inherently slower mass transfer rates. The basic understanding of the mechanism of mass transfer during osmotic dehydration will play an important role in devising novel applications of this potential technique in food processing by future researchers. With the advent of newer technologies involving adsorbence and membranes, the problem of handling used osmotic solution may find newer means of resolution, which will further increase the economical feasibility of this technology.

Nomenclature ␣ 1/A2 a, b, c As, Bs Aw, Bw Cn Ccn Cpn Dew and Des Fow and Fos Jo(qcn), J1(qcn) mo, m⬁ and mt Mr and Sr qn, s qcn, s qpn, s so, s⬁ and st

ratio of volume of solution to that of each piece (1/a2) ⫹ (1/b2) ⫹ (1/c2) half length of the sides of rectangular parallelepiped (m) coefficients for solute diffusion in Equation (5) coefficients for water diffusion in Equation (6) 2␣(1 ⫹ ␣)/(1 ⫹ ␣ ⫹ ␣2qn2 ) 4␣(1 ⫹ ␣)/(4 ⫹ 4␣ ⫹ ␣2q2cn ) 2␣ (1 ⫹ ␣)/(1 ⫹ ␣ ⫹ ␣2q2pn ) effective diffusivity of water and solute (m2/s) Fourier numbers for moisture and solute diffusion roots of Bessel function of zero and first order moisture concentrations initially, at equilibrium and at any time (kg/kg) moisture and solute ratio non-zero positive roots of the equation tan qn ⫽ ⫺␣qn non-zero positive roots of the equation ␣qcnJo(qcn) ⫹ 2J1(qcn) ⫽ 0. non-zero positive roots of the equation tan qpn ⫽ ⫺␣qpn moisture concentrations initially, at equilibrium and at any time (kg/kg)

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Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours K S M S Raghavarao1, Naveen Nagaraj1, Ganapathi Patil1, B Ravindra Babu1 and K Niranjan2 1

Department of Food Engineering, Central Food Technological Research Institute, Mysore, India 2 School of Food Biosciences, The University of Reading, Reading, Berkshire, UK Liquid foods and natural colours are concentrated in order to reduce the costs of storage, packaging, handling and transportation. However, both liquid foods and natural colours are sensitive to temperature and concentration by conventional methods, such as evaporation, results in product deterioration. Alternative processes, such as freeze concentration, have the drawback with respect to the maximum achievable concentration (only up to 40–45°Brix). In recent years membrane processes such as microfiltration, ultrafiltration and reverse osmosis are gaining importance for the concentration of liquid foods and natural colours. These existing membrane processes have limitations of concentration polarization, membrane fouling, shear damage (in the case of protein) and maximum achievable concentration (only up to 25°Brix). Recently, technological advances related to the development of athermal membrane processes such as osmotic membrane distillation and direct osmosis have shown the potential to overcome the above limitations. Furthermore, these processes can be employed as a pre-concentration step to reduce water load on subsequent processing steps and can be easily scaled up. Recent advances and developments in these athermal membrane processes used for concentration of liquid foods and natural colours along with theoretical aspects are discussed in this chapter.

1 Introduction Liquid foods such as fruit juices are of high nutritive value as they are naturally enriched with minerals, vitamins and other beneficial components required for human health. Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

10

252 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

In recent years, food colours derived from natural sources (plant/marine) are gaining importance over their synthetic counterparts as food additives since they are non-toxic and non-carcinogenic. When extracted from their sources, both fruit juices and natural colours have low solid content, colour strength and high water load. Water, being the major constituent of liquid foods and natural colours, contributes to the growth of the microorganisms. Removal of water helps to reduce the microbial load, thereby favouring an increase in the shelf-life of the liquid foods and natural colours. Hence, it is desirable to concentrate these liquid foods and natural colours to improve shelf-life, stability and to reduce storage/transportation costs (Philip, 1984; Petrotos and Lazarides, 2001). This chapter, besides briefly considering the existing concentration methods such as evaporative concentration, freeze concentration and membrane concentration discusses the newer athermal membrane processes like osmotic membrane distillation (OMD) and direct osmosis (DO) for the concentration of liquid foods and natural colours. Apart from merits and demerits, suggestions for future work and the possibilities of integrating the newer membrane processes (OMD/DO) with the existing processes are also addressed.

2 Existing methods The concentration of liquid foods constitutes the major aspect of the food processing industry. The following processes are currently in use for the concentration of liquid foods and natural colours.

2.1 Evaporative concentration Evaporation is one of the oldest methods employed for concentrating liquid foods and natural colours. Evaporation is defined as the removal of water by vaporization from the solution to produce a concentrated solution. Selection of the proper evaporator is necessary and depends upon many factors such as the properties of the feed material, quality of the product, operating conditions and operating economy. Some evaporators that are commonly used for the concentration of liquid foods and natural colours are discussed in following sections (McCabe et al., 2001). 2.1.1 Open pan evaporators

These are the simplest commercial available evaporators and their low cost makes them popular. Open pan evaporators consist of a container open to the atmosphere in which fluid is heated by a flame or by steam through a coil or external jacket. The pans may be closed to permit vacuum operation. Stirring increases the rate of heat transfer and reduces risk of product ‘burn on’. These are used in tomato pulp concentration, soup and sauce preparations and in jam and confectionary boiling. Small-jacketed pans are very useful, but with large capacities the ratio of heat transfer surface to liquid volume falls and the heating becomes less effective. Internal heating coils fitted in large units can interfere with the liquid circulation, so affecting the heat transfer rate. In general when larger capacities are required other types of evaporators are preferred.

Existing methods 253

2.1.2 Plate evaporators

This type of evaporator consists of a set of plates distributed in units in which vapour condenses in the channel formed between the plates. The heated liquid boils on the surface of the plates and forms a film. The good heat transfer and short residence time make the evaporator more useful to concentrate heat-sensitive products. Plate evaporators are mainly used to concentrate coffee, soup broth and citrus juices. 2.1.3 Rising film evaporator

These types of evaporator have tubes 3–10 m in length with diameters of 25–50 mm. Liquid preheated to near boiling is introduced at the bottom of the tube assembly. Expansion due to vaporization causes high velocity vapour bubbles and carries away the liquid which continues concentrating as it rises upward. Under optimum conditions the vapour lifts a thin film of rapidly concentrating liquid up the walls of the tubes. The leaving vapor–liquid mixture passes into a separator where vapour is removed. The concentrated liquid may be used directly, mixed with fresh feed and recirculated, or passed to a second evaporator for further concentration. Residence time in a climbing film evaporator is short which makes it useful for concentrating heat-sensitive materials. 2.1.4 Falling film evaporator

This is similar to a rising film evaporator but the pre-heated liquid feed enters at the top of the tube assembly. As the evaporation proceeds the vapour passes down the tubes as a central, high velocity core, dragging a film of liquid with it. Since a hydrostatic liquid head is absent, a uniform and low boiling temperature may be maintained. Residence times are short so this unit is excellent for the concentration of heat-sensitive materials. It is widely used with citrus fruit juices where high rates of evaporation are obtained at temperatures as low as 50–60°C under vacuum operation. 2.1.5 Agitated thin-film evaporators

These are essentially large diameter jacketed tubes in which the product is vigorously agitated and continuously removed from the tube wall by scraper blades (or wipers) with a shaft rotating inside the tube. Thus the material processed is continuously spread as a thin film. The horizontal, vertical and inclined type of agitated thin-film evaporators are more common. Concentration of liquid foods and natural colours by a thermal process like evaporation results in a loss of flavours/aroma, colour degradation and a ‘cooked taste’ leading to a low quality end product. Another serious drawback of the evaporation process is the high-energy requirement.

2.2 Freeze concentration Freeze concentration is another method employed for the concentration of liquid foods such as fruit juices. Freeze concentration involves partial freezing of the product and removal of ice crystals, thus leaving behind all the non-aqueous constituents in the

254 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

concentrated phase. In freeze concentration two distinctive steps are involved, i.e. ice crystallization and ice separation from the concentrate. In the former, fruit juice is super-cooled below its freezing point to allow water to form ice crystals. In the latter, the ice crystals are separated from the concentrated juice by centrifugation. The major advantages that the freeze concentration process offers over evaporation is that it can concentrate the fruit juices without appreciable loss in taste, aroma, colour and nutritive value. Furthermore, freeze concentration can eliminate self-oxidation problems and can produce higher quality juice than that obtained from an evaporation process. However, the major drawback of freeze concentration is the maximum achievable concentration, which is much lower than that achieved during the evaporation process. Because of low process temperatures and high viscosities of the fruit juices, most of the liquid foods cannot be concentrated beyond 40–45°Brix. The high viscosity of the fruit juice retards the rate of crystallization, furthermore, it makes the pumping of the juice concentrate and washing of ice crystals increasingly difficult. In addition, this technique is not suitable to handle liquid foods with a high pulp content. Moreover, the energy requirement for the formation of ice crystals during freeze concentration is high (Despande et al., 1982).

2.3 Membrane processes Membrane processing is a technique that permits concentration and separation of macro- and micromolecules based on molecular size and shape. Membrane processing is fast emerging among various unit operations available for separation processes, especially in the field of chemical engineering, biotechnology and food processing. Better process economy, higher yield, improved product quality, utilization of byproducts and a solution to some environmental problems, can all be achieved by using membrane processing. In recent years, membrane processes such as microfiltration (MF), ultrafiltration (UF) and reverse osmosis (RO) are gaining importance for processing liquid foods and natural colours. Concentration of liquid foods and natural colours has been widely explored after the discovery of asymmetric membranes by Loeb and Sourirajan in the early 1960s. These processes normally operate at ambient temperature, thereby reducing the thermal damage to the product and retaining the colour, flavour/aroma and nutritive components of the product. Studies have shown that membrane applications can be less energy intensive than evaporation and freeze concentration.

2.3.1 Microfiltration

In this process, large molecules (e.g. fat globules) and suspended particles are held by the membrane while the remaining components of the solution pass through the membrane. MF resembles conventional coarse filtration and can selectively separate particles with molecular weights greater than 200 kDa. The pore sizes of microfiltration membranes are in the range of 0.05–10
m and the porosity of the membrane is

Existing methods 255

about 70 per cent. Membrane thickness is in the range of 10–150
m. In microfiltration the applied pressure is in the range of 0.1–2 bar. Microfiltration finds application in cell harvesting, clarification of fruit juice, wastewater treatment, separation of casein and whey protein and separation of oil–water emulsions (Petrus and Nijhuis, 1993).

2.3.2 Ultrafiltration

This process is mainly used for clarification, concentration and purification of fruit juices, natural colours etc. The membranes are made up of polysulphone, polyvinyldene fluoride and cellulose acetate of pore size 1–100 nm. The applied pressure is in the range of 1–10 bar (Mulder, 1998). Ultrafiltration is also used for the separation of high molecular components from low molecular components having applications in the food, dairy, pharmaceutical, textile, chemical, metallurgy, paper and leather industries. The various applications in the food and dairy industry are concentration of milk, recovery of whey proteins, recovery of potato starch and proteins, concentration of egg products and clarification of fruit juices and alcoholic beverages (Mulder, 1998).

2.3.3 Reverse osmosis

In this process the concentration of solute in the solution (feed) will increase by the flow of water (or solvent) across the membrane to a dilute solution. This can be accomplished by applying the pressure in excess of the osmotic pressure (10–100 bar) of the solution. RO removes most of the organic compounds and up to 99 per cent of all ions. This process achieves rejection of 99.9 per cent of viruses, bacteria and pyrogens and was the first cross flow membrane separation process to be widely commercialized. The RO involves dense membranes having pore size of ⬍2 nm. The porosity of the membrane is about 50 per cent. The separation mechanism is based on solution diffusion across the membrane. Membranes are made up of cellulose triacetate, polyether urea and polyamide. RO finds application in the concentration of liquid foods such as fruit juices, milk, etc. and desalination of brackish and seawater (Girard and Fukomoto, 2000). The limitations of these membrane processes (MF, UF, RO) are concentration polarization, membrane fouling, shear damage (in proteins) and constraints on the maximum attainable concentration (only up to 25–30°Brix). Even the new membrane process, membrane distillation (MD), is not without limitations as it suffers from membrane wetting, temperature polarization and loss of volatiles (Lee et al., 1982; Lawson and LLoyd, 1997; Girard and Fukomoto, 2000). Recently, technological advances related to the development of new membrane processes and improvements in process engineering have enabled the above limitations to be overcome to a large extent. Newer membrane processes such as osmotic membrane distillation (OMD) and direct osmosis (DO) have the potential to concentrate liquid foods and natural colours at ambient temperature and pressure without product deterioration and are discussed in detail in the following section.

256 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

3 Osmotic membrane distillation 3.1 Fundamentals of osmotic membrane distillation Osmotic membrane distillation (OMD) is a novel athermal, non-pressure driven membrane process capable of concentrating liquid foods (fruit and vegetable juices, non-food aqueous solutions) and natural colours under ambient temperature and pressure, thus avoiding thermal degradation of the product. OMD is a separation process in which a microporous hydrophobic membrane separates the two aqueous solutions (feed and osmotic solution) having different solute concentrations. The driving force in OMD is vapour pressure difference across the membrane generated as a result of a difference in concentration. Water evaporates from the surface of the solution having higher vapour pressure (feed); the vapour passes through the pores of the membrane and condenses on the surface of the solution of the lower vapour pressure (osmotic agent, OA) as shown in Figure 10.1. This results in the concentration of the feed and dilution of the OA solution. OMD is also known as osmotic evaporation, membrane evaporation and isothermal membrane distillation or gas membrane extraction. It can be employed to achieve maximum concentration of up to 70°Brix without product damage (Hogan et al., 1998). In OMD, the vapour pressure of flavour/fragrance components due to low concentration (relative to that of water) is substantially depressed, thereby reducing the driving force for transmembrane transport of these solutes. The solubilities of these lipophilic solutes are substantially lower in concentrated saline (OA) solutions than in pure water. As a consequence, the vapour pressure of these solutes, when present even in trace concentration in such solutions, is much higher than that over water at the same concentration. Thus, the vapour pressure driving force for vapour phase transfer of these solutes from feed to the strip is far lower. Furthermore, due to the higher molecular weights of these solutes, their diffusive permeabilities through the membrane are Feed

Membrane

Pore

Figure 10.1

Mechanism of osmotic membrane distillation.

OA

Osmotic membrane distillation 257

lower. The overall result of all these factors makes OMD an attractive complementary or alternative process for the concentration of liquid foods with high flavour retention. Some of the OMD processes which have been carried out by various researchers are listed in Table 10.1. Furthermore, OMD can also be employed as a pre-concentration step prior to lypholization (freeze drying) of temperature-sensitive biological products such as vaccines, hormones, enzymes and proteins to obtain the product in powder form without product deterioration.

3.2 Mathematical models 3.2.1 Mass transfer

In OMD, water transport across the membrane involves evaporation of water at the surface of the solution, diffusion of water vapour through the membrane and condensation of the water vapour on the osmotic agent (OA) side. The basic model used to describe the water transport in the system that relates the mass flux (J) to the driving force is given by: J ⫽ K ⌬P b

(1)

where K is the overall mass transfer coefficient which accounts for all three resistances (feed, membrane and OA side) for water transport. 3.2.2 Mass transfer through the membrane

Mass transfer in OMD occurs by diffusive transport of water vapour across the microporous hydrophobic membrane. The mode of diffusion for water vapour through the stagnant gas phase of the membrane pore can be described either by the Knudsen diffusion or molecular diffusion mechanism depending on the pore size (Geankoplis, 1993). When the membrane pore size is lower than the mean molecular free path, the molecules tend to collide more frequently with the pore wall. Under these conditions, the mode of diffusion is by Knudsen diffusion and the equation for water flux can be written as (Schofield et al., 1987):  re JK ⫽ 1.064 ␹␦  

 M 0.5     (P ⫺ P )  RT   1 2 

(2)

where the first term of RHS in Equation (2) is the Knudsen diffusion coefficient corrected for membrane porosity as well as pore tortuosity and the second term accounts for the driving force. When the membrane pore size is relatively large, the collisions between the gas molecules themselves are more frequent and the mode of diffusion is called molecular diffusion. Water flux across the membrane is represented by (Sherwood et al., 1975):  1 De M   (P ⫺ P ) Jm ⫽   1 2 ␹␦ Y RT   ln

(3)

258 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

Table 10.1 Work carried out by various researches on osmotic membrane distillation Type

Juices/water/ Colorants

Osmotic agent

Membranes and their operating conditions

Fluxes (l/m2/h)

References

OMD

Orange juice, seawater

Seawater MgSO4

Module: Hollow fibre PP membrane; r 700 A; S 0.18 m2, ␧ 50%

0.4–7.9

Lefebvre (1988)

OMD

Orange, apple, NaCl grape juices

Module: Syrinx plate and frame PTFE membrane; r 0.2
m; S 0.7 m2; ␦ 100 ␮m Temperature: 29–40°C

0–2.2

Sheng et al. (1991)

OMD

Water

NaCl

Module: Lewis cell (stirred cell) a) Millipore PVDF (GVHP) r 0.2␮m; ␧ 70%; ␦ 125 ␮m; S 0.00275 m2 b) Millipore PTFE (FHLP) r 0.2 ␮m; ␧ 70%; ␦ 175 ␮m; S 0.00275 m2 c) Gelman PTFE (TF-1000) r 1 ␮m; ␧ 80%; ␦ 178 ␮m; S 0.00275 m2 d) Gelman PTFE (TF-450) r 0.45 ␮m; ␧ 80%; ␦ 178 ␮m; S 0.00275 m2 e) Gelman PTFE (TF-200) r 0.2 ␮m; ␧ 80%; ␦ 178 ␮m; S 0.00275 m2

0–0.5

Mengual et al. (1993)

OMD

Water

NaCl, MgCl2

Module: Capillary modules (LM2P06, MD020CP2N) in shell-tube configuration a) Accurel PP Q3/2; r 0.2 ␮m; ␧ 70%; SI 0.04 m2 b) Accurel PP S6/2; r 0.2␮m; ␧ 70%; SI 0.104 m2 Temperature: 25–50°C

0.2–2.5

Gostoli (1999)

OMD

Water

CaCl2

Module: Flat sheet membrane (Co-current) a) Pall-Gelman TF200 r 0.2 ␮m; ␦ 165 ␮m: ␧ 60%; S 0.004 m2 b) Pall-Gelman TF450 r 0.45 ␮m; ␦ 178 ␮m: ␧ 60%; S 0.004 m2 Temperature: 25°C

4–12

Courel et al. (2000a)

OE

Passion fruit juice

CaCl2

Module: Hollow fibres module PP membrane; r 0.2 ␮m; S 10.2 m2 Temperature: 30°C

0.5–0.75

Vaillant et al. (2001)

OMD

Water

Glycerol, NaCl, CaCl2

Module: Stirred cell PP membrane; r 0.1 ␮m; ␦ 90 mm; ␧ 55%; S 0.00113 m2 Temperature: 20–45°C

0.4–3.2

Alves and Coelhoso (2002)

OMD

Water, sugarcane juice

NaCl, K2HPO4, CaCl2

Module: Flat membrane test cell a) PP membrane; r 0.05 ␮m; ␦ 90 ␮m; S 0.045 m2 b) PP membrane; r 0.2 ␮m; ␦ 150 ␮m; S 0.045 m2 c) PTFE membrane; r 0.025 ␮m; S 0.045 m2 Acoustic field: 1.2 MHz Temperature: 25–60°C

0.4–0.93

Narayan et al. (2002)

OMD

Phycocyanin

CaCl2, K2HPO4

Module: Flat membrane test cell a) PP membrane; r 0.05 ␮m; ␦ 90 ␮m; S 0.0115 m2 b) PP membrane; r 0.2 ␮m; ␦ 150 ␮m; S 0.0115 m2 Temperature: 25°C

1.4–1.9

Naveen et al. (2003a)

OE

Water

Brine

Module: Ceramic tubular membrane r 0.2 and 0.8 ⫻ 10⫺6 m Temperature: 25–35°C

0.15–1.4

Brodard et al. (2003)

Osmotic membrane distillation 259

Both these models are useful for predicting the mass transfer through the membrane, each of them having its own limitations. The Knudsen model requires details of membrane pore geometry (pore radius, membrane thickness and tortuosity), whereas the molecular diffusion model is not valid at low partial pressure of the air (as Yln tends to zero) (Schofield et al., 1987).

3.2.3 Mass transfer through the boundary layers

The boundary layers of concentrated feed and dilute brine solution are present on either side of the membrane. This results in significant resistance to mass transfer which cannot be neglected. Mengual et al. (1993) proposed a model to study the influence of the boundary layer on transmembrane flux during OMD in a stirred cell, which explains the dependency of flux on bulk concentration and on stirring rate. In the case of a specially designed cross flow/stirred membrane cell, the liquid mass transfer coefficient in the boundary layer (ki) has been obtained by using the following correlations (Courel et al., 2000b; Alves and Coelhoso, 2002; Naveen et al., 2003b): (4)

Sh ⫽ b1Reb2 Scb3 where Sh ⫽

k iL , Dw

Re ⫽

uL␳


and

Sc ⫽

␮

Dw

(5)

where Dw is the water diffusion coefficient and Ki is liquid mass transfer coefficient.

3.2.4 Heat transfer

The water transport in OMD is a simultaneous heat and mass transfer process. Evaporation cools the feed and condensation warms up the brine (OA). The resultant temperature gradient across the membrane translates into a lower vapour pressure gradient, which in turn results in reduction of the driving force. The total heat transferred across the membrane is given by: Q ⫽ H⌬T

(6)

where H is the overall heat transfer coefficient which accounts for all three resistances (feed, membrane, and OA) (Courel et al., 2000b).

3.2.5 Heat transfers through boundary layers

Heat transfer across the boundary layer influences the rate of mass transfer and mainly depends on the physical properties as well as the hydrodynamic conditions of the solution. So far, no study has been published regarding the magnitude of the heat transfer through the boundary layers. However, the boundary layer heat transfer coefficients

260 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

can be estimated from empirical correlations involving dimensionless numbers, like Nusselt (Nu), Reynolds (Re) and Prandtl (Pr) numbers and are given by: Nu=b1Reb2Pr b3

(7)

where Nu ⫽

hLL kT

and

Pr ⫽


CP kT

3.3 OMD membranes The membranes are made up of synthetic polymers such as polytetrafluoroethylene (PTFE), polypropylene (PP) and polyvinylidene difluoride (PVDF), which are hydrophobic in nature and can be employed for OMD processes (Kunz et al., 1996). The membrane employed in the OMD process should be highly porous (60–80 per cent) and as thin as possible (0.1–1
m) since the flux is directly proportional to the porosity and inversely to the membrane thickness (pore length). Furthermore, it should be highly conductive so that the energy of vaporization of the feed can be supplied by conduction across the membrane at a low temperature gradient, thereby making the process essentially isothermal. The hydrophobicity of the membrane is a decisive parameter to make the OMD process viable. However, quantifying this parameter on porous material is not easy, as it is not supported by any theory. The method of estimating the contact angle by accounting the surface energy of smooth dense material does not apply for porous membranes. The pressure variable can be included in the wettability definition via the liquid entry pressure represented by the Laplace equation (Courel et al., 2001): ⌬Pentry ⫽

⫺2B␥L Cos ␪ ␥max

(8)

where ⌬Pentry is the liquid entry pressure, B is geometric factor, ␥L is liquid surface tension, ␪ is liquid–solid contact angle and ␥max is the largest pore radius. Once pressure drop across the vapour–liquid interface ⌬Pinterface exceeds penetration pressure ⌬Pentry, the liquid can penetrate into the membrane pores and the membrane is termed ‘wetted’. Hence, wettability of OMD membranes can be better defined by a critical surface tension combined with operating pressure conditions rather than by contact angle measurements. Development work is currently underway to attempt to produce hollow fibre microporous membranes from more hydrophobic membranes such as PTFE or PVDF or to make laminate membranes that prevent liquid intrusion without impeding vapour transport (Hogan et al., 1998; Michaels, 1999). Recently, it has been found that an amorphous copolymer of a certain perfluorinated dioxole monomer, namely perfluoro2,2-dimethyl-1,3-dioxole (PDD), can be formed into non-porous gas membranes, which provide acceptable transmission rates for OMD. These types of membranes can concentrate pulpy fruit juices and limonene-containing juices, such as orange juice, at high flux for long durations between membrane cleanings. Additionally, less

Osmotic membrane distillation 261

contamination of the OA solution into the feed side occurs, thus providing a high quality concentrate (Bowser, 2001). The membrane having relatively larger pore sizes at the surface showed higher organic volatiles retention per unit water removal than those with smaller openings. Accordingly pores with larger diameters at the membrane surface allow greater intrusion of the feed and OA streams, which provides an extended boundary layer. This extended layer offers extra resistance through which the diffusion of volatile components occurs. The above study helps to understand the utilization of membranes with larger surface pore diameters when the retention of volatiles and flavour/fragrance components are desirable for product quality (Barbe et al., 1998). More recently, Brodard et al. (2003) successfully employed hydrophobic ceramic tubular membranes in the osmotic evaporation process. Ceramic tubular membranes have been obtained by grafting siloxane compounds on alumina porous supports. Ceramic membranes have the advantage of physical and chemical stability when compared with polymeric membranes.

3.4 Effect of various process parameters The effect of various process parameters, such as type of osmotic agent, concentration, flow rate, temperature and membrane pore size, on transmembrane flux are discussed in the following sections. 3.4.1 Type of osmotic agent

The OMD process involves the use of a concentrated osmotic agent (OA) solution at the downstream side of the membrane. The rate of water (solvent) transport increases as the solvent vapour pressure on the OA side is reduced. In order to maintain the required driving force (vapour pressure difference), generally salts of high water solubility and low equivalent weights such as NaCl, CaCl2, MgCl2, MgSO4, K2HPO4, KH2PO4 are suitable as OAs in OMD. Potassium salts of ortho- and pyrophosphoric acid offer several advantages, including high water solubility, low equivalent weight, steep positive temperature coefficients of solubility and safe use in foods and pharmaceuticals. 3.4.2 Concentration

The feed and osmotic agent concentrations influence the OMD flux. The effect of OA concentration on transmembrane flux was studied in considerable detail for a model system (water as feed) as well as for real systems (fruit juices) (Mengual et al., 1993; Courel et al., 2000a; Alves and Coelhoso, 2002; Narayan et al., 2002; Ravindra Babu, 2003). It was observed that the transmembrane flux was increased with an increase in OA concentration in all cases. This is mainly due to an increase in vapour pressure difference (driving force) across the membrane. The transmembrane flux decreases with an increase in feed concentration and it strongly depends on the osmotic pressure difference between the two aqueous solutions (feed and OA). When osmotic pressure difference is decreased from 416 atm (dilute feed) to 280 atm (concentrated juice), a fivefold decline in flux was observed (Sheng et al., 1991).

262 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

3.4.3 Flow rate

The effect of OA flow rate on transmembrane flux was observed for model as well as for real systems (Courel et al., 2000a, b; Naveen et al., 2003b). In all these cases, transmembrane flux was increased with an increase in OA flow rate, which can be attributed to reduction in the concentration polarization layer. 3.4.4 Temperature

The effect of temperature on transmembrane flux has been studied by various researchers (Mengual et al., 1993; Gostoli, 1999; Courel et al., 2000a; Alves and Coelhoso, 2002; Narayan et al., 2002). The transmembrane flux increased with an increase in temperature. The rise in temperature provides an additional driving force that works synergistically with the driving force generated due to the concentration gradient. It is not difficult to appreciate the strong dependence of flux on temperature, which follows the Arrhenius dependency. 3.4.5 Membrane pore size

The effect of membrane pore size on transmembrane flux was studied (Mengual et al., 1993) and not much change in flux was observed. Recently, Brodard et al. (2003) have employed ceramic (inorganic) membranes made up of alumina having pore sizes of 0.2 mm and 0.8 m. The water transport was independent of pore size and followed molecular diffusion. Furthermore, the water fluxes obtained were much lower than those obtained by Courel et al. (2000b).

3.5 Process design and economics The essential design parameters which affect the OMD process performance for the concentration of liquid foods and natural colours are: 1 2 3 4 5

plant capacity solute concentrations in feed and concentrate water vapour pressure/concentration relationship for the feed stream water vapour pressure/concentration relationship for the OA solution and intrinsic water vapour permeability of the OMD membrane.

The potential and compatibility of OMD for concentrating liquid foods and natural colours have been proved beyond doubt. OMD like any other membrane process has low flux and production cost is higher than the thermal evaporation process. In order to overcome this problem, attempts have been made to enhance the transmembrane flux by the application of an acoustic field in a lab-scale membrane cell (Figure 10.2). The application of an acoustic field disturbs the hydrodynamic boundary layer of the solution, thereby reducing the effect of concentration polarization (Narayan et al., 2002). Another serious problem associated with commercial application of OMD is management of the diluted osmotic agent solution. It is essential to reuse the OA solution for better economics of the process. Corrosion and scaling make it expensive to

Direct osmosis 263

Cover Amicon cell (50 ml capacity)

Stirrer guide Vent Strip solution Teflon tube

Feed inlet Membrane (hydrophobic)

Feed solution (pure water)

Acoustic waves

Transducer Magnetic stirrer

Figure 10.2

Application of an acoustic field in membrane cell (lab-scale).

reconcentrate the diluted OA solutions. Evaporation, solar ponds and reverse osmosis could be used for re-concentration of OA solutions.

4 Direct osmosis 4.1 Fundamentals of direct osmosis Direct osmosis (DO) is another non-pressure driven membrane process capable of concentrating liquid foods and natural colours at ambient conditions without product deterioration. The concept is similar to that used by Eastern European farmers for the concentration of fruit juices, wherein a bag filled with juice was immersed in a brine solution (Cussler, 1984). Initially this process could not be exploited commercially due to low flux. In recent years, DO is gaining importance for the concentration of liquid foods and natural colours (Popper et al., 1964; Bolin et al., 1971; Loeb and Bloch, 1973; Rodriguez et al., 2001) and desalination of seawater (Kravath and Davis, 1975). DO, which is also known as direct osmosis concentration (DOC), uses a semipermeable dense hydrophilic membrane which separates two aqueous solutions (feed and OA solution) having different osmotic pressures (Figure 10.3). The driving force is the difference in osmotic pressure across the membrane (Beaudry and Lampi, 1990). The transfer of water occurs from lower to higher solution concentration until the osmotic pressures of both the systems become equal. DO also offers similar advantages as OMD with respect to energy and thermoliable component retention during the concentration of liquid foods and natural colours and concentration up to about 45–60°Brix could be achieved (Wong and Winger, 1999). Some of the DO processes as carried out by various researchers are shown in Table 10.2.

264 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

4.2 Mathematical models Various models have been proposed to explain mass transport through the membrane in the DO process. 4.2.1 Mass transfer through the membrane

The water, along with solute, tends to diffuse through the porous support of the membrane and solute concentration increases at the surface of the active membrane skin Feed

Membrane

OA

Pore Figure 10.3

Mechanism of direct osmosis.

Table 10.2 Work carried out by various researchers on direct osmosis Type

Juices/water/ colorants

Osmotic agent

Membranes and their operating conditions

Fluxes (l/m2h)

References

DOC

Grape juice

NaCl

2.5

Popper et al. (1964)

DO

HFCS

0.9–1.4

Wrolstad et al. (1993)

5–6

Herron et al. (1994)

DOC

Red raspberry juice Orange, raspberry, tomato juices Tomato juice

0.37–3.1

Petrotos et al. (1998)

DOC

Tomato juice

NaCl CaCl2 Ca(NO3)2 Sucrose PEG Brine

4.5

Petrotos and Lazarides (2001)

DOC

Red radish

HFCS

Module: Plate and frame membrane module Cellulose acetate membrane Module: Osmotek DOC cell S 0.14 m2 Module: Osmotek DOC module Cellulose triacetate membrane MWCO 100 Da, ␦ 90 ␮m Module: Tubular membrane module AFC99 aromatic polyamide thin film composite reverse osmosis membrane ␦ 500 and 600 ␮m Module: Flat sheet membrane module Commercial reverse osmosis membrane, ␦ 260 ␮m Module: Pilot plant (Osmoteck Inc., Corvalis OR)

0.5–2

Rodriguez-Saona et al. (2001)

DOC

PEG, HFCS

Direct osmosis 265

layer and it continues to rise until a steady state is reached resulting in internal polarization. The increase in solute concentration can cause back diffusion of the solute. Hence, the water flux will be more when the solute back diffusion rate is high, in other words the solute resistivity in the membrane porous substructure is low. Thus, in direct osmosis the porous substructure has a large significance on water flux and is given by (Loeb et al., 1997): Jw ⫽

1  ⌸ OA  Ds ln ⫽ K  ⌸ Feed  t

   ⌸ OA  ln    ⌸Feed 

(9)

where K is the resistivity of the porous substructure to water transfer, DS is the diffusivity of the solute, t is the thickness of the porous substructure, Feed and OA are the osmotic pressure of the feed and OA solutions, respectively. The preferential sorption capillary flow (PSCF) model offers a better visualization of the factors implied in transport across a reverse osmosis (RO) membrane and the same was preferred to explain the transport through a DO membrane (Ghiu et al., 2002). This model considers the surface layer of the membrane to be microporous and heterogeneous. The mechanism of separation is dictated by surface phenomena and pressure driven transport through capillary pores. When there is a pressure difference, the solute and solvent tend to permeate through the membrane, but water is adsorbed into the pores, whereas solute is rejected (due to physiochemical nature of the surface layer). However, due to the difference in chemical potential, eventually the solute is transported by diffusion through the pores and the flux is proportional to the concentration difference across the membrane. The solute flux through the membrane is given by: dm (10) Js ⫽ dt ⭈ S D K  Js ⫽  SM S  (C SM ⫺ C SDI)  ␦ 

(11)

where Js is solute flux through the membrane, S is membrane surface area, CSM is solute concentration of feed at the membrane surface, CSDI is solute concentration in feed solution, ␦ is the membrane thickness, DSM is diffusion coefficient of the salt in the membrane and KS is the solute partition coefficient between solution and membrane. The first term in equation (10) is known as the salt permeability and it is ideally the same for both RO and DO and was simplified as (DSM KS/␦) in order to calculate the salt permeability, by making the assumption that the solute concentration of feed at the membrane surface is the same as the concentration in the feed solution and is given by:   DSMK S   ⌬m   1    ⫽     ␦   S   0.5(A ⫹ B)T 2 ⫺ C SO T   

(12)

where ⌬m is the moles of solute transported through the membrane at time T, A and B are changes in solute concentration on the OA and feed side respectively.

266 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

4.3 DO membranes DO uses membranes similar to those used for RO, however, the osmotic pressure difference across the membrane is the driving force in the former, while it is hydraulic pressure in the latter (Beaudry and Lampi, 1990). The DO membranes are generally hydrophilic and asymmetric in nature, having a low molecular weight cutoff (100 Da). The hydrophilic nature provides a continuous water link between feed and OA. An asymmetric membrane structure is one that has a very thin active layer (skin), which facilitates the rejection of smaller polar compounds, and a much thicker and porous layer that provides strength to the membrane. The most suitable membranes for the DO process are cellulose acetate, cellulose diacetate, cellulose triacetate, polyamide and polysulphone membranes. Asymmetric cellulose-based membranes have shown better performance due to their water absorbing characteristics. Furthermore, these membranes are more resistant to fouling than many commercially available dense RO membranes (Herron et al., 1994; Wong and Winger, 1999).

4.4 Effect of various process parameters 4.4.1 Type of osmotic agent

The selection of the OA is one of the most important parameters, as it affects the DO performance. An osmotic agent should be highly soluble in water, hygroscopic, nontoxic, inert towards the flavour, odour and colour of the foodstuffs and should not pass through the membrane. Generally, the higher the concentration of the dissolved solids and the lower the molecular weight of the dissolved solids, the higher is the osmotic pressure. The most commonly employed osmotic agents are sodium chloride, sucrose, glycerol, cane molasses and corn syrup. Studies show that the transmembrane flux obtained for polyethylene glycol 400 (PEG 400) and carbohydrate solutions were less when compared to those of other solutions such as sodium chloride (NaCl), calcium chloride (CaCl2) and calcium nitrate (CaNO3). This is mainly due to the physical properties of the OA solutions such as viscosity (high in the case of PEG 400 and carbohydrate) and diffusivity. The lower viscosity offers less resistance to mass transfer through the concentration polarization layer (Petrotos et al., 1998). 4.4.2 Concentration

The extent of the concentration achievable in the DO process depends on the osmotic pressure of the osmotic agent (OA). Also, higher OA concentration results in higher flux and better relative rejection of salt ions (which otherwise leads to cross contamination). It is desirable to have the highest possible OA concentration, however, physical limits usually constrain achieving this. The OA solutions must have an osmotic pressure greater than that of the concentrated feed. For example, the osmotic pressure of a 74°Brix high fructose corn syrup is about 270 bar which is greater than the 90 bar for 42°Brix pulpy orange juice. The transmembrane flux decreases with an increase in feed side concentration due to the decrease of the osmotic pressure difference between the OA solution and the feed.

Direct osmosis 267

4.4.3 Temperature

An increase in temperature increases the osmotic pressure difference, which in turn increases transmembrane flux. This is mainly due to the reduction in viscosity and increase in diffusion coefficients. The increase in temperature reduces the mass transfer resistance during DO. 4.4.4 Flow rate

The flux in the DO process increased with an increase in the flow rate of the solutions. This increase in flux is due to a reduction in the resistance offered to mass transfer by the concentration polarization layer adjacent to the membrane. However, the effect of flow rate on transmembrane flux is less in DO when compared to OMD. Petrotos et al. (1998) reported that an increase in flow rate (4.6 fold) resulted in a marginal flux increase (only 32 per cent) during concentration of tomato juice. 4.4.5 Membrane thickness

The transmembrane flux increases with a decrease in membrane thickness. Petrotos et al. (1998) observed that the fluxes increased exponentially with a decrease in thickness of the backing material (600–400
m). Meanwhile, Beaudry et al. (1990) observed it to be linear. Even Loeb et al. (1997), who have studied permeation fluxes in DO process for non-food systems, observed that the porous fabric of the membranes clearly decreased the osmotic permeation. Some further detailed studies are required in this regard.

4.5 Process design and economics The design parameters which affect the DO performance are: 1 2 3 4 5

volume of feed to be concentrated final concentration required osmotic pressure difference between feed and OA physical properties of feed and OA and characteristics of membrane (thickness, water permeability, etc).

Even though DO can be employed for the concentration of dilute liquid foods, there are still some constraints which limit its full commercial application. Water flux is only one-fifth when compared to RO, but the ability to concentrate without prefiltering provides an advantage in DO. DO has a wide range of applications for the concentration of various liquid foods and natural colours. The economics of DO operation is dependent on effective OA management. The cost of DO operation depends mainly on the re-concentration method chosen. Re-concentration processes for the OA include solar evaporation, thermal evaporation and RO, but salts at high concentrations are corrosive to the metals which are used in the evaporators. Sugar solutions employed as OA can be concentrated by thermal evaporation or RO.

268 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

Figure 10.4

Stirred membrane cell (controlled conditions).

5 Membrane modules The membrane configurations normally employed for operating OMD/DO are stirred membrane cell, plate and frame, tubular, spiral wound and hollow fibre. The stirred membrane cell (Figure 10.4) is suitable for bench-top feasibility studies. However, the flux data obtained in stirred cells are not a good indication of the flux that can be obtained with larger modules. A majority of the laboratory scale modules are designed for use with flat sheet membranes (Figure 10.5), as these membrane modules are more versatile when compared with tubular or hollow fibre membrane modules. Flat sheet membranes are easier for examination and cleaning. As a result the same membrane module can be used to test many different types of membranes. Tubular membrane modules are usually operated under turbulent flow conditions, which help in the reduction of the concentration polarization effect. Tubular membranes do not require a support, which results in lower boundary layer resistances compared to flat sheet membrane modules. However, the main disadvantage of the tubular unit is the low surface area to volume ratio and hence the requirement for high floor space. Another disadvantage is the high hold-up volume within these units. The hollow fibre membrane configuration consists of a membrane in the form of a self-supporting tube and has the advantage of a ‘back-flushing’ provision. However, the disadvantage of the hollow fibre module is the cost of membrane replacement. This is mainly because even if one

Applications 269

Figure 10.5

Flat membrane module.

single membrane fibre ruptures, the entire membrane cartridge needs to be replaced. The spiral wound module is one of the most compact and inexpensive designs available today. These modules are basically flat sheets arranged in parallel to form a narrow slit to fluid flow. The main advantage of the spiral wound module is its surface area to volume ratio, which is fairly high and results in a lower floor area. The other advantages of spiral wound module are low capital cost and low power consumption (Herron et al., 1994; Kunz et al., 1996; Lawson and Lloyd, 1997; Hogan et al., 1998; Petrotos et al., 1999; Wong and Winger, 1999; Shaw et al., 2001; Vaillant et al., 2001).

6 Applications 6.1 OMD Most of the work in OMD has been carried out at lab scale to concentrate numerous fruit juices, vegetable juices, natural food colours, proteins and other aqueous solutions. Only a few reports are available at pilot scale. OMD can be employed as a pre-concentration step prior to relatively costlier processes such as lypholization, in the case of thermally-sensitive products like enzymes/ proteins, natural food colours, etc. Another potential application is de-alcoholization of fermented beverages (wine or beer). The most common application of the OMD process is to concentrate fruit juices up to 70°Brix without product damage. The use of pressure driven membrane processes such as UF, RO, NF for the concentration of phycocyanin (natural food colourant/protein) may result in shear damage to

270 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

the product as well as membrane fouling (Jaoquen et al., 1999). In order to overcome these drawbacks, experiments using OMD have been carried out at CFTRI, Mysore, India. C-phycocyanin obtained from freshly harvested biomass (initial concentration of 0.9 mg/ml) was concentrated by employing the OMD process in a flat membrane module using a hydrophobic polypropylene membrane. The concentration of C-phycocyanin increased by around 220 per cent without product damage (confirmed by spectrophotometric analysis). Furthermore, the concentrated C-phycocyanin was lyophilized to obtain it in a powdered form, which can be readily used for food applications (Naveen et al., 2003a). The newer membrane OMD processes suffer from low flux, which limits their full commercial application. Also, using the RO process, the maximum achievable concentration is only up to 25–30°Brix due to osmotic pressure limitation. In order to overcome these drawbacks and to improve the product quality and process economics, concentration of liquid foods and natural colours by integrated membrane processes appears very attractive. Therefore, an integrated membrane process involving clarification by MF/UF, pre-concentration by RO and final concentration by OMD yields a high quality product with a significant reduction in production cost. This area has scientific potential and can provide an efficient scalable alternative athermal process for the concentration of liquid foods and natural colours without product deterioration. Table 10.3 summarizes some of the integrated membrane processes employed for clarification and concentration of various liquid foods (such as orange juice, grape juice, passion fruit juice, carrot juice and coconut water) and some of them are discussed below. Many researchers have carried out concentration of fruit juices (orange, passion fruit and grape) involving microfiltration (MF)/ultrafiltration (UF) followed by an OMD process on a pilot scale level. These studies demonstrated the feasibility of integrating OMD with MF to concentrate fruit juices to an intermediate concentration degree with high flavour quality (Bailey et al., 2000; Shaw et al., 2001). More recently, a three-stage hybrid membrane process for the concentration of ethanol-water extracts of the Echinacea plant (which is used as immunostimulant) has been investigated. This resulted in a highly concentrated product suitable for marketing in capsule form (Johnson et al., 2002). Table 10.3 Summary of integrated membrane processes Feed

Integrated processes

References

Grape juices

UF/RO and OD are integrated and fresh fruit juices were concentrated up to 65–70°Brix without product deterioration or loss of flavours

Hogan et al. (1998)

Orange and passion fruit juices

A pilot scale process involving MF and OE. The juices (orange and passion) were concentrated up to 33.5°Brix and 43.5°Brix, respectively. The quantitative analysis shows about 32–36% average loss of volatile components

Shaw et al. (2001)

Citrus (orange and lemon) and carrot juices

The integrated membrane process (UF, RO and OMD) was used. The citrus and carrot juices were concentrated up to 60–63 gTSS/100 g. Total antioxidant activity, aroma, colour and quality of the juices were better preserved during concentration

Cassano et al. (2003a)

Coconut water

The RO concentrated coconut water (20–25°Brix) was further concentrated up to 56°Brix by using OMD. The sensory analysis shows that there is not much significant difference between fresh coconut water and final concentrate

Rastogi et al. (2003)

Suggestions for future work 271

An integrated membrane process for the production of concentrated kiwi fruit juice has been evaluated. Fresh kiwi fruit juice, after enzymatic treatment was subsequently clarified/concentrated by UF and OD. The UF clarified juice was concentrated by an OD process up to about 60°Brix. A small reduction of total antioxidant (TAA) was observed and the vitamin C content was well preserved in the final concentrate (Cassano et al., 2003b). Possible integration of aqueous two-phase extraction (ATPE) with membrane processes such as OMD/DO is being explored for the purification and concentration of food colours (especially when there are proteins). The use of ATPE will enable desired products (enzyme/protein) to partition to one of the phases, thus purifying and reducing the volume of the process stream to be handled. Furthermore, the OMD/DO process can be used as pre-concentration step to reduce the water load on subsequent processing steps such as freeze drying or subsequent purification steps such as electrophoresis, chromatography, etc.

6.2 DO DO can be employed to improve the quality of grape wines by concentrating a low quality grape juice into a product with increased soluble solids. It can be used to reduce the alcohol content in wine or beer products. Speciality products and pharmaceutical products such as flavouring agents and aloe vera, which are used in many cosmetic applications, have been concentrated using DO (Wong et al., 1999). Anthocyanin extracted from red radish extract was concentrated by conventional evaporative technology followed by direct osmosis (Rodrigrez-Saona et al., 2001). Another novel application (integrated with electrodialysis and RO) has been in the recovery of about 97 per cent water from wastewater. The relative non-fouling behaviour of DO allows processing of wastewater containing oils and soap scum that quickly foul the RO system (Beaudry and Herron, 1997). Currently, one plant in the USA has gone commercial to concentrate vegetable juices (cited by Wong et al., 1999).

7 Suggestions for future work In this chapter, the mechanism of water transport, effect of process and membrane related parameters in both OMD/DO processes have been discussed along with the advantages and potential applications of these processes both at lab scale and pilot scale. However, there is still ample scope for future research and development. Efforts are required to develop and evaluate hybrid processes on a larger scale involving MF/UF, RO and OMD/DO (Figure 10.6). If OMD/DO is to be made a commercially viable option, development of suitable membranes with improved diffusional characteristics, selectivity, better pore geometry and stability and membranes with longer life cycles needs to be undertaken at affordable costs. Another major constraint for the wide spread commercial application of OMD/DO process is the management of spent OA solution. An effective and

272 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

Feed reservoir

Reconcentrated OA solution

OA solution reservoir OMD/DO module

Condenser

MF/UF module

RO module

RO concentrate

Heat exchanger

Condensate To waste or recovery RO permeate OMD/DO concentrate

Evaporator

Lypholization (in case of natural colours)

Figure 10.6

Integrated concentration process for liquid foods and natural colours.

environmentally benign re-concentration technique for spent OA needs to be developed. Like any other membrane processes OMD/DO have the problem of low flux which needs to be addressed by the application of an acoustic field on a larger scale. Plant/marine algae constitute the major source of different natural colourants that differ in their colour and stability. Hence, efforts are required to carry out detailed studies in order to develop simple, efficient and economic methods for the processing of these natural colours and to develop possible process integration involving aqueous two-phase extraction (ATPE) with OMD/DO. In the view of the scientific and industrial potential of OMD/DO, even if some of these aspects, namely development of tailor-made membranes, optimization of process parameters and integration of process steps are addressed in greater depth by future researchers, it is certain that both OMD/DO processes will find their applications in the food and allied industry in the years to come.

8 Conclusions The potential advantages of athermal membrane processes (osmotic membrane distillation/direct osmosis) for the concentration of liquid foods and natural colours have

Nomenclature 273

been successfully demonstrated, with respect to maximum achievable concentration, improved product quality, ease of scale up and low energy consumption. Furthermore, efforts are required in the development of suitable membranes with improved diffusional characteristics, selectivity and with longer life cycles in order to make osmotic membrane distillation/direct osmosis viable options. Improvements of process engineering in terms of module design as well as process design and optimization are required in order to overcome the drawbacks like low flux. Integrated membrane processes involving microfiltration/ultrafiltration, reverse osmosis and osmotic membrane distillation/direct osmosis are expected to gain prominence in the near future for processing of liquid foods and natural colours. The integrated membrane processes allow improvements in the process efficiency and economy. Efforts are required also to develop possible integration of osmotic membrane distillation/direct osmosis with aqueous two-phase extraction for extraction, purification and concentration of natural colours and biomolecules. Due to the ever increasing cost of energy, it can be easily anticipated that the athermal membrane processes will be the technology of the future in the food and allied industries.

Acknowledgements The authors thank Dr V Prakash, Director, CFTRI, Mysore, for his encouragement and keen interest in the research work on athermal membrane processes. Financial assistance from the Department of Science and Technology, New Delhi, is gratefully acknowledged. Naveen Nagaraj and Ganapathi Patil thank the Council of Scientific and Industrial Research for Senior Research Fellowships.

Nomenclature B cp CSDI CSM D Dm h H J k K K KS kT

pore shape geometry factor heat capacity (J/kg/K) solute concentration in feed tank (mol/l) solute concentration in the membrane (mol/l) diffusion coefficient (m2 /s) moles of solute transported through the membrane in time dt (mole) liquid heat transfer coefficient (W/m2/K) total heat transfer coefficient (W/m2/ K) flux, mass (kg/m2/h), molar (mol/m2/s), volume (m3/m2/s) mass transfer coefficient in boundary layer (m/s) resistivity of the membrane (m2/h/kg) overall mass transfer coefficient (kg/m2/h/Pa) solute partition coefficient between adjacent solution and membrane thermal conductivity (W/m/K)

274 Athermal Membrane Processes for the Concentration of Liquid Foods and Natural Colours

L M P Q R r S t T u xs Yln ␧ ␦ ⌬ ␥ ␮ ␹ ␪ ␳ ⌸

length of the membrane (m) molecular weights of constituents (kg/mol) vapour pressure (Pa) heat flux (W/m2) gas constant (kJ/mol/K) membrane pore radius (m) membrane surface area (m2) thickness of porous substructure (m) temperature (K or °C) velocity of the fluid (m/s) osmotic agent molar agent mole fraction of air (log-mean) [⫺] porosity [⫺] membrane thickness (m) difference surface tension (N/m) viscosity of the fluid (Pa/s) tortuosity factor [⫺] contact angle (ds) density of the fluid (kg/m3) osmotic pressure (bar)

Hydrodynamic dimensionless numbers Nu Nusselt Pr Prandtl Re Reynolds Sc Schmidt Sh Sherwood Subscripts 1 feed side 2 OA side k knudsen diffusion L liquid M molecular diffusion S solute W water or vapour Abbreviations ATPE aqueous two-phase extraction DO direct osmosis DOC direct osmosis concentration MD membrane distillation MF microfiltration OA osmotic agent

References 275

OE OMD PP PTFE PVDF RO TSS UF

osmotic evaporation osmotic membrane distillation polypropylene polytetrafluoroethylene polyvinylidenedifluoride reverse osmosis total soluble solids ultrafiltration

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Naveen N, Chethana S, Jayaprakashan SG et al. (2003a) An improved process for the extraction, separation, purification and concentration of natural food colourant. IP No 195/NF/03. Naveen N, Ganapathi P, Ravindrababu B, Hebbar U, Raghavarao KSMS, Sanjay N (2003b) Modeling of mass transfer in osmotic membrane distillation. In Proceedings of Filtech Europa-2003, Dusseldorf, Germany, 2, 435–439. Petrotos KB, Quantick P, Petropakis H (1998) A study of the direct osmotic concentration of tomato juice in tubular membrane – module configuration. I. The effect of certain basic process parameters on the process performance. Journal of Membrane Science, 158, 110. Petrotos KB, Quantick P, Petropakis H (1999) Direct osmotic concentration of tomato juice in tubular membrane – module configuration. II. The effect of using clarified tomato juice on the process performance. Journal of Membrane Science, 160, 1–177. Petrotos KB, Lazarides HN (2001) Osmotic concentration of liquid foods. Journal of Food Engineering, 49, 201–206. Petrus CF, Nijhuis HH (1993) Application of membrane technology to food processing. Trends in Food Science and Technology, 4, 277–282. Philip T (1984) Purification and concentration of natural colourants by membranes. Food Technology, 38 (12), 107–108. Popper K, Camirand WM, Nury F, Stanley WL (1964) Dialyzer concentrates beverages. Food Engineering, 38 (4), 102–104. Rastogi NK, Raghavarao KSMS, Naveen N, Subramanium R, Maya P (2003) A novel athermal process for the concentration of tender coconut water, Patent No:177/NF/03. Ravindra BB (2003) Osmotic membrane distillation for the processing of liquid foods and natural colours. M.Tech Thesis, Visveswaraiah Technological University, Belgaum, India. Rodriguez-Saona LE, Giusti MM, Durst RW, Wrolstad RE (2001) Development and process optimization of red radish concentrate extract as potential natural red colourant. Journal of Food Processing Preservation, 25, 165–182. Schofield RW, Fane AG, Fell CJD (1987) Heat and mass transfer in membrane distillation. Journal of Membrane Science, 33, 299–313. Shaw PE, Lebrun M, Dornier M, Ducamp MN, Courel M, Reynes M (2001) Evaluation of concentration orange and passion fruit juices prepared by osmotic evaporation. Lebensmittel -Wissen und Technologie, 34, 60–65. Sheng J, Johnson RA, Lefebvre MS (1991) Mass and heat transfer mechanism in the osmotic distillation process. Desalination, 80, 113–121. Sherwood TK, Pigford RL, Wilke CR (1975) Mass Transfer. New York: McGraw-Hill. Vaillant F, Jeanton E, Dornier M, O’Brien G, Reynes M, Decloux M (2001) Concentration of passion fruit juice on an industrial pilot scale using osmotic evaporation. Journal of Food Engineering, 47, 195–202. Wong M, Winger RJ (1999) Direct osmotic concentration for concentrating liquid foods. Food Australia, 51 (5), 200–205. Wrolstad RE, McDaniel MR, Durst RW, Micheals N, Lampi KA, Beaudry EG (1993) Composition and sensory characterization of red raspberry juice concentration by direct-osmosis or evaporation. Journal of Food Science, 58 (3), 633–637.

High Intensity Pulsed Light Technology Luigi Palmieri and Domenico Cacace Stazione Sperimentale per l’Industria delle Conserve Alimentari (Experimental Station for the Food Preserving Industry), Angri (SA), Italy

Pulsed light technology (PLT) is an innovative method of purification and sterilization of food items by using very high-power and very short-duration pulses of light emitted by inertgas flashlamps. Such a technology, known since the 1980s, was approved by the Food and Drug Administration (FDA) in 1996 and recently has been widely investigated in view of possible commercial applications. In this chapter, the main topics about PLT are discussed, including the theoretical and technical principles, the mechanisms of microbial inactivation, the process critical parameters and the optimization criteria. In addition, some engineering details about pulsed light (PL) systems and some examples of experimental plants for treatment of food items are presented. Finally, the results of a number of experimental studies about the effects of PLT on microorganisms and food properties are reported. These results confirm that PLT could be a rapid, low-energy and low-thermally damaging technique for food microbial inactivation but also note that it should be limited to treatments of very transparent foods or surface treatments of foods and food packages.

1 Introduction Pulsed light technology (PLT) involves the use of inert-gas flashlamps which convert short-duration and high-power electric pulses, as those used in pulsed electric fields technology, into short-duration and high-power pulses of radiation included in the spectra of ultraviolet (UV), visible (VL) and infrared (IR) light. The large amount of power provided by the PLT can be used for a wide range of purposes, including achieving a rapid and effective purification or sterilization of foods and food-related items. The bactericidal effect of continuous UV light, formally discovered by Gates (1928), has been fully discussed and confirmed (Jagger, 1967; Smith, 1977) and today using UV light is a well-established technique of sterilization, particularly for food packaging films (Cerny, 1977). While many different PL devices were developed before 1970 for different industrial purposes, the use of inert-gas flashlamps generating intense and brief pulses of UV light as a technique of microbial inactivation definitely started during the late 1970s in Japan and was patented by Hiramoto (1984). In 1988 the PurePulse Technologies®, Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

11

280 High Intensity Pulsed Light Technology

a subsidiary of Maxwell Technologies®, acquired the Hiramoto patent and carried out extended experimentation which resulted in a new broad-spectrum PL process, patented by Dunn et al. (1989) and named Pure Bright®. During the subsequent years there were a lot of developments in PLT, including patents of different types of devices and equipment such as those by Wek-tec® and Xenon Corporation®, scientific publications about the features and the effects of PL treatments and some applications. A great increase in the research and development of PLT in the food industry occurred in 1996 when the FDA approved the use of PLT ‘for production, processing and handling of foods’ and recommended some conditions for such use. Some possible PLT applications concern semiconductor and DVD manufacture (Panico, 2000), cosmetic, dermatological and medical treatments (skin treatments, hair removal, vascular therapy), decontamination and sterilization of various instruments (medical or dental instruments), devices, packages, surfaces or atmospheres of laboratories, hospitals and any environments requiring a high degree of cleanliness (Barbosa-Canovas et al., 1997; Anonymous, 2003). In the food industry, PLT can be applied to sterilize, sanitize or reduce microbial load in foods, food packaging materials, as well as surfaces, environments, plants, devices and media (water, air) involved in food processes.

2 Principles of pulsed light technology The electromagnetic radiations are emitted and propagated by means of waves which differ in wavelength (␭), frequency (␯) and energy (E). The term light is generally used to mean radiations having ␭ ranging about from 180 to 1100 nm, which includes ultraviolet rays (UV, ␭ ⫽ 180–400 nm, roughly subdivided into UVA, 315–400 nm, UVB, 280–315 nm, UVC, 180–280 nm), visible light (␭ ⫽ 400–700 nm) and infrared rays (IR, ␭ ⫽ 700–1100 nm). Light can be emitted from different sources by different mechanisms, due to the spontaneous transition of some atoms from an excited state to a condition of lower energy. When a light radiation of energy E0 hits the surface of a material body, part of its energy (rE0, where r is the reflection coefficient of the material) is reflected by the surface, part of it is absorbed by the material layers through which it penetrates and part of it is transmitted to the inner layers (Figure 11.1). The energy E(x) of light transmitted to a distance x below the surface of a material body decreases with x according to the Lambert-Beer law (Dunn et al., 1989): E(x) ⫽ (1 ⫺ r) E0e⫺[␣]x

(1)

where ␣ is the extinction coefficient, which measures the transparency or the opacity of the material for each given ␭. Most solids are opaque (␣ : ⬁) and do not transmit radiations, while a lot of liquids and all gases are transparent (␣ : 0) and do not absorb any part of the energy; usually, in most materials (including foods), light intensity rapidly decreases while penetrating into the bulk.

Principles of pulsed light technology 281

Incident radiation of energy E0

Reflected radiation of energy rE0

Transmitted energy E(x) Absorbed energy Ed

Figure 11.1

x d

A schematic diagram of reflection, transmission and absorption of light radiations.

The energy Ed absorbed by a layer of depth d below the distance x is: Ed ⫽ E(x)(1 ⫺ e⫺[␣]d)

(2)

The absorbed light energy is generally dissipated as heat, resulting in a temperature increase equal to: ⌬T ⫽

Ed ␳cp Ad

(3)

where ␳ and cp are the density and the specific heat of the material and A is the surface area. Such ⌬T causes a temperature gradient between the outer and the inner layers of the material that gives rise to a conductive heat transfer within the material. The rate of both the heat transfer and the material temperature increase depends on the intensity and duration of the incident radiation and on the thermal properties of the material. The effect of a radiation upon a material body can be better evaluated using the energy density or fluence F, defined as the energy delivered to a unit material surface and measured by kJ/m2. Light can be delivered either continuously or in the form of pulses. Several quantities have to be introduced in order to understand better the energy transfer to a material by means of a series of pulses of light. If a process consists of a number n of pulses and each of them has a duration t and energy density or fluence F, the total duration is ttot ⫽ n ⭈ t, the frequency f ⫽ 1/t, the total fluence Ftot ⫽ n ⭈ F. Finally, a pulse power density or fluence rate Fr ⫽ F/t, can be defined and measured by kW/m2. The most important feature of delivering energy in the form of pulses is that apart from the number and duration of pulses, power provided by the pulses is greater than that provided by a continuous light radiation of equivalent total energy; total energy being equal, the shorter the duration of each pulse, the higher the pulse power (Figure 11.2). For this reason, if compared with continuous light radiations, light pulses show a much higher penetrating capability through the materials (Dunn et al., 1989).

282 High Intensity Pulsed Light Technology

1.0

E ⫽ 1 kJ t ⫽ 1 ms P ⫽ 1 MW

Power (MW)

Light pulse of 1 ms

Light pulse of 2 ms

0.5

E ⫽ 1 kJ t ⫽ 2 ms P ⫽ 0.5 MW

Continuous light

0.001

E ⫽ 1 kJ t ⫽ 1000 ms P ⫽ 0.001 MW 0

1

2

1000 Time (ms)

Figure 11.2 Power delivered by continuous light and light pulses of different duration, having equal energy content.

Another important consequence of the short duration of light pulses is the reduced time availability for thermal conduction inside the material. This results in a very rapid heating of a thin surface layer up to a temperature much higher than the steadystate temperature achieved by a continuous light radiation of equivalent total energy, without significantly increasing the bulk temperature (Dunn et al., 1989; Dunn, 1996). The development of a PL system mainly involves the generation of high-power electrical pulses and their transformation into high-power light pulses. In a general PL system (Figure 11.3), continuous low-power electric energy is: 1 2 3 4 5

collected from a primary energy source accumulated and temporarily stored rapidly released and converted into pulsed high-power electric energy which is then converted into pulsed high-power light energy and finally delivered to the desired target.

The details of this process are explained below. 1 An electrical power supplier is generally used to convert line low-voltage AC power into high-voltage DC power. 2 Energy storage is normally performed by using a capacitor bank, i.e. a number of high-voltage capacitors connected in parallel, which accumulate energy from the electrical power supplier during the charge phase and release it during the

Principles of pulsed light technology 283

Line

Low-power low-voltage low AC continuous electric current

Electric energy supply (converter)

Low-power high-voltage low DC continuous electric current

Electric energy storage (capacitors)

Low-power high-voltage high DC continuous electric current

Electric pulse forming (switches)

High-power high-voltage high DC pulsed electric current

Pulsed light source (inert-gas flashlamps)

High-power pulsed light

Target

Figure 11.3

A flowsheet of a general pulsed light system.

discharge phase, thus supplying large amounts of current. As an alternative, Marx generators can be used, which differ from capacitor banks only during the discharge phase when all the capacitors are temporarily connected in series; therefore the Marx generators also work as voltage amplifiers supplying large amounts of higher voltage current (Pai and Zhang, 1995).

284 High Intensity Pulsed Light Technology

3 The conversion of the continuous low- into the pulsed high-electric power is obtained by means of special switches capable of handling very high power and performing opening/closing cycles of very short duration, by instantaneously passing from a perfect insulating condition to a perfect conducting condition. The action of the switches is regulated by a controller that determines the pulse shape and the electrical operating conditions in order to yield the optimum PL wavelength for a particular application (Pai and Zhang, 1995). 4 The high-power pulsed electric energy delivered by the switches is usually converted into high-power light pulses by means of gas-filled flashlamps or other PL sources. The current associated with the high-power electric pulses passes through the gas in the lamp transferring energy to some atoms of the gas which are carried in an ‘excited-state’; afterwards, they tend to go back spontaneously in conditions of lower energy giving off energy in the form of intense pulses of light. 5 The obtained energy is finally delivered to the target by various systems depending on the different applications.

3 Effects of pulsed light on food products 3.1 Effects of PL on microorganisms The extremely high power provided by PLT has been fully demonstrated to be able to inactivate microorganisms to various extents. Many different causes and mechanisms have been proposed to explain the inactivation effect of PL on food microorganisms (Barbosa-Canovas et al., 2000). To date, the most accepted hypothesis for such an effect consists of a combination of both a photochemical mechanism, involving lethal effects of light pulses on some constituents of microbial cells, and a photothermal mechanism, due to a temperature increase determined by heat dissipation of light pulses penetrating the product. The main photochemical effect can be attributed to the well-known action of UV light on the DNA of microbial cells (Farkas, 1997). DNA (deoxyribonucleic acid), which is essential for the reproduction of all microorganisms, absorbs UV light energy mainly through the highly conjugated carbon-to-carbon double bonds (Jay, 1996). Such absorbed energy is able to break organic molecular bonds, causing DNA rearrangement, cleavage and destruction and to activate both electronic and photochemical reactions that can produce some substances inhibiting DNA reproduction, including pyrimidine dimers and pyrimidine (6–4) pyrimidone photoproducts, as well as thymine dimers (Hariharan and Gerutti, 1977; Franklin et al., 1985; Glickman et al., 1986). It was reported (Anonymous, 2000) that samples of DNA extracted from E. coli after exposure to PL showed a large number of random double- and single-stranded cleavages, while a lot of thymine dimers were formed on the surviving strands. On the other hand, it was also found (Anonymous, 2002) that exposure at approximately 260 nm PL caused formation of pyrimidone dimers in DNA. These modifications finally result in

Effects of pulsed light on food products 285

mutations, damage to the genetic information, impairment of replication and gene transcription and then in the death of the microorganism cells. It is known (Friedberg, 1985) that the DNA molecule shows a remarkable capability of reacting to modifications and damage by means of a self-repairing action carried out by some enzymes. It has been noted that the great inactivating effect of PL is mainly due to the almost total absence of any enzymatic repairing action in DNA damaged molecules (Anonymous, 2000), contrary to what happens after application of continuous UV radiations (Block, 1991), whose effects are quite reversible and therefore less effective. This can be attributed (Barbosa-Canovas et al., 2000) both to the much higher power levels of PL, which may cause either an extent of damage too large for the repair mechanisms to be effective or the destruction of the DNA repair system itself, and to the much shorter duration of PL, which allows no opportunity for cell adaptation mechanisms. The greater inactivation effect of PL was confirmed by Dunn et al. (1995) who found that under certain experimental conditions more than 7 logs of Aspergillus niger spores inactivation were achieved by a few light pulses, while using continuous UV light, even a strong treatment in time and energy was not able to obtain more than 3–5 logs of inactivation. Many authors (Barbosa-Canovas et al., 2000; Morgan, 1989) stressed that damage to DNA is mainly due to shorter wavelengths in the UVC range, which have higher energy levels. As a demonstration of this, Wekhof et al. (2001) reported that by eliminating UVC from the spectrum of light pulses, the inactivation of Aspergillus niger spores inoculated on a polyethylene terephtalate (PET) surface in given experimental conditions, was from three to five times less effective, depending on the intensity of the treatment. Concerning the photothermal mechanism of microbial inactivation, it has been already introduced that most energy of the light pulses that penetrate through a food product is absorbed by the layers nearest to the surface and dissipated as heat, causing in such thin layers a certain increase in temperature. Since microbial cells have a higher absorption of the pulsed light than that of the surrounding medium (water), this determines a localized rapid heating of microorganisms, which at very high pulse power values can reach temperatures (about 130°C) sufficient to cause their overheating, rupture and death and the extremely short pulse duration prevents the microorganism cell surface from being cooled by the surrounding medium (Wekhof, 2000). Wekhof et al. (2001) measured the temperature increase of E. coli cells on a polymeric surface when exposed to UVPL in different conditions and found that no temperature increase was detected until a fluence threshold was exceeded, after which temperature rapidly took off up to values higher than 120°C. The localized nature of such overheating was confirmed, according to Wekhof et al. (2001), by the fact that no damage to the polymeric material surface, which had a melting temperature of about 120°C, was detected. The different contributions of the spectral intervals of PL to microbial inactivation was also studied by Wekhof et al. (2001), who exposed Aspergillus niger spores on a PET surface to light pulses having different spectral distribution and different fluence values and reported that at lower fluence values (around 10–30 kJ/m2), at which temperature increase is not high enough to thermally inactivate microorganisms, the inactivation

286 High Intensity Pulsed Light Technology

effect was mainly (80–90 per cent or more) due to the photochemical effect of UVC light, while at higher fluence values (around 50–60 kJ/m2), the contribution of photothermal action of UVB and UVA light increased up to 40–60 per cent. In addition, Dunn et al. (1989) found a threshold relationship between fluence and inactivation of E. coli in a microbiological medium when using only wavelengths greater than 300 nm, which confirmed that in the absence of UVC light, the inactivation depends only on the heating of the product surface and the inactivation is not significant until the fluence threshold required for such heating is reached. Nevertheless, it is interesting to note that according to the photothermal overheating model, inactivation of microorganisms is due to the entire PL spectrum, including IR and visible light and it justifies an effective PL treatment even by using a UVC-free light spectrum. Therefore, it may be summarized that to date PL-induced microbial inactivation can be explained by a combination of both a lethal photochemical effect of UV (mainly UVC) light pulses on the DNA of microbial cells and a lethal photothermal effect of the broad spectrum light (including IR and visible light) pulses on the structure of microbial cells. In the past few years, the inactivation effect of PLT on various microbial species has been widely studied by many researchers in different foods and food-related items and over a wide range of experimental conditions. In Tables 11.1–11.4, a summary of selected scientific works published up to 2003 about PLT in foods and food-related items, including experimental conditions, is presented.

3.2 PL process optimization 3.2.1 General considerations

From the results in Tables 11.1–11.4, some generalizations can be made about the effects of PL on microorganisms and about the parameters affecting such effects and some suggestions for the optimization of PL treatments can be obtained. For optimizing the PL treatments, a preliminary requirement is provided by the FDA, which recommends that ‘food treated with pulsed light shall receive the minimum treatment reasonably required to accomplish the intended technical effect’. In designing a PL treatment for food items, both source (as light wavelength, energy density, duration and number of the pulses, interval between pulses) and target (as product transparency, colour, size, smoothness and cleanliness of surface) parameters are critical for process optimization, in order to maximize the effectiveness of product microbial inactivation and to minimize product alteration. Such alteration can be mainly determined by an excessive increase of temperature causing thermal damage to foods but also by an excessive content of UVC light which could result in some undesired photochemical damage to foods or food packaging materials. As already discussed, the temperature increase of products exposed to PL is much lower and localized in a thinner surface layer than that of an equivalent continuous light (CL) treatment, due to the short duration of pulses. For this reason, the food (except for a very superficial layer) is not heated to a temperature that can significantly alter its characteristics. For example, Dunn et al. (1989) found that in hard crusted bread

Effects of pulsed light on food products 287

Table 11.1 A summary of selected scientific works about effects of PL on microorganisms in solid food items Item

Experimentals

Results/remarks

Reference

Shrimp

BSPL F ⫽ 1–2 J/cm2 n ⫽ 4–8

1–3 lcr a of Listeria (inoc.b), resulting in a shelf-life extension of 1 week versus untreated samples

Dunn et al. (1989)

Curds of dry cottage cheese

BSPL F ⫽ 16 J/cm2, n ⫽ 1–2

1.5 lcr of Pseudomonas sp.c

Dunn et al. (1989)

Freshly baked cakes packaged in clear plastic containers

BSPL F ⫽ 16 J/cm2 n⫽3

Absence of moulds in treated Dunn et al. (1989) samples after storage at room temperature for 11 days, while untreated samples were very mouldy

Hard crusted white bread rolls

BSPL F ⫽ 16 J/cm2 n ⫽ 1–2

1.5 lcr of mould sp. with n ⫽ 1 and 2.7 lcr of mould sp. with n ⫽ 2

Dunn et al. (1989)

Packed white bread slices

BSPL

Fresh appearance for more than 2 weeks, without surface mould formation, while untreated samples were very mouldy

Rice (1994)

Meat

BSPL

Reduction of Listeria and Salmonella population

Rice (1994)

Chicken wings

BSPL

2 lcr of Salmonella (inoc.)

Dunn et al. (1995)

Frankfurters

BPSL F up to 30 J/cm2

2 lcr of Lysteria innocua (inoc.)

Dunn et al. (1995)

Retail meat

BSPL

1–3 lcr of total aerobic, lactic, enteric bacteria and Pseudomonas

Dunn et al. (1995)

Commercial or raw eggs

BSPL F ⫽ 0.5 J/cm2, n ⫽ 8

Up to 8 lcr of Salmonella enteriditis (inoc.) Inactivation effect observed on eggshells and a little extended into the egg pores

Dunn (1996)

Wax-coated strawberries

BSPL F ⫽ 0.5 J/cm2, n ⫽ 4

No mould growth after 2 weeks storage at room temperature

Dunn et al. (1996)

HDPE Prepackaged catfish fillets

BSPL F ⫽ 0.25–0.50 J/cm2 n ⫽ 2–4

Psychrotropic (PPC) and coliform (TCC) bacteria were not reduced initially by any treatment. After one week of storage, PPC were 1 (in treated

Shuwaish et al. (2000)

(Continued)

288 High Intensity Pulsed Light Technology

Table 11.1 (Continued) Item

Experimentals

Results/remarks

Reference

samples with F ⫽ 0.25) or 2 (in treated samples with F ⫽ 0.50) log cfu/g lower than untreated samples, TCC were reduced from about 50 to less than 10 cfu/g Eggshells

BSPL F ⫽ 1.5 J/cm2 n ⫽ 1–6

lcr of Bacillus subtilis sp. (inoc.) ranging from 3 to 6 with n ranging from 2 to 6

Mimouni (2000)

Cake

BSPL F ⫽ 1.5 J/cm2 n ⫽ 1–16

lcr of Aspergillus niger sp. (inoc.) ranging from 3 to 6 with n ranging from 2 to 6 Shelf-life increased from 26 days for untreated samples to 6 months for treated samples

Mimouni (2000)

Packed slices of bread

BSPL F ⫽ 1.5 J/cm2

Shelf life increased from 16 days for untreated samples to 5 months for treated samples

Mimouni (2000)

Corn meal

UVPL

Up to 5 lcr of fungal sp. of Aspergillus niger

Jun et al. (2003)

Clover honey

UVPL F ⫽ 5.6 J/cm2 n ⫽ 15–540

Samples 2 mm deep: reduction of sp. of Clostrydium sporogenes (inoc.) ranging from 39.5 to 73.9% with n ranging from 135 to 405 Samples 8 mm deep: reduction of sp. of Clostrydium sporogenes (inoc.) ranging from 0 to 89.4% with n ranging from 15 to 540

Hillegas and Demirci (2003)

a

⫽ log cycle reduction; b ⫽ inoculated; c ⫽ spores.

Table 11.2 A summary of selected scientific works about effects of PL on microorganisms in liquid food items Item

Experimentals

Results/remarks

Reference

Potable and ingredient water

BPSL

6–7 lcra/ml of Klebsiella terrigena with F ⫽ 0.5 and n ⫽ 2 6–7 lcr/ml of Cryptosporidium parvum with F ⫽ 1 and n ⫽ 1

Dunn et al. (1995)

Effects of pulsed light on food products 289

Table 11.2 (Continued) Item

Experimentals

Results/remarks

Reference

Liquids in plastic recipients

BSPL F ⫽ 4.5 J/cm2 n⫽2

About 6 lcr of a variety of inocb. sp.c and veg.d microorganisms

Mimouni (2000)

Water in plastic bottles

BSPL

About 5 lcr of Aspergillus niger (inoc.) and Bacillus stearothermophilus (inoc.) with Ftot ⫽ 3

Mimouni (2000)

Washing water

BSPL

⬎7 lcr of a microbial population (inoc.) with Ftot ⫽ 6

Mimouni (2000)

Flowing water

BSPL F ⫽ 0.25 J/cm2 n⫽1

4.6 lcr of Cryptosporidium parvum (inoc.) 7.5 lcr of Klebsiella terrigena (inoc.) 4.9 lcr of Simian rotavirus SA11 (inoc.) 6.2 lcr of Poliovirus type 1 (inoc.) 4.3 lcr of Bacteriophage MS-2 (inoc.) 5.4 lcr of Bacteriophage PRD-1 (inoc.) 7.7 lcr of Pseudomonas fluorescens (inoc.) 6.0 lcr of Bacillus stearothermophilus (inoc.)

Anonymous (2000)

Physiological solutions in PE pouches

BSPL n⫽8

⬎6 lcr of Clostridium sporogenes sp. (inoc.) and Bacillus pumilus sp. (inoc.)

Clark et al. (2003)

Water in PE containers

BSPL F ⫽ 1 J/cm2, n ⫽ 10–20

⬎6 lcr of Aspergillus niger (inoc.), Bacillus subtilis sp. (inoc.), Bacillus pumilus sp. (inoc.)

Clark et al. (2003)

Transparent model liquids

BSPL n ⫽ 1–8

6–8 lcr of E. coli, Staphylococcus aureus and E. faecalis with n ⫽ 1 lcr of ascospores of Aspergillus niger ranging from 3 to 4 with n ranging from 1 to 2 lcr of Bacillus subtilis sp. ranging from 5 to 6 with n ranging from 1 to 2

Tonon and Agoulon (2003)

a

⫽ log cycle reduction; b ⫽ inoculated; c ⫽ spores; d ⫽ vegetative.

Table 11.3 A summary of selected scientific works about effects of pulsed light on microorganisms in food-related items Item

Experimentals

Results/remarks

Plastic lids and cup surfaces

BSPL No growth of Staphylococcus aureus F ⫽ 0.25–3 J/cm2 (inoc.b) with F ⬎1 n⫽1 No growth of Bacillus cereus sp.c (inoc.) with F ⬎1.75 No growth of Aspergillus niger (inoc.) with F ⬎2

Reference Dunn et al. (1989)

(Continued)

290 High Intensity Pulsed Light Technology

Table 11.3 (Continued) Item

Experimentals

Results/remarks

Reference

Dry PE surfaces

BSPL F up to1.5 J/cm2 n⫽1

lcra of Bacillus pumilus sp. (inoc.) ranging from 1 to 5.5 with F ranging from 0.26 to 1.5 lcr of Bacillus subtilis sp. (inoc.) ranging from 1 to 5.5 with F ranging from 0.28 to 1.5 lcr of Bacillus stearothermophilus sp. (inoc.) ranging from 1 to 5 with F ranging from 0.29 to 1.5 lcr of Aspergillus niger sp. (inoc.) ranging from 1 to 7 with F ranging from 0.12 to 1

Anonymous (2000)

PET pieces and glass plates

BSPL–VPL F up to 5 J/cm2 n up to 5

BSPL: lcr of Aspergillus niger sp. ranging from 2 to 6 with F ranging from 1 to 5 and n ⫽ 1 VPL: lcr of Aspergillus niger sp. ranging from 0.2 to 1.3 with F ranging from 1 to 5 and n ⫽ 1 BSPL: lcr of Bacillus subtilis sp. ranging from 1.7 to 4.6 with F ranging from 0.25 to 5 and n ⫽ 1 VPL: lcr of Bacillus subtilis sp. ⬍1 with F ranging from 1 to 5 and n ⫽ 1

Wekhof et al. (2001)

White plastic packaging materials

BSPL F ⫽ 0.34–1.3 J/cm2 n ⫽ 1–40

No survivors of inoc. population of Aspergillus niger with F ⫽ 1.3 and n ⫽ 1, 0.48 log survivals with F ⫽ 1 and n ⫽ 1, 0.5 log survivals with F ⫽ 0.75 and n ⫽ 7, 1.67 log survivals with F ⫽ 0.53 and n ⫽ 20, 1.73 log survivals with F ⫽ 0.3 and n ⫽ 40

Clark et al. (2003)

a

⫽ log cycle reduction; b ⫽ inoculated; c ⫽ spores.

Table 11.4 A summary of selected scientific works about effects of pulsed light on microorganisms in microbiological media Item

Experimentals

Results/remarks

Reference

Microbiological media

BSPL-VPL F ⫽ 0.05–12 J/cm2 n ⫽ 1–35

10 lcra of Escherichia coli (inoc.b) with Dunn et al. (1989) F ⫽ 1.5 and n ⫽ 2 or with F ⫽ 4 and n ⫽ 1 10 lcr of Bacillus subtilis veg.d (inoc.) with F ⫽ 1 and n ⫽ 4 or with F ⫽ 4 and n ⫽ 2 10 lcr of Bacillus subtilis sp.c (inoc.) with F ⫽ 1.5 and n ⫽ 2 or with F ⫽ 4 and n ⫽ 1

Effects of pulsed light on food products 291

Table 11.4 (Continued) Item

Experimentals

Results/remarks

Reference

8 lcr of Staphylococcus aureus (inoc.) with F ⫽ 0.2 and n ⫽ 4, 10 lcr with F ⫽ 0.75 and n ⫽ 2 10 lcr of Saccharomyces cerevisiae (inoc.) with F ⫽ 0.4 and n ⫽ 4 10 lcr of Aspergillus niger sp. (inoc.) with F ⫽ 4 and n ⫽ 4 or with F ⫽ 12 and n ⫽ 1 VPL was far less effective in all cases, requiring much more F and n for comparable lcr Microbiological media

PL with ␭ ⫽ 200–530 nm F ⫽ 3 J/cm2 n ⫽ 1–512

lcr of Escherichia coli and Listeria monocytogenes ranging from 1 to 5 with n ranging from 64 to 512 lcr of Escherichia coli O157:H7 ranging from 1 to 5 with n ranging from 16 to 512

MacGregor et al. (1998)

Microbiological media

BSPL with either low or high content of UV n ⫽ 100–200

lcr of Escherichia coli, Listeria monocytogenes, Salmonella enteriditis, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus ranging from 3 to 6 with n ranging from 50 to 300 with high-UV light No significant lcr with any n of low-UV light

Rowan et al. (1999)

Microbiological media

UVPL n ⫽ 1–4

lcr of Bacillus subtilis sp. (inoc.) ranging from about 2 to about 5 with n ranging from 1 to 4

Sonenshein (2003)

Microbiological media

BSPL

3–5 lcr of Cryptosporidium oocysts with F ⫽ 0.11 and n ⫽ 2 or with F ⫽ 0.22 and n ⫽ 1

Dunn et al. (2001)

a

⫽ log cycle reduction; b ⫽ inoculated; c ⫽ spores; d ⫽ vegetative.

roll samples treated by broad spectrum light pulses of 160 kJ/m2, the surface temperature increase was negligible by using 1 pulse and about 5°C by using 2 pulses. Nevertheless, if the intensity of light or number or duration of pulses is relatively high, the temperature increase of the product may be greater than desirable and it can cause a real burning of the surface layers of food. This was confirmed by Hillegas and Demirci (2003) who treated samples of clover honey with a large number of 56 kJ/m2 UV pulses and observed that the sample surface temperature increased from 20°C to up 80–100°C when exposed to more than 50–100 pulses. In consideration of this, according to Dunn et al. (1989), the treatment should be limited so as to maintain the surface temperature increase to less than 50°C and preferably to less than 15°C, at least 10 seconds after such treatment.

292 High Intensity Pulsed Light Technology

3.2.2 Spectral distribution and treatment intensity

The most important ‘source’ parameters in PLT are the spectral distribution and the total fluence delivered by the light pulses. They are strictly cross-related and should be selected depending on the type of product and the extent of treatment required to achieve the desired microbial inactivation objective without adversely affecting product quality. With regard to spectral distribution, depending on the specific application, it is possible to use either the broad light spectrum, including UV, visible and IR radiations, or a selected range of wavelengths. Since the distribution of wavelengths in the spectrum of light emitted by a flashlamp strongly depends on the density of current exciting the lamp gas, the selected wavelength range can be obtained by properly adjusting the current density of the electrical pulses generated by the switches (Anonymous, 2003). The same result can be achieved by using some proper filters which absorb the radiations of undesired wavelengths and prevent them reaching the product (Dunn et al., 1989). According to the described microbial inactivation mechanisms of PL, the UV wavelengths are the main cause of microbial inactivation, since they are only responsible for the photochemical effect and most responsible for the photothermal effect. This was widely confirmed by several researchers. Dunn et al. (1989) obtained 10 log reductions of Aspergillus niger spores by 4 pulses of 40 kJ/m2 or 1 pulse of 120 kJ/m2 using broad spectrum pulsed light (BSPL), while such conditions resulted in a substantially lower inactivation using visible pulsed light (VPL); in order to obtain 10 log reductions by VPL, at least 15 pulses of 80 kJ/m2 were required. Similarly, 10 log reductions of Bacillus subtilis spores were achieved by 2 pulses of 15 kJ/m2 or 1 pulse of 40 kJ/m2 of BSPL, while the same inactivation using VPL required at least 30 pulses of 80 kJ/m2. Rowan et al. (1999) reported the results of inactivation of surface inoculated E. coli using up to 300 pulses obtained by means of two light sources containing either a low- or a high-UV content (Figure 11.4). Low-UV PL resulted in a very poor

Cell number (log 10 cfu/plate)

9 8 7 6 5 4 3 2 0

50

150

100

200

250

300

Number of pulses High UV

Low UV

Figure 11.4 Inactivation of surface-inoculated E. coli using up to 300 pulses of either high- or low-UV content light (from Rowan et al., 1999).

Effects of pulsed light on food products 293

inactivating effect (1 log), while by using high-UV PL, about 1 to 6 decimal reductions were achieved when the number of pulses ranged from about 15 to 300. Finally, Wekhof et al. (2001) studied the effect of progressively eliminating UV wavelength ranges from the spectrum of PL on the population of Aspergillus niger and Bacillus subtilis spores inoculated on PET surfaces. BSPL resulted in a much faster and higher inactivation (6 log reduction by 1 pulse of 40 kJ/m2), while UVCfree light pulses were much less effective, particularly when UVB and UVA were also filtered out from the spectra (respectively 1.7, 1.2 and 1 log reduction by 1 pulse of 40 kJ/m2). These experimental findings show that UV-alone or UV-rich pulsed light results either in a greater microbial inactivation, power delivered being equal, or in an equivalent inactivation by using a reduced light power (as required by FDA) and then minimizing the heating of the product and its degradation. This led to the development of systems which, by increasing the current density of electrical pulses (Wekhof, 2000), use only or preferentially the wavelengths corresponding to the UV light (UVPL). However, because of the undesired photochemical damage due to UVC light, in some situations, when increasing the UV content in the spectral distribution of PL, it could be necessary to filter UVC light out from the spectrum by simply using flash lamps made of Pyrex tubes so that UVC photons are absorbed by the lamp walls (Wekhof, 2002). Moreover, according to Wekhof (2002), since some organic cells of foods absorb the UV light just as effectively as microorganisms, a light excessively rich in UV could result in a competitive pulse UV energy absorption by such cells and result in a less effective microbial inactivation. The determination of the intensity of the PL treatment for food items, in terms of total fluence delivered in the fixed spectral range, once again mainly involves a balance between the desired photothermal microbial inactivation effect and the undesired overheating of the product. According to FDA recommendations, ‘the total cumulative treatment shall not exceed 120 kJ/m2 which is more than sufficient to achieve a high inactivation of a wide range of microorganisms including bacterial and fungal spores’. In experimental food applications, 1 to more than 100 pulses having fluence 0.1–500 kJ/m2 were used, corresponding to power up to 107 kW/m2, due to the very short pulse duration. Higher total fluences generally result in higher levels of microbial inactivation. As an example, the effect of Ftot on the microbial inactivation can be evaluated by observing the data reported by Mimouni (2000) about the results of inactivation of spores of Bacillus subtilis inoculated on eggshells exposed to 1–6 pulses of 15 kJ/m2, resulting in total fluence of 15–90 kJ/m2 (Figure 11.5). The number of surviving cells decreased from the initial value of 7 106 to about 1, when Ftot increased from 15 to 90 kJ/m2 by a rate that resulted in a negative linear trend in a log chart. Such behaviour can suggest an interesting theoretical analogy with the well-known models and parameters of thermobacteriology (Anonymous, 2000). If the data of survivors for a given microorganism after exposure to single light pulses having different levels of pulse fluence F are plotted, graphs similar to that in Figure 11.6 can be obtained. In thermal sterilization, decimal reduction time DT is the time required to reduce a microbial population of 1 log at a given temperature T and can be obtained by graphs showing survivors versus different times of thermal treatment. Similarly, by applying a linear regression to the data in such logarithmic graphs as in Figure 11.6, the fluence

Number of survivors (Spores/eggshell)

294 High Intensity Pulsed Light Technology

10 000 000 1 000 000 100 000 10 000 1000 100 10 1

0

10

20

30

40

50

60

70

80

90

100

Total fluence (kJ/m2) Figure 11.5 Pulsed light-induced inactivation of inoculated spores of Bacillus subtilis on eggshells (reproduced from Mimouni et al., 2000). 1 000 000

Number of survivors

100 000 10 000 1000 100 10 1

0

2

4

6 DF11

8

10

12

14

16

Fluence of a single light pulse (kJ/m2) Figure 11.6 Example of relationship between pulsed light-induced microbial inactivation and fluence of a single light pulse.

required to reduce the microbial population of 1 log (that is 90 per cent) using a single light pulse can be defined as ‘decimal reduction fluence’ DF1 (kJ/m2). DF1 values can be used to compare resistance to PL of different microorganisms: the higher the DF1 value, the more resistant to PL the microorganism is. The test carried out by PurePulse Technologies (Anonymous, 2000) on a clean dry surface estimated DF1 values of 1.2, 2.6, 2.8, 2.9 kJ/m2 respectively for spores of Aspergillus niger, Bacillus pumilis, Bacillus subtilis and B. stearothermophilus, showing that Aspergillus niger spores were relatively more sensitive and then less resistant to PL treatments. The same result was obtained by Dunn et al. (1989), in which treatments having minimum

Effects of pulsed light on food products 295

Ftot ⫽ 120 kJ/m2 were required in order to achieve 10 log reduction of Aspergillus niger spores, while a treatment at 8–40 kJ/m2 was sufficient to achieve the same microbial inactivation of E. coli, Bacillus subtilis vegetative cells and spores, Staphylococcus aureus and Saccharomyces cerevisiae. Furthermore, Mimouni (2000) found DF1 values of 7.9 kJ/m2 for Bacillus subtilis spores in eggshell, 36.6 kJ/m2 for ascospores of Aspergillus niger in some snacks, 2.9 kJ/m2 for Bacillus subtilis spores on plastic PA/PE films. The above results confirm that each microbial species has a different resistance to PL. Rowan et al. (1999) reported that Gram-positive bacteria are more resistant to the effects of UV light (both continuous and pulsed) than Gram-negative ones. According to Dunn et al. (1991), mould spores are more resistant to PL than bacterial spores. Wekhof (2002) tried to correlate the dimensions of microorganisms and the effect of PL and suggested that smaller microorganisms were more resistant than the larger ones and advanced the hypothesis that this was due to the fact that smaller objects cool faster because of their higher volume/surface ratio. As a possible development in modelling PL-induced microbial inactivation, DFn values could be defined as the fluences required to reduce the microbial population of 1 log using n light pulses. In thermobacteriology, zT is the temperature increment necessary to reduce DT by 1 log; a similar parameter zF could be introduced as the increment in the number of light pulses necessary to reduce DFn by 1 log, i.e. to increase ten times the effectiveness of process at the same fluence value. If a logarithmic relationship is found, zF values can be estimated by carrying out tests at different number n of light pulses for a given F value and by estimating the slope of a log plot of DFn versus n. Once the value of Ftot is fixed, single values for F and n can be found by experimental tests. In fact, a desired effect of microbial inactivation can be achieved either by a higher number of lower fluence pulses or by a lower number of pulses having a higher fluence, but no exact relationships between these two parameters were reported. Clark et al. (2003) demonstrated that a treatment consisting of a single pulse of a higher fluence was far more effective in sterilizing than that consisting of a higher number of pulses of a lower fluence. They showed that on the surface of a white plastic packaging material inoculated with Aspergillus niger, no survivors were detected after a treatment of just one pulse of 13 kJ/m2 (Ftot ⫽ 13 kJ/m2), while 1.73 log survivals were found after a treatment of 40 pulses of 3.4 kJ/m2 (Ftot ⫽ 136 kJ/m2). On the contrary, Dunn et al. (1991) observed that 4 pulses of 10 kJ/m2 resulted in 10 log reduction of vegetative cells of Bacillus subtilis in a microbiological medium, while when using the same Ftot obtained by 1 pulse of 40 kJ/m2, only 6.5 log reduction was achieved. In some cases, a threshold value for the number of pulses was observed, below which no effect of PL is achieved. For example, MacGregor et al. (1998) found that in given conditions treatments of up to 32 light pulses did not achieve any reduction of cell survivors of E. coli and Lysteria monocytogenes, while an increase in the number of pulses up to 512 resulted in a corresponding increase of up to 5 log cycles reduction.

3.2.3 Time parameters

Concerning the time-related parameters, in most experimental applications, the duration of pulses ranged from 1 ␮s to 0.1 s (FDA recommends ⬍2 ms), so that even low

296 High Intensity Pulsed Light Technology

total energy density amounts resulted in a very high-power light pulses, while typical pulse frequencies were 1 to 20 pulses per second. When using a single flashlamp (or more than one lamp flashing simultaneously), the maximum pulse frequency is limited by the features and efficiency of the lamp cooling system, which is necessary to avoid lamp overheating and to increase lamp lifetime. However, the effective pulse frequency may be increased by employing multiple lamps which are sequentially flashed and by providing relative movement between the lamp and the product being treated (Dunn et al., 1989). Moreover, the longer the interval between pulses, the higher the amount of heat dissipated from the product surface. Therefore, in order to avoid undesired effects due to product overheating, such an interval should be as long as possible. Nevertheless, Dunn et al. (1989) suggested that it should range generally from 0.1 to 5 s and preferably less than about 2 s in order to allow the multiple pulses to have a cumulative effect.

3.2.4 Target parameters

Regarding ‘target’ parameters, in order to obtain an effective PL treatment for microbial inactivation, a food item has to be highly ‘transparent’ to the desired light wavelengths. This first means that the product surface should reflect light as little as possible, i.e. its reflection coefficient should be as low as possible; apart from this, the higher the product absorption coefficient, the more efficient the inactivating effect and the higher the product transmission coefficient, the deeper the layer within which such effect is achieved. Many fluids, such as water, have a high degree of transparency to a broad range of wavelengths including visible and UV light, while other liquids, such as sugar solutions and wines, exhibit a more limited transparency. Normally, in an aqueous solution, the higher the solute concentration, the lower the transparency and therefore the less effective the PL treatment is. In order to examine the penetration of light through liquids having different degrees of transparency, Tonon and Agoullon (2003) reported that the intensity of a 260 nm radiation at a 50 mm of depth became 63.8 per cent of its initial value in a transparent model liquid and only 0.1 per cent in a complex model liquid. As a demonstration of the effect of product absorptivity on VPL-induced inactivation, Dunn et al. (1989) found that 4 flashes of 120 kJ/m2 caused more than 6 log reductions in an E. coli culture seeded on a white surface, while no inactivation was detected in similar conditions when the surface was coloured black by india ink. In order to increase the light absorption of materials being treated by PL, some absorptionenhancing agents can be used (Dunn et al., 1989). These agents are photon-sensitive substances, such as dyes, having a very high optical absorption coefficient at the desired wavelength. These agents can be sprayed, vaporized or spread in the form of powder on the product surface or applied as a dissolved liquid. Mixtures of two or more components, having high absorption coefficients at different wavelengths, may be used to increase the optical absorption over the desired spectrum. Moreover, since the light absorption of a given microbial cell depends on the light wavelength, using agents which selectively enhance absorption of a specific wavelength can offer interesting applications for selective inactivation of microorganisms on foods or

Effects of pulsed light on food products 297

packaging materials (Mertens and Knorr, 1992). Although the absorption-enhancing agents may be easily removed from the product after processing, food items require usage of edible enhancing agents, generally based on approved food colorants such as carotene, lime green, black cherry, as well as natural cooking oils (Dunn et al., 1989). When using PLT for treating packaged foods or food packaging materials, the above considerations about transparency are referred to the packaging materials. For example, materials such as glass, polystyrene and PET, which allow visible light to penetrate through the container, are not transparent to the UV wavelengths that are essential for microbial inactivation and therefore they are not suitable for PL treatments. On the other hand, polymers such as polyethylene, polypropylene, polybutylene, EVA, nylon, Aclar and EVOH, transmit UV light and hence meet the requirements for PLT very well (Anonymous, 2000). In addition, ink printed labels or drawings could interfere with the light absorption of the treated item and should be avoided on the surface of packaging materials. Besides the intrinsic transparency of the material, for the success of a PL process it is very critical that the ‘condition’ of the item to be treated is suitable for the penetration of the light. This means that the product surface should be smooth, clear and without roughness, pores and grooves which could ‘shadow’ the microbial cells from the light, causing less complete light diffusion and thus reducing process effectiveness; for the same reason, the item to be treated should be clean and free of contaminating particulates. In addition, items having a complex geometry could have areas hidden from the light and could require a more accurate design of the treatment chamber in order for the light pulses to reach each point of the product surface. As reported by Dunn et al. (1995), PL treatments that achieved high levels of microbial inactivation on relatively simple surfaces, generally showed only 1–3 log reduction on complex surfaces such as meats. Since light absorption depends on the distance through which the light is passing, the thickness of the product and/or the package strongly affects the efficiency of the treatment and consequently the total fluence required to achieve the same microbial inactivation as the thinner the food item, the more efficient the PL treatment can be. The treatment can be further enhanced by cutting food products into thin slices or pieces so that light can penetrate almost through the entire product. The effect of product thickness on PL-induced microbial inactivation was studied by Hillegas and Demirci (2003) who treated both 2 mm and 8 mm thick clover honey samples by PL and showed that a treatment of 135 pulses of 56 kJ/m2 caused a 39.5 per cent reduction of C. sporogenes in 2 mm samples, while it had no effect in 8 mm samples; in the mean time 405 pulses of the same fluence resulted in 73.9 per cent reduction in the thinner samples and 14.2 per cent reduction in the thicker ones. Finally, in order to achieve almost 1 log reduction of such microorganisms in these latter samples, at least 540 pulses were required. Similar results were obtained by Tonon and Agouillon (2003) in milk. In samples treated with 4 pulses of 60 kJ/m2, an initial bacterial population was reduced to 28 per cent at a depth of 1 mm, 43 per cent at a depth of 2 mm, while no bacterial reduction was observed for depth greater than 4 mm.

298 High Intensity Pulsed Light Technology

3.3 Effects of PL on enzymes and food properties While microbial effects of PL have been largely investigated, relatively few studies have been carried out on the effects of PL treatments on food enzymes and the nutritional and sensory properties of foods. 3.3.1 Enzymes

PLT has been demonstrated (Dunn et al., 1989) to be effective in significantly reducing the activity of a wide variety of enzymes (oxidoreductases, hydrolases, lipases, isomerases, proteinases, etc.) present in some food product (fruit, vegetables, meats, fish and shellfish) surfaces within a layer of 0.1 mm deep. Concerning polyphenol oxidase (PPO), which causes enzymatic browning in lots of fruits and vegetables, Dunn et al. (1989) treated potato slices by 2–5 flashes of broad spectrum PL at a fluence of 30 kJ/m2 and observed that they retained their colour after a prolonged storage, while the untreated samples began to brown through the action of PPO in a few minutes; however, the opposite unexposed surfaces of treated samples showed visible browning. Similar effects have been demonstrated when treating slices of bananas and apples. In the same work, Dunn et al. (1989) reported that the activity of alkaline phosphatase, which catalyses the hydrolysis of phosphatase esters, was reduced by 60–70 per cent with a single pulse of broad spectrum light at fluence of 10 kJ/m2 and completely inactivated by 5 pulses at the same fluence. Experimental data from Tonon and Agouillon (2003) about the effects of PL on the activity of some enzymes suggest a possible mechanism similar to the competitive inhibition for alkaline phosphatase and a non-competitive inhibition model for trypsine. 3.3.2 Nutritional properties

The nutritional analysis (testing protein, riboflavin, nitrosamine, benzopyrene and vitamin C) carried out by Dunn et al. (1995) on frankfurters exposed to up to 300 kJ/m2 of PL total treatment showed that no differences between treated and untreated samples were observed. While a strong loss of riboflavin is observed in foods because of heat, oxygen and light, Dunn et al. (1995) reported that even strong PL treatments did not influence its concentration in beef, chicken and fish. A similar behaviour was found by Tonon and Agouillon (2003) for both riboflavin and vitamin E, whose content was reduced only to 95 per cent of its initial value by 4 light pulses and to 85 per cent by 8 light pulses. Figueroa-Garcia et al. (2002) found that fresh catfish fillets treated by 2–4 pulses of 2.5–5 kJ/m2 showed a slightly increased oxidative rancidity (thiobarbituric acid oxidation values) after 2 days of refrigerated storage, but such values did not differ from those of untreated samples thereafter and did not reach typical levels of rancidity for seafoods. 3.3.3 Sensory properties

For the effect of PLT on sensory properties of foods, few data are available. Sensory analysis was performed by Dunn et al. (1989) on various samples of summer flounder

Systems for pulsed light technology 299

fillets after exposure to 1–5 pulses having 20–100 kJ/m2, which is sufficient to lower significantly the surface coliform and psychrotroph microbial populations. They reported that after 7 days of refrigerated storage, the PL treated samples showed slightly less sensory acceptance if compared with the untreated samples, while after 15 days of refrigerated storage, odour, appearance, taste and texture of the PL treated samples scored better (score of 2.4) than those of the untreated ones (score of 1.4). Dunn et al. (1989) reported that no visible discoloration and no changes in taste were caused by 1–2 pulses of 20 kJ/m2 in dry cottage cheese. Shuwaish et al. (2000) found that neither Hunter colour values nor shear force values significantly changed in HDPE packaged catfish fillets treated by 2–4 pulses of 2.5–5 kJ/m2; the same treatments carried out by Figueroa-Garcia et al. (2002) on fresh catfish fillets did not cause any change in initial moisture and water-holding capacity, but such parameters slightly decreased during refrigerated storage.

4 Systems for pulsed light technology 4.1 Description of PL systems As described previously, a typical PL system for treatment of food items basically consists of an electrical unit that provides the high-power electrical pulses, a lamp unit that converts them into high power light pulses, a treatment chamber where the power is delivered to the item to be treated and some auxiliary equipment such as a data acquisition system, control and cooling systems. It is interesting to note that PLT is substantially similar to the pulsed electrical field technology (PEFT), which involves discharge of electrical power directly on to the product, without passing through the lamps. Therefore, the most distinguishing element of PLT is the conversion of electrical pulses into light pulses and for this reason some technological and engineering aspects of lamp systems will be discussed. Generally a flashlamp consists of a tube made of various materials (mainly quartz) and arranged in various forms (spherical, spiral, etc.) with two sealed electrodes usually made of tungsten (Dunn et al., 1989). The flashlamps are filled with inert gases at various pressures, which should have a high efficiency (about 45–50 per cent) of conversion of electrical energy into optical and include mainly xenon, krypton or a mixture of noble gases (Dunn et al., 1989). The lamp unit consists of one or more lamps; in the latter case, the lamps can flash either simultaneously or sequentially by means of an internal controller. As the spectral distribution and the total energy density of light pulses are the key parameters for a successful PL treatment, these parameters should be instantaneously and continuously monitored, controlled and verified during a PL process. Since these parameters mainly depend on the current intensity, lamps are equipped with a system of photodetectors (Clark et al. 2003), which provide data for adjusting the lamp current density in order to obtain the desired spectral distribution. Moreover, they allow monitoring of the performance of the lamp unit on a real-time basis. When the fluence in the

300 High Intensity Pulsed Light Technology

desired spectrum falls below a predetermined value, the preset conditions should be recovered by a feed-back system, otherwise the process would be suspended and the lamps could require replacement. Depending on the number of pulses delivered, the lifetime of the lamp ranges usually from six months to one year (Anonymous, 2000). In order to design an efficient process, it is essential to take maximum care in concentrating most light pulses produced by the lamp towards the target and thus minimizing any light reflection; this can be achieved by equipping the lamp with one or more reflectors usually consisting of parabolic-shaped surfaces with high reflective sidewalls (Dunn et al. 1989). For further optimization of light usage, Clark et al. (2003) suggest enclosing both the PL source and the product to be treated in a ‘cavity’ made by a highly reflective material; in this way, PL which initially did not directly hit the product could be reflected and ‘recycled’ within the reflective cavity until it interacts with the product. In order to avoid excessive heating of the lamp electrodes by the high power pulses, a cooling system using water or air is usually installed in the PL system. Plants are often equipped with a system of flowing air in order to remove ozone produced by oxygen in UV-induced reactions (Anonymous, 2000).

4.2 Examples of PL experimental plants Although many companies already produce PL plants for different non-food applications and some plants for food items treatment have been patented, PL is not yet applied on an industrial scale in the food sector. In the following, some examples of patented experimental PL plants for food treatment are presented. Dunn et al. (1989) patented a system for sterilizing pumpable foods such as water or fruit juices. The fluid food flows through an annular treatment chamber between an inner cylinder containing the lamps which emit the light pulses and an outer cylinder made of a highly reflective material which drives the light back through the food product. The flow rate of the product through the chamber is controlled by a pump according to the pulse frequency required to deliver the selected number of pulses to the product (Figure 11.7a). A plant for achieving sterilization of a flexible film for aseptic packaging while manufacturing the container can be arranged as follows (Dunn et al., 1989). A reel of the material is driven by rollers through a solution of an absorption-enhancing agent and the film is subsequently formed into a longitudinally sealed tube that moves surrounding an inner tube along which one or more flashlamps are distributed; such lamps provide the light pulses to sterilize the entire inner surface of the sealed packaging material tube. Then the tube is filled with a sterile food product flowing out from the inner tube and transversally sealed. A flow of sterile air is used both to cool the flashlamp and to remove any possible photochemical by-products from the tube (Figure 11.7b). A system for sterilizing preformed containers was patented by Dunn et al. (1989). The containers, optionally sprayed with an absorption enhancing agent solution, progressively move through a number of flashlamp treatment stations where the lamps

Figure 11.7

(c)

Flashlamps

(b)

Final containers

Product filling system

Product circulation pump

Preformed containers

In

Reflective surface

Reel of packaging material

Solution of absorption-enhancing agent

Some examples of layouts of possible pulsed light systems for treatment of food items (from Dunn et al., 1989).

Absorption-enhancing agent spraying system

Sterile air inlet

(a)

Out

Flashlamps

Treatment chamber

Final packages

Final sealing system

Product outlet and filling system

Inner support tube

Outer tube (packaging material)

Flashlamp

Longitudinal sealing system

Product inlet

Systems for pulsed light technology 301

302 High Intensity Pulsed Light Technology

are introduced above or into the container openings and provide the light pulses required to sterilize the internal surface of the containers. Then the sterilized containers are filled with a pre-sterilized food product and sealed by a lid, which can also be sterilized using light pulses (Figure 11.7c). A possible application of PLT to sterilize continuous inner surfaces of preformed containers, such as bottles, plastic jars or paper packs for milk and juices, was described by Wekhof (2002). The containers, located on a conveyer belt, pass under the lamp that dives into them and flashes. Afterwards the lamp returns to its initial position and the container is moved on. A different plant arrangement was proposed by Clark et al. (2003), in which the containers move on a conveyer belt and are lifted by means of a transfer device and placed in a treatment chamber made of four lamps simultaneously flashing and four reflectors arranged in a quadricilindrycal shape in order to maximize the light absorption; afterwards, the processed container is transferred to a second conveyer belt and a new container is moved into the chamber.

5 Conclusions Short-duration high-power (either UV or broad spectrum) light pulses have been sufficiently demonstrated to be a powerful tool in order to inactivate microorganisms in food items by a combination of both photochemical and photothermal mechanisms without apparently affecting product properties. Compared with conventional thermal sterilization, PLT allows achievement of an effective microbial inactivation requiring much lower processing time and energy, thus preserving more food nutritional and sensory properties at the same time, particularly if the photothermal temperature increase is properly controlled. Compared with some alternative techniques which use chemical preservatives or ionizing radiations, PLT does not involve toxic chemicals or photolytic by-products (the used wavelengths are too long to cause ionization of small molecules) that strongly reduce consumer acceptability of such systems. Furthermore, if compared with conventional continuous UV light treatment (UVCL), PL, due to its higher power and short duration, allows more effective microbial inactivation to be achieved requiring a relatively lower energy input, penetrating opaque or thick materials better than UVCL and causing significantly lower product thermal damage. In addition, it is claimed by the manufacturers that PL systems have relatively low operating costs and do not significantly contribute to the environmental impact because they do not produce volatile organic compounds or suspended airborne particulates and generate little liquid and solid wastes. Finally, for PL processes, a realtime process monitoring with treatment level verification can be easily realized. Apart from the high investment costs (€300 000–800 000), which limit possible PLT applications to high value added products and particular market situations, the major limitation of PLT is due to the intrinsically poor penetrating power of light and to the requirements of transparency and surface smoothness for the product to be

Nomenclature 303

treated. For this reason, PLT can be successfully used only either for bulk treatments of extremely transparent materials or for surface treatments of less transparent ones. Therefore PLT, can be applied to the food industry in the following areas:

• unpackaged solid foods requiring only a (primary) surface sterilization or decontamination • packaged solid foods requiring (terminal) sterilization or decontamination through PL-compatible packages • unpackaged liquid foods flowing through treatment chambers • liquid foods packaged in PL-compatible containers. Actually, the most interesting application of PLT could be in sterilizing films of packaging materials, particularly those used in the aseptic technology, as an important alternative to the use of hydrogen peroxide, whose residuals are not fully acceptable in foods and to conventional UV-continuous treatments, which can cause some undesired oxidative reactions in such materials. However, any possible commercial applications of PLT need further experimental confirmation from independent nonindustrial researchers. More extended studies on the resistance to specific PL treatments of the most common and critical food microorganisms and a more accurate validation of microbial inactivation effectiveness of such treatments are also required, as well as investigations about any potential undesired effects of PL, such as formation of toxic by-products. A valid prospect in PLT application could be obtained by combining it with other methods of preservation, such as high pressure or pulsed electrical fields technology.

Acknowledgements The authors are extremely grateful to their co-workers Rosaria D’Antonio, Maria de Rosa and Gian Paolo Gentile, whose contribution of expertise and care was invaluable throughout all the stages of the preparation of this chapter.

Nomenclature A BSPL CL cp d DF1 DFn DT E

surface area (m2) broad spectrum pulsed light continuous light specific heat (J/kg°C) layer depth (m) decimal reduction fluence using a single pulse (kJ/m2) decimal reduction fluence using n pulses (kJ/m2) decimal reduction time (s) energy (J)

304 High Intensity Pulsed Light Technology

E0 Ed f F Fr Ftot IR n P PEFT PL PLT r t T ttot UV UVA UVB UVC UVCL UVPL VL VPL x zF zT ␣ ⌬T ␭ ␯ ␳

energy of an incident radiation (J) energy absorbed by a layer of depth d (J) pulse frequency (1/s) energy density or fluence (of a single pulse) (kJ/m2) power density or fluence rate (kW/m2) total energy density or fluence (of n pulses) (kJ/m2) infrared number of pulses pulse power (kW) pulsed electric fields technology pulsed light pulsed light technology reflection coefficient duration of a single pulse (s) temperature (°C) total duration of n pulses (s) ultraviolet part of UV part of UV part of UV UV continuous light UV pulsed light visible light visible pulsed light distance below a surface (m) increment of n required to reduce DF by 1 log temperature increment to reduce DT by 1 log (°C) extinction coefficient temperature increase (°C) wavelength (nm) wave frequency (Hz) specific gravity (kg/m3)

References Anonymous (2000) PureBright® Sterilization Systems. PurePulse Technologies, now Maxwell Technologies, San Diego, USA. Anonymous (2002) Photochemical sterilization by pulsed light. Xenon Corporation, Woburn, USA. Anonymous (2003) Sterilization and decontamination using high energy UV light. Xenon Corporation, Woburn, USA. Barbosa-Canovas GV, Pothakamury UR, Palou E, Swanson BG (1997) Application of light pulses in the sterilization of foods and packaging materials. In Nonthermal Preservation of Foods, New York: Marcel Dekker Inc., pp. 139–159.

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Barbosa-Canovas GV, Schaffner DW, Pierson MD, Zhang QH (2000) Pulsed light technology. Journal of Food Science, Supplement, 65, 82–85. Block SS (2000) Disinfection, Sterilization and Preservation. Philadelphia: Williams & Wilkins Publishers. Cerny G (1977) Sterilization of packing materials for aseptic packaging 2. Investigation of the germicidal effects of UV-C rays. Verpackungs-Rundschau, 28 (10), 77–82. Clark WR, Lierman JC, Lander D, Dunn JE (2003) Parametric control in pulsed light sterilization. PurePulse Technologies Inc., San Diego, USA. US Patent 65 66659. Dunn J (1996) Pulsed light and pulsed electric field for foods and eggs. Poultry Science, 75 (9), 1133–1136. Dunn JE, Clark WR, Asmus JF et al. (1989) Methods for preservation of foodstuffs. Maxwell Laboratories Inc., San Diego, USA. US Patent 4871559. Dunn JE, Clark WR, Asmus JF et al. (1991) Methods for preservation of foodstuffs. Maxwell Laboratories Inc., San Diego, USA. US Patent 5034235. Dunn J, Clark W, Ott T (1995) Pulsed-light treatment of food and packaging. Food Technology, 49 (9), 95–98. Dunn JE, Clark WR, Bushnell AH, Salisbury KJ (2001) Deactivation of organisms using high-intensity pulsed polychromatic light. PurePulse Technologies Inc., San Diego, USA. US Patent 6228332. Farkas J (1997) Physical methods of food preservation. In Food Microbiology. Fundamentals and Frontiers (Doyle MP, Beauchat LR, Montville TJ, eds). Washington: ASM Press, pp. 497–519. Figueroa-Garcia JE, Silva JL, Kim T, Boeger J, Cover R (2002) Use of pulsed-light to treat raw channel catfish fillets. Journal of the Mississippi Academy of Sciences, 47 (2), 114–120. Franklin WA, Doetsch PW, Haseltine WA (1985) Structural determination of the ultraviolet light-induced thymine-cytosine pyrimidine-pyramidone [6-4] photo-product. Nucleic Acid Research, 13, 5317–5325. Friedberg EC (1985) DNA repair. New York: W. H. Freeman. Gates FL (1928) On nuclear derivatives and the lethal action of UV light. Science, 68, 479–480. Glickman BW, Schaaper RM, Haseltine WA, Dunn RL, Brash DE (1986) The C-C [6-4] UV photoproduct is mutagenic in Escherichia coli. Proceedings of the National Academy of Sciences, USA, 83, 6945–6949. Hariharan PV, Gerutti PA (1977) Formation of products of the 5–6 dihydroxy dihydrotymine type by ultraviolet light in HeLa cells. Biochemistry, 16 (12), 2791–2795. Hillegas SL, Demirci A (2003) Inactivation of Clostridium sporogenes in clover honey by pulsed UV-light treatment. Agricultural Engineering International: the CIGR Journal of Scientific Research and Development, V, December. Hiramoto T (1984) Method of sterilization. Ushio Denki Kabushikikaisha, Tokyo, Japan. US Patent 4464336. Jagger J (1967) Ultraviolet photobiology. Englewood Cliffs: Prentice-Hall Inc. Jay JJ (1996) Modern food microbiology. New York: Chapman & Hall. Jun S, Irudayraj J, Demirci A, Geiser D (2003) Pulsed UV-light treatment of corn meal for inactivation of Aspergillus niger. International Journal of Food Science and Technology, 38, 883–888.

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MacGregor SJ, Rowan NJ, McIlvaney L, Anderson JG, Fouracre RA, Farish O (1998) Light inactivation of food-related pathogenic bacteria using a pulsed power source. Letters in Applied Microbiology, 27, 67–70. Mertens B, Knorr D (1992) Developments of nonthermal processes for food preservation. Food Technology, 46 (5), 129. Mimouni A (2000) Application de la lumiere pulsee en agroalimentaire. Industries Alimentaires & Agricoles, October, 37–39. Morgan R (1989) Green light disinfection. Dairy Industries International, 54, 33–35. Pai ST, Zhang Q (1995) Energy Storage (ch. 2) and Switch (ch. 5). In Introduction to high power pulse technology. Singapore: World Scientific Publishing. Panico RL (2000) Pulsed UV curing for DVD production. Xenon Corporation, Woburn, USA. Rice J (1994) Sterilizing with light and electrical impulses. Food Processing, July, 66. Rowan NJ, MacGregor SJ, Anderson JG, Fouracre RA, McIlvaney L, Farish O (1999) Pulsed-light inactivation of food-related microorganisms. Applied and Environmental Microbiology, 65 (3), 1312–1315. Shuwaish A, Figueroa JE, Silva JL (2000) Pulsed-light-treated prepackaged catfish fillets. 2000 IFT Annual Meeting, 10–14 June, Dallas, USA. Smith K (1977) The Science of Photobiology. New York: Plenum Press. Sonenshein AL (2003) Killing of Bacillus spores by high-intensity ultraviolet light. Xenon Corporation, Woburn, USA. Tonon F, Agoulon A (2003) Lumiere pulse, principe et application au cas des solutions liquides. Industries Agro-alimentaires, la conservation de demain, 4e edition, 20 November 2003, Talence, France. Wekhof A (2000) Disinfection with flash-lamps. PDA Journal of Pharmaceutical Science & Technology, 54 (3), 264–276. Wekhof A (2002) Sterilising packaging and preserving foodstuffs with pulsed light. Newsletter of International UV Association, 4 (5). Wekhof A, Trompeter FJ, Franken O (2001) Pulsed UV disintegration (PUVD): a new sterilisation mechanism for packaging and broad medical-hospital applications. The First International Conference on Ultraviolet Technologies, 14–16 June 2001, Washington, USA.

Non-thermal Processing By Radio Frequency Electric Fields David J Geveke US Department of Agriculture, Eastern Regional Research Center, Wyndmoor, PA, USA

Radio frequency electric fields (RFEF) processing is relatively new and has been shown to inactivate bacteria in apple juice at moderately low temperatures. Key equipment components of the process include a radio frequency power supply and a treatment chamber that is capable of applying high electric fields to liquid foods. The process is similar to the pulsed electric fields process, except that the power supply is continuous rather than pulsed; therefore, the capital costs may be less. Using an 80 kW RFEF pilot plant unit, Escherichia coli K12 in apple juice flowing at 1.0 l/min was exposed to an electric field strength of 20 kV/cm at a frequency of 21 kHz. RFEF processing reduced the population of E. coli by 2.7 log at 60°C and a hold time of 3 s, whereas conventional heating at the same conditions had no effect. The electrical cost of the RFEF processing was $0.0011 per litre of apple juice. Increasing the electric field strength, number of treatment steps and temperature enhance the microbiological inactivation.

1 Introduction Consumers are drinking freshly squeezed fruit and vegetable juices in greater and greater quantities (Dahm, 2000). There is a growing demand for juices that have freshlike qualities. In addition, consumers are insisting that these beverages contain no pathogens. Although conventional thermal pasteurization assures the safety of these products, it can also affect sensorial and nutrient attributes. Therefore, alternative pasteurization processes are actively being sought. The use of radio frequency electric fields (RFEF) as a pasteurization method has been studied for more than 60 years. Although reports often claimed pasteurization had been achieved at non-thermal conditions and with low electric field strengths, these assertions could never be confirmed. Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

12

308 Non-thermal Processing By Radio Frequency Electric Fields

Decareau (1985) provided an excellent review of the early work on the effects of RFEF on microorganisms. Fleming (1944) concluded that, in the range of 11–350 MHz, inactivation of Escherichia coli was easy to obtain even with a small amount of power of 10 W. However, Brown and Morrison (1954) were unable to repeat Fleming’s work. Nyrop (1946) reported 99.5 per cent kill of E. coli at
40°C by applying a 0.23 kV/cm electric field at 20 MHz for a total of 7 s. However, details of the experimental apparatus and procedures were not described. Nyrop also claimed inactivation of foot and mouth virus by applying the same field strength for 10 s at
36°C, whereas inactivation using heat required 60 h at 37°C. Ingram and Page (1953), aware of the work of Nyrop, studied the effects of RFEF at 10 and 20 MHz on E. coli, tobacco mosaic virus, baker’s yeast and bacteriophage. The samples were maintained below 30°C and subjected to electric fields up to 2 kV/cm. No significant effects were observed under these conditions. More recently, Ponne et al. (1996) studied the effect of RFEF on liposomes, yeast and bacteria. These materials were exposed to frequencies ranging from 1 to 100 MHz and an electric field strength of
0.0034 V/cm while being maintained at
40°C. Although exposure of liposomes to frequencies of 27 and 100 MHz resulted in increased lysis of vesicles, no effects were demonstrated when low intensity RFEF was applied to S. cerevisiae and Erwinia carotovora. This led Ponne et al. (1996) to conclude that there was no non-thermal effect of RFEF on microbial inactivation. In summary, several studies have claimed that low intensity RFEF inactivate microorganisms by non-thermal means. The economic benefits of pasteurizing liquid foods with a low electric field strength and low power would be enormous. However, there is no evidence that this technique is being practised today by the food industry (Decareau, 1985; Mertens and Knorr, 1992). Some of the studies that disclaimed the existence of non-thermal effects suggested that erroneous temperature measurements were the cause of the ‘positive’ results reported by others. In order to test for the existence of low intensity RFEF non-thermal inactivation, a system that accurately and precisely controlled temperature was assembled (Geveke et al., 1999; Brunkhorst et al., 2000). When an electric field strength of 0.5 kV/cm at a frequency of 18 MHz was applied to apple cider, beer, deionized water and tomato juice, thermal effects on E. coli K-12, Listeria innocua and yeast were observed, but non-thermal effects were not detected (Geveke et al., 2002a). High intensity pulsed electric fields (PEF) were applied to suspensions of vegetative bacteria and yeast cells at room temperature (Sale and Hamilton, 1967). A minimum electric field strength of 5 kV/cm was necessary to achieve non-thermal inactivation. Also, increasing the electric field increased inactivation and yeasts were more sensitive than bacteria. Inactivation is thought to occur by electroporation (Chang et al., 1991). Cell membranes have electrical resistance and capacitance. In an electric field, a voltage is formed across the membrane. As the voltage is increased, the opposite charges on either side of the membrane are attracted to each other with greater force and the membrane becomes thinner. At a sufficiently high voltage, pores are formed in the membrane and the cell ruptures (Zimmermann, 1986). PEF processing of foods has attracted widespread attention (Dunn and Pearlman, 1987). Kotnik et al. (1998) reported on the time course of transmembrane voltage induced by RFEF. There is a lag between the applied voltage and the induced voltage across

Radio frequency electric fields equipment 309

the membrane due to its capacitance. Although they stated that it is generally very difficult to predict the peak value of the induced transmembrane voltage, it is clear from their analysis that the voltage is significantly reduced as the frequency is increased from 100 kHz to 1 MHz. Geveke et al. (2000, 2002a) hypothesized that non-thermal RFEF inactivation might be achieved if a field strength of 5 kV/cm and/or a frequency of 18 MHz was applied. A novel RFEF system was designed and assembled that was capable of applying a field strength of 30 kV/cm over a frequency range of 20–100 kHz to suspensions of S. cerevisiae in water (Geveke et al., 2002b). The combination of lower frequency and higher field strength resulted in the reduction of S. cerevisiae by 3.8 log or 99.98 per cent at 35°C (Geveke and Brunkhorst 2003a). The remainder of this chapter will cover RFEF equipment, additional results and recent developments, costs and the outlook for the future.

2 Radio frequency electric fields equipment High electric fields are produced by placing a liquid between two electrodes that are in close proximity to each other and applying a high voltage to the electrodes. The voltage can be applied by several different means. One method is to use direct current (DC), however, a disadvantage of this method is that undesirable electrolysis reactions may occur (Qin et al., 1994). Another shortcoming with DC is that charged particles in the liquid may form a layer on the anode that would require periodic cleaning. Using either bipolar waveforms or alternating current (AC) overcomes these problems. Bipolar waveforms are extensively used in PEF processing where a charging power supply produces a high voltage and a high speed electrical switch delivers the stored energy to the electrodes. The power supply must then be recharged which results in pulsed processing. The high cost of the pulse generator is a problem confronting the industrial application of PEF processing (Jeyamkondan et al., 1999). At a high pulse frequency and large scale of operation for industrial applications, the charging power supply and high speed electrical switch are the major costs of the pulse generator (Zhang et al., 1995a). In RFEF processing, an AC power supply continuously provides the high voltage. This potentially simpler method of generating high electric fields may have lower capital and operating costs than those associated with PEF processing. Several types of AC power supplies for non-thermal RFEF processing have been reported in the literature. Geveke et al. (2002b) designed and constructed a power supply that consisted of four 1 kW RF amplifiers (Industrial Test Products, Port Washington, NY, model 1000A) and four step up transformers (Industrial Test Products). A photograph of the system is presented in Figure 12.1. These were connected in series and produced a peak voltage of 4.0 kV over a frequency range of 20–100 kHz. A function generator (Tektronix, Beaverton, OR; model AFG 310) drove the amplifiers. The voltage and current were measured using a voltage divider (Ross Engineering, Campbell, CA; model VD15-8.3-A-KB-A), a current probe (Pearson Electronics, Palo Alto, CA, model 411) and an oscilloscope (Tektronix, model TDS210).

310 Non-thermal Processing By Radio Frequency Electric Fields

Oscilloscope 4 kW power supply

Function generator

Pump Figure 12.1 RFEF experimental system including pump, function generator, 4 kW power supply, oscilloscope, and data acquisition system.

Uemura and Isobe (2002) used a power supply consisting of a PC controlled signal generator (Hewlett-Packard, model HP8904A) and an amplifier (NF Circuit Block, model 4510). These produced a voltage of 0.3 kV at a frequency of 20 kHz. Recently, Geveke and Brunkhorst (2003b) have scaled up the RFEF process to achieve greater treatment capacity. The power supply that was conceived and put together consisted of an 80 kW RF power supply (Ameritherm, Scottsville, NY, model L-80) and a custom designed matching network that enabled the RF energy to be applied to a resistive load (Ameritherm) over a frequency range of 21.1–40.1 kHz. The nominal maximum voltage applied was limited to 5.0 kVpeak in order to control the temperature rise of the liquid. The voltage and currents were measured using a voltage divider (Ross Engineering, model VD15-8.3-A-KB-A), current probes (Pearson Electronics, CA, model 411) and an oscilloscope (Tektronix, model TDS224). Equal in importance to the power supply is the treatment chamber where the high electric fields are applied to the liquids. Several different designs have been published. Geveke et al. (2002b) designed and fabricated a novel cross-field treatment chamber made of Teflon. The liquid flowed through a bore with a diameter of 6.4 mm (Figure 12.2). Two stainless-steel electrodes were inserted into the Teflon perpendicular to and in contact with the liquid flow. The electrodes were cylindrical with a 6.4 mm diameter and their ends were rounded and polished. At their closest proximity the electrodes were 1.6 mm apart. The output of the RFEF power supply was connected to the electrodes such that the electric flux lines were approximately perpendicular to the direction of the liquid flow. Uemura and Isobe (2002) also used a treatment chamber in which the flux lines were perpendicular to the flow (Figure 12.3). Liquid flowed through four pairs of electrodes

Radio frequency electric fields equipment 311

Electrode

Liquid

Electrode

1.6 mm

Insulation

Figure 12.2 Cross-section of cross-field treatment chamber including Teflon insulation and rounded stainless steel electrodes.

Flow

m, Cp, T1

Electrodes

W

h AC d

T2

(a)

Liquid flow

Liquid flow

AC

Teflon spacer Titanium electrodes (b)

Thermocouple

Figure 12.3 (a) Schematic of cross-field treatment chamber including one pair of flat electrodes. (b) Schematic of chamber including all four pairs of electrodes and Teflon insulation (Uemura and Isobe, 2002).

312 Non-thermal Processing By Radio Frequency Electric Fields

in series. The electrodes were made of small titanium strips separated by a 0.2 mm gap. The electrodes were sandwiched between Teflon plates that contained a hole for the liquid to flow through. AC voltage was applied to each pair of electrodes in parallel. A treatment chamber has also been used in which the electric flux lines were approximately parallel to the direction of the liquid flow (Geveke and Brunkhorst, 2003b, 2004). The treatment chamber was fabricated of Rexolite, a transparent crosslinked polystyrene copolymer (C-Lec Plastics, Philadelphia, PA). It was designed to converge the liquid into a narrow flow area in order to reduce the power requirement (Matsumoto et al., 1991; Sensoy et al., 1995). Liquid entered and exited the Rexolite chamber through the annuli of cylindrical stainless steel electrodes (Swagelok, Solon, OH, part no. SS-400-1-OR) as shown in Figure 12.4. The electrodes were separated by a thin partition, with a channel of circular cross-section through the centre. The diameter and length of the channel were 1.2 mm and 2.0 mm, respectively (Geveke and Brunkhorst, 2003b). A 9.0 mm space between the end of each of the electrodes and the central channel prevented arcing. To increase inactivation, the liquid flowed in series through two treatment chambers joined by stainless steel tubing. The inner electrodes between the treatment chambers were connected to the RFEF power supply in parallel. The outer electrodes were grounded. The experimental system employed by Geveke and Brunkhorst (2003b) included a stainless steel feed tank and a progressing cavity pump (Moyno, Springfield, OH; model 2FG3) that supplied the liquid to the RFEF treatment chambers at a flow rate of 1.4 l/min through stainless steel tubing (Figure 12.5). Multiple treatment chambers and turbulent flow within the treatment chambers (Reynolds number 18 000) improved the processing uniformity. The liquid was exposed to intense RFEF in the 2 mm

Liquid

Food

1.2 mm

SS Rexolite 9 mm Figure 12.4 Cross-section of converged co-field treatment chamber including Rexolite insulation and stainless steel electrodes. RFEF treatment chambers

Feed tank

Sample Heat exchanger Feed pump Figure 12.5

Schematic diagram of continuous RFEF process.

Cooler

Modelling of radio frequency electric fields 313

chambers for a total duration of 190 s. At a frequency of 21.1 kHz, the liquid was exposed to 2 AC cycles of RFEF in each chamber. A back pressure of 1 atmosphere gauge minimized arcing. The inlet temperature to the first treatment chamber was controlled using a 0.24 m2 stainless-steel heat exchanger (Madden Manufacturing, Elkhart, IN; model SC0004) and a temperature controller (Cole-Parmer, model CALL 9400). During RFEF processing, the temperature of the liquid rose due to ohmic (resistance) heating. The temperatures of the liquid immediately before and after the RFEF treatment chambers were measured with 3.2 mm diameter chrome-constantan thermocouples (Omega Engineering, Inc., Stamford, CT). The temperatures were continuously logged to a data acquisition system (Dasytec USA, Amherst, NH, Dasylab version 5.0). The liquid was quickly cooled to less than 25°C using a stainless-steel heat exchanger after exiting the last treatment chamber (Madden Manufacturing, model SC0004). The time for the liquid to travel from the treatment chamber to the sample cooler was 2 s (Geveke and Brunkhorst, 2003b). Controls were performed to determine the effect of temperature alone. In order to ensure that the control liquid received the same time and temperature history as the treated liquid, the pair of converged treatment chambers was replaced with an ohmic heating chamber. The chamber consisted of two stainless steel electrodes (Swagelok, Solon, OH, part no. SS-400-1OR) inserted into a 102 mm length of 6.4 mm ID plastic tubing. The ohmic heater quickly brought the liquid temperature up to the desired temperature. The control liquid was identically held for 2 s before cooling to less than 25°C.

3 Modelling of radio frequency electric fields The anisotropic electric field strengths within the treatment chamber can be modelled with finite element analysis software such as QuickField™ (Tera Analysis Ltd, Svendborg, Denmark, version 5.0). Figure 12.6 presents the model’s results for an electric field strength of 20 kV/cm within the treatment chamber shown in Figure 12.4.

20 Rexolite

15 10 5

Rexolite 0 kV/cm Figure 12.6 Figure 12.4.

Modeled anisotropic AC electric field strength within the treatment chamber shown in

314 Non-thermal Processing By Radio Frequency Electric Fields

The liquid flows through the electrode and enters a field-free region. It then flows into the central channel where the field is quickly raised to 20 kV/cm. The field within the channel is nearly uniform which ensures that all of the liquid is treated equally. The uniformity improves the energy efficiency of the process. By minimizing the regions within the treatment chamber where the electric field is too low to inactivate bacteria and only heats the liquid, approximately less than 5 kV/cm, the energy loss is minimized. Similarly, by minimizing the regions where the field is higher than needed to inactivate bacteria, the energy loss is minimized. Thus, the outlet temperature is lessened and the liquid is not overly treated.

4 RFEF non-thermal inactivation of yeast Non-thermal processing of liquids with high intensity radio frequency electric fields (RFEF) has only been studied for the past several years. Microbial inactivation using low intensity RFEF, with field strengths less than 5 kV/cm, was shown to be due to heat alone (Geveke et al., 2002a). Geveke et al. (2002b) designed, fabricated and assembled a 4 kW RFEF power supply and treatment chamber that were capable of applying a 30 kV/cm electric field to liquids. RFEF processing successfully inactivated suspensions of Saccharomyces cerevisiae in water at non-thermal conditions. The extent of microbial inactivation was dependent on the electric field strength, number of treatments and temperature. A series of experiments were performed at 20 kHz to determine the effect of electric field strength and temperature (Geveke and Brunkhorst, 2003a). The population of S. cerevisiae was reduced by 3.1 log after being exposed to a 30 kV/cm electric field at a treatment chamber inlet temperature of 26°C and outlet temperature of 45°C. When the electric field was eliminated and the inlet temperature was raised to match the outlet temperature of 45°C, the reduction was only 0.3 log. Lowering the inlet temperature to achieve an outlet temperature of 40°C, with the same electric field of 30 kV/cm, reduced S. cerevisiae by 2.1 log. The non-thermal inactivation is believed to be due to dielectric breakdown of the cells (Zimmermann et al., 1974). At 55°C with a hold time of 3 s, S. cerevisiae was reduced by 4.7 log. Inactivation was significantly greater using a field of 30 kV/cm rather than 20 kV/cm for outlet temperatures of 40–55°C. These results proved that

Table 12.1 Effect of repetitive RFEF treatments on the inactivation of S. cerevisiae in water at 35°C, 20 kHz and 30 kV/cm. Means of two replicate experiments Number of treatment steps

Inactivation (log cfu/ml)

1 2 3

0.8 2.2 3.8

Bench scale RFEF inactivation of bacteria and spores 315

the electric field strength plays an important role in inactivation in addition to temperature. The effects of frequency and multiple treatments were studied (Geveke and Brunkhorst, 2003a). Experiments were conducted at frequencies of 20, 40 and 60 kHz. A 20 kV/cm electric field strength at an outlet temperature of 50°C was applied to S. cerevisiae. The microbial reductions varied from 1.8 to 2.0 log and were not significantly different across the limited range of frequencies. Inactivation increased significantly with increasing number of treatments as shown in Table 12.1. A single treatment of a 30 kV/cm electric field at 35°C reduced the population of S. cerevisiae by 0.8 log, whereas three treatments resulted in a 3.8 log reduction.

5 Bench scale RFEF inactivation of bacteria and spores Uemura and Isobe (2002) used a 20 kHz RFEF apparatus to study inactivation of microorganisms in saline water. Application of a 14 kV/cm electric field reduced Escherichia coli by 5 log at 74°C with a hold time of less than 1 s. A higher electric field at the same temperature produced a larger decrease of E. coli as shown in Figure 12.7. RFEF strengths of greater than 7 kV/cm were needed to inactivate E. coli. Using a similar RFEF apparatus, Uemura and Isobe (2003) applied a 16.3 kV/cm field to orange juice containing Bacillus subtilis spores. RFEF processing at 121°C, under pressurized conditions to elevate the boiling point, reduced the viable Bacillus subtilis spores in orange juice 4 log in 1 s of treatment. Only 10 per cent of the original ascorbic acid in the orange juice was destroyed after RFEF treatment. RFEF processing, using a converged treatment chamber, successfully inactivated E. coli in apple juice at non-thermal conditions (Geveke and Brunkhorst, 2004). The

Survived E. coli (cfu/ml)

1.0E07 1.0E06

0.5% 0.3% 0.2%

1.0E05

0.1% 0.5%

1.0E04

0.3% 0.2%

1.0E03

0.05%

0.15%

1.0E02 1.0E01

0.1% 0.08%

1.0E00 0

5

10

15

20

Electric field (kV/cm) Figure 12.7 Inactivation of E. coli as a function of electric field, outlet temperature and saline concentration. ▲, 65°C and ■, 70°C. The hold time was less than 1 s. The values at each point show the saline concentration (Uemura and Isobe, 2002).

316 Non-thermal Processing By Radio Frequency Electric Fields

Table 12.2 Effect of electric field strength on the inactivation of E. coli in apple juice at 20 kHz and 45°C with a 4 s hold time using a 4 kW RFEF system (Geveke and Brunkhorst, 2004). Means of two replicate experiments Electric field (kV/cm)

Inactivation (log cfu/ml)

9 12 16 18 20 22 24

0.4 1.0 1.4 1.4 1.3 1.4 1.4

extent of microbial inactivation was dependent on the electric field strength (up to 16 kV/cm), number of treatment stages, frequency and temperature. A series of experiments was performed at 20 kHz and 45°C to determine the effect of electric field strength on inactivation and the results are presented in Table 12.2 (Geveke and Brunkhorst, 2004). The population of E. coli was reduced by 1.4 log after being exposed to a 24 kV/cm peak electric field. When the field was eliminated and the inlet temperature was raised to match the outlet temperature of 45°C, the reduction was 0.1 log. Inactivation increased significantly as the electric field strength increased up to 16 kV/cm. However, inactivation remained constant with field strength above 16 kV/cm. Jayaram et al. (1992) applied pulsed electric fields (PEF) to Lactobacillus brevis and observed similar behaviour. Inactivation of L. brevis greatly increased with field strength up to 15 kV/cm, whereas at higher fields, inactivation remained constant at temperatures between 30 and 45°C. Wouters et al. (1999) reported similar results for PEF treatment of Listeria innocua except that the threshold field strength was higher at 30 kV/cm and was observed between 45 and 60°C. The effect of frequency on the inactivation of E. coli in apple juice was investigated (Geveke and Brunkhorst, 2004). The juice was treated with a 20 kV/cm electric field strength at frequencies of 15–70 kHz at 50°C with a 4 s hold time. Significantly greater inactivation occurred at frequencies less than or equal to 20 Hz as shown in Table 12.3. The cause of this has yet to be determined. Geveke and Brunkhorst (2003a) applied RFEF to S. cerevisiae in water at frequencies of 20, 40 and 60 kHz and concluded that frequency had no effect on inactivation. The variation in results may be due to the use of different microorganisms, media, or treatment chambers. The effect of initial concentration on inactivation was studied (Geveke and Brunkhorst, 2004). A 17 kV/cm electric field strength at a temperature of 45°C was applied to E. coli having initial concentrations of 4.3, 5.4 and 6.2 log cfu/ml, respectively. The inactivations varied from 1.0 to 1.1 log and were not significantly different across the range of initial concentrations studied. These results are in agreement with those of Zhang et al. (1995b) for PEF treatment of E. coli in ultra-filtrated simulated milk. Initial concentration, which ranged from 3 to 8 log cfu/ml, had no effect on inactivation. However, earlier Zhang et al. (1994) found that PEF inactivation of S. cerevisiae in apple

Pilot scale RFEF inactivation of bacteria 317

Table 12.3 Effect of frequency on the inactivation of E. coli in apple juice at 20 kV/cm and 50°C with a 4 s hold time using a 4 kW RFEF system (Geveke and Brunkhorst, 2004). Means of two replicate experiments Frequency (kHz)

Inactivation (log cfu/ml)

15 20 30 40 50 60 70

2.2 2.2 1.7 1.8 1.6 1.6 1.6

juice was inversely affected by the initial concentration over the span of 4–6 log cfu/ml. These results were attributed to a cluster protection mechanism. Recently, Damar et al. (2002) inactivated E. coli in peptone solution at initial concentrations between 3 and 8 log cfu/ml with PEF. Inactivation was found to be inversely proportional to initial concentration and was presented as further support of a cluster protection mechanism. Once again, the discrepancy in results may be due to differences in the process parameters, microorganisms or media used. Inactivation increased with increasing number of treatment stages (Geveke and Brunkhorst, 2004). The juice was exposed to RFEF for approximately 170 s during each stage. A single treatment of an 18 kV/cm electric field at 50°C reduced the population of E. coli by 1.8 log, whereas 3 stages of treatment resulted in a 3.0 log reduction. Inactivation of recycled material was less than that of untreated material. This may be due to the more-RFEF-sensitive E. coli having already been eliminated in the first treatment stage.

6 Pilot scale RFEF inactivation of bacteria The RFEF process has been successfully scaled up. Using an 80 kW RFEF system, E. coli in apple juice was inactivated at non-thermal conditions (Geveke and Brunkhorst, 2003b). The 80 kW system was capable of treating 1.4 l/min, whereas a 4 kW RFEF system had been limited to a flow rate of 0.55 l/min. The extent of microbial inactivation was dependent on the electric field strength, treatment time, frequency and temperature. A series of experiments was performed at 21.1 kHz to determine the effects of electric field strength and temperature on inactivation. The population of E. coli was reduced by 2.2 log after being exposed to a 20 kV/cm peak electric field at a treatment chamber inlet temperature of 26°C, treatment time of 270 s, outlet temperature of 55°C and hold time of 3 s (Table 12.4). Applying the same field at 60°C resulted in a reduction in E. coli of 2.7 log. When the juice was ohmicly heated to the same

318 Non-thermal Processing By Radio Frequency Electric Fields

outlet temperature of 60°C and held for the same time of 3 s, the population of E. coli was unaffected. Increasing the flow rate to 1.4 l/min and hence decreasing the treatment time to 190 s, lessened the treatment effectiveness somewhat. The population of E. coli was reduced by 2.1 log after being exposed to a 20 kV/cm field at a treatment chamber outlet temperature of 60°C and hold time of 2 s. Increasing the field strength to 25 kV/cm at the same temperature resulted in a reduction in E. coli of 2.3 log. Using a 4 kW RFEF system, E. coli in apple juice was reduced by 1.9 log at 55°C, relative to the control, at a flow rate of 0.55 l/min and a hold time of 4 s (Geveke and Brunkhorst, 2004). Using the 80 kW RFEF pilot plant, the RFEF process was successfully scaled up to 1.4 l/min. The effect of frequency on inactivation using the 80 kW RFEF pilot plant has been investigated. The inactivation of E. coli in apple juice was substantially increased as the frequency was decreased from 40.1 kHz to 21.1 kHz as shown in Table 12.5. In a previous work, significantly greater inactivation of E. coli in apple juice occurred at frequencies of 15 and 20 kHz compared to frequencies of 30–70 kHz. (Geveke and Brunkhorst, 2004). These results are extremely interesting, not only because they indicate that the RFEF process could be more efficient at even lower frequencies,

Table 12.4 Effects of temperature and electric field strength on the inactivation of E. coli at a 270 ␮s treatment time and 3 s hold time (1.0 l/min flow rate) using an 80 kW RFEF system. Means of two replicate experiments Temperature (°C)

Electric field (kV/cm)

Inactivation (log cfu/ml)

55 55 55 60 60 60 65 65 65

1 (control) 15 20 1 (control) 15 20 1 (control) 15 20

0.2 1.6 2.2 0.0 1.3 2.7 0.2 2.3 3.1

Table 12.5 Effects of frequency and temperature on the inactivation of E. coli at 20 kV/cm, 270 ␮s RFEF treatment time and 3 s hold time using an 80 kW RFEF system. Means of two replicate experiments Temperature (°C)

Frequency (kHz)

Inactivation (log cfuU/ml)

55 55 55 60 60 60

21.1 29.5 40.1 21.1 29.5 40.1

2.2 1.4 0.7 2.7 2.1 1.1

Conclusions 319

but also because RFEF equipment costs should be significantly less at lower frequencies as well.

7 Electrical costs The energy costs of alternative pasteurization processes are an important factor in determining whether the new technologies will be commercialized. The electrical costs were calculated for the case of RFEF processing of apple juice at 15 kV/cm, 65°C and 2 s. At these conditions, the population of E. coli was reduced by 2.5 log. Based on the flow rate and the voltage and current measured by an oscilloscope, 3.0 kVpeak and 1.2 Apeak, respectively, the energy applied was 77 J/ml. From the inlet temperature, 50°C, the energy calculated to raise the temperature of the apple juice to 65°C is 63 J/ml. This is in fairly good agreement with the energy calculated using the current and voltage. The estimated energy required for a 5 log reduction using pulsed electric fields (PEF) ranges from 100 to 400 J/ml (Barsotti and Cheftel, 1999; Schoenbach et al., 2002). It is probable that the RFEF electrical costs for a 5 log reduction will be similar to those of PEF as they are both considered electroporation processes (Geveke and Brunkhorst, 2004). Based on the US Department of Energy’s data for the average industrial electric price for the first ten months of 2003 of $0.050/kWh, the energy cost for the RFEF process was $0.0011 per litre of apple juice. For comparison, conventional thermal pasteurization, with heat regeneration or recovery, costs only $0.0005 per litre or less.

8 Conclusions The radio frequency electric fields (RFEF) process has been shown to reduce significantly the population of Escherischia coli in apple juice at 45°C. Inactivation is dependent upon the electric field strength, number of treatment stages and temperature. Significantly better inactivation has been observed at radio frequencies near 20 kHz compared to frequencies near 40 kHz. Inactivation is independent of initial microbial concentration in the range of 4–6 log cfu/ml. The RFEF process has successfully been scaled up from 0.55 l/min to 1.4 l/min using an innovative pilot plant consisting of an 80 kW power supply and novel matching network. The calculated electrical cost was $0.0011 per litre of apple juice. Although remarkable progress has recently been made in the development of the non-thermal RFEF process, more research needs to be done before it can be commercialized. The RFEF process needs to be further scaled up to be of commercial interest. In addition, a 5 log reduction is desirable. This should be achievable by adding several more treatment chambers in series. The stability of the equipment, including the metal electrodes, at longer operational times must be studied. The relative costs and merits of the RFEF and pulsed electric fields processes must be examined

320 Non-thermal Processing By Radio Frequency Electric Fields

(Geveke, 2003). Finally, RFEF processing at much lower frequencies, where the efficiency may be enhanced, deserves attention.

Acknowledgements The author thanks C. Brunkhorst of Princeton University for discussions on electrical engineering aspects. Note: Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

References Barsotti L, Cheftel JC (1999) Food processing by pulsed electric fields. II. Biological aspects. Food Reviews International, 15 (2), 181–213. Brown GH, Morrison WC (1954) An exploration of the effects of strong radio-frequency fields on micro-organisms in aqueous solutions. Food Technology, 8 (8), 361–366. Brunkhorst C, Ciotti D, Fredd E, Wilson JR, Geveke DJ, Kozempel M (2000) Development of process equipment to separate non-thermal and thermal effects of RF energy on microorganisms. Journal of Microwave Power and Electromagnetic Energy, 35 (1), 44–50. Chang DC, Gao PQ, Maxwell BL (1991) High efficiency gene transfection by electroporation using a radio-frequency electric field. Biochimica et Biophysica Acta, 1092 (2), 153–160. Dahm L (2000) Refreshing the juice category. Food Processing, 61 (1), 42–47. Damar S, Bozoglu F, Hizal M, Bayindirli A (2002) Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields. World Journal of Microbiological Biotechnology, 18 (1), 1–6. Decareau RV (1985) Microwaves in the Food Processing Industry. Orlando: Academic Press. Dunn JE, Pearlman JS (1987) Methods and apparatus for extending the shelf life of fluid food products. US Patent 4695472. Fleming H (1944) Effect of high frequency fields on micro-organisms. Electrical Engineering, 63 (1), 18–21. Geveke DJ (2003) Inactivation of microorganisms in liquids by high electric fields. Journal of the Association of Food and Drug Officials, 67 (4), 48–51. Geveke DJ, Brunkhorst C (2003a) Inactivation of Saccharomyces cerevisiae using radio frequency electric fields. Journal of Food Protection, 66 (9), 1712–1715. Geveke DJ, Brunkhorst C (2003b) Radio frequency electric fields inactivation of Escherichia coli in apple juice using an 80 kW power supply. In Proceedings of the American Institute of Chemical Engineers Annual Meeting, San Francisco, CA, Paper 123b.

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Geveke DJ, Brunkhorst C (2004) Inactivation of Escherichia coli in apple juice by radio frequency electric fields. Journal of Food Science, 69 (3), 134–138. Geveke DJ, Brunkhorst C, Kozempel M (2002b) Preliminary results of radio pasteurization (RAP). In Proceedings of the American Society of Agricultural Engineers Annual International Meeting, Chicago, IL, Paper 026005. Geveke DJ, Kozempel M, Brunkhorst C (1999) Development of equipment to separate non-thermal and thermal effects of radio frequency energy on microorganisms. In Proceedings of the American Institute of Chemical Engineers Annual Meeting, Dallas, TX, Paper T3015. Geveke DJ, Kozempel M, Scullen OJ, Brunkhorst C (2000) The combined effects of RF energy and thermal energy on microorganisms. In Proceedings of the American Society of Agricultural Engineers Annual International Meeting, Milwaukee, WI, Paper 6104. Geveke DJ, Kozempel M, Scullen OJ, Brunkhorst C (2002a) Radio frequency energy effects on microorganisms in foods. Innovative Food Science and Emerging Technologies, 3 (2), 133–138. Ingram M, Page LJ (1953) The survival of microbes in modulated high-frequency voltage fields. Proceedings of the Society for Applied Bacteriology, 16, 69–87. Jayaram S, Castle GSP, Margaritis A (1992) Kinetics of sterilization of Lactobacillus brevis cells by the application of high voltage pulses. Biotechnology and Bioengineering, 40 (11), 1412–1420. Jeyamkondan S, Jayas DS, Holley RA (1999) Pulsed electric field processing of foods: A review. Journal of Food Protection 62 (9), 1088–1096. Kotnik T, Miklavcic D, Slivnik T (1998) Time course of transmembrane voltage induced by time-varying electric fields – A method for theoretical analysis and its application. Bioelectrochemistry and Bioenergetics, 45 (1), 3–16. Matsumoto Y, Satake T, Shioji N, Sakuma A (1991) Inactivation of microorganisms by pulsed high voltage application. In Proceedings of the IEEE Industry Applications Society Annual Meeting, Dearborn, MI. pp. 652–659. Mertens B, Knorr D (1992) Developments of non-thermal processes for food preservation. Food Technology, 46 (5), 124, 126–133. Nyrop JE (1946) A specific effect of high-frequency electric currents on biological objects. Nature, 157, 51. Ponne CT, Balk M, Hancioglu O, Gorris LGM (1996) Effect of radio frequency energy on biological membranes and microorganisms. Lebensmittel Wissenschaft und Technologie, 29 (1/2), 41–48. Qin BL, Zhang QH, Barbosa-Canovas GV, Swanson BG, Pedrow PD (1994) Inactivation of microorganisms by pulsed electric fields of different voltage waveforms. IEEE Transactions on Dielectrics and Electrical Insulation, 1 (6), 1047–1057. Sale AJH, Hamilton WA (1967) Effects of high electric fields on microorganisms. I. Killing of bacteria and yeasts. Biochimica et Biophysica Acta, 148, 781–788. Schoenbach KH, Katsuki S, Stark RH, Buescher ES, Beebe SJ (2002) Bioelectrics – new applications for pulsed power technology. IEEE Transactions on Plasma Science, 30 (1), 293–300.

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Sensoy A, Zhang QH, Sastry SK (1995) A high voltage electric field treatment system for preservation of electrically conductive foods. In IFT Annual Meeting Conference Proceedings, pp. 150. Uemura K, Isobe S (2002) Developing a new apparatus for inactivating Escherichia coli in saline water with high electric field AC. Journal of Food Engineering, 53 (3), 203–207. Uemura K, Isobe S (2003) Developing a new apparatus for inactivating Bacillus subtilis spore in orange juice with a high electric field AC under pressurized conditions. Journal of Food Engineering, 56 (4), 325–329. Wouters PC, Dutreux N, Smelt JPPM, Lelieveld HLM (1999) Effects of pulsed electric fields on inactivation kinetics of Listeria innocua. Applied Environmental Microbiology, 65 (12), 5364–5371. Zhang Q, Monsalve-Gonzalez A, Qin BL, Barbosa-Canovas GV, Swanson BG (1994) Inactivation of Saccharomyces cerevisiae in apple juice by square-wave and exponential-decay pulsed electric fields. Journal of Food Process Engineering, 17 (4), 469–478. Zhang Q, Barbosa-Canovas GV, Swanson BG (1995a) Engineering aspects of pulsed electric field pasteurization. Journal of Food Engineering, 25 (2), 261–281. Zhang Q, Qin BL, Barbosa-Canovas GV, Swanson BG (1995b) Inactivation of E. coli for food pasteurization by high-strength pulsed electric fields. Journal of Food Processing and Preservation, 19 (2), 103–118. Zimmermann U (1986) Electrical breakdown, electropermeabilization and electrofusion. Reviews of Physiology, Biochemistry and Pharmacology, 105, 176–256. Zimmermann U, Pilwat G, Riemann F (1974) Dielectric breakdown of cell membranes. Biophysical Journal, 14, 881–899.

Application of Ultrasound Timothy J Mason1, Enrique Riera2, Antonio Vercet3 and Pascual Lopez-Buesa3 1

Coventry University, Sonochemistry Centre, School of Science and the Environment, Coventry, UK 2 Instituto de Acústica, CSIC, Ultrasonics Department, Madrid, Spain 3 Universidad de Zaragoza, Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Zaragoza, Spain

Nowadays, power ultrasound is considered to be an emerging and promising technology for food processing in industry. In this chapter a review of the most recent uses of power ultrasound in the food industry will be presented. The potential use of this novel technology to produce permanent changes in the material will be discussed in liquid systems (by the production of intense cavitation) and in gases (by the generation of high-intensity acoustic fields). Mechanical, chemical and biochemical effects produced by the propagation of highintensity ultrasonic waves through the medium will be discussed. The inactivation of microorganisms and enzymes for food preservation or decontamination by ultrasonic irradiation will demonstrate the benefits of ultrasound (alone or combined with heat and high-pressure techniques) as a food preservation tool. In addition, the increasing number of industrial processes that employ power ultrasound as a processing aid will be described including the mixing of materials; foam formation or destruction; agglomeration and precipitation of airborne powders; the improvement in efficiency of filtration, drying and extraction techniques in solid materials and the enhanced extraction of valuable compounds from vegetables and food products. Finally, the effect of ultrasound on food properties such as flavour, colour and texture will be analysed.

1 Introduction Probably the first question that might be asked about applications of ultrasound in food technology is, why use ultrasound? For the answer to this we need only think of two properties of sound to appreciate the possibilities. The first is the use of sound as a diagnostic tool, e.g. in non-destructive evaluation and the second is the use of sound as a source of energy, e.g. in sonochemistry. These applications involve different frequency ranges of ultrasound (Figure 13.1) and the uses of both ranges within the food industry continue to be an active subject for research and development (Povey and Mason, 1998). Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

13

324 Application of Ultrasound

0

Figure 13.1

10

102

103

104

105

106

Human hearing

16 Hz–18 kHz

Conventional power ultrasound

20 kHz–100 kHz

Extended for special applications

20 kHz–1 MHz

Diagnostic ultrasound

5 MHz–10 MHz

107

Frequency ranges of sound.

Table 13.1 Some uses of power ultrasound in food processing Mechanical effects crystallization of fats, sugars etc degassing destruction of foams extraction of flavourings filtration and drying freezing mixing and homogenization precipitation of airborne powders tenderization of meat Chemical and biochemical effects bactericidal action effluent treatment modification of growth of living cells alteration of enzyme activity sterilization of equipment

Until recently the majority of applications of ultrasound in food technology involved non-invasive analysis with particular reference to quality assessment. Such applications use techniques that are similar to those developed in diagnostic medicine, or non-destructive testing, using high frequency (⬎1 MHz) low power (⬍1 W/cm2) ultrasound (Mulet et al., 2002). Examples of the use of such technologies are to be found in the location of foreign bodies in food (Cho and Irudayaraj, 2003), the analysis of droplet size in emulsions of edible fats and oils (Coupland and McClements, 2001) and the determination of the extent of crystallization in dispersed emulsion droplets (Hindle et al., 2002). In recent years food technologists have discovered that it is possible to employ a more powerful form of ultrasound (⬎5 W/cm2) at a lower frequency (generally around 40 kHz). This is usually referred to as power ultrasound and its history can be traced

Fundamentals of ultrasound 325

back to 1927 when a paper was published entitled ‘The chemical effects of high frequency sound waves: a preliminary survey’, which described the development of power ultrasound for use in a range of processing including emulsification and surface cleaning (Richards and Loomis, 1927). By the 1960s the uses of power ultrasound in the processing industries were well accepted and this interest has continued to develop (Abramov, 1998; Mason, 2000; Mason and Lorimer, 2002). In this chapter we will concentrate on possible applications of power ultrasound in the food industry, an indication of the breadth of which is shown in Table 13.1.

2 Fundamentals of ultrasound 2.1 The physics and chemistry of ultrasound 2.1.1 Power ultrasound in liquid systems

The major mechanical effects of ultrasound are provided when the power is sufficiently high to cause cavitation. Like any sound wave, ultrasound is propagated via a series of compression and rarefaction waves induced in the molecules of the medium through which it passes. At sufficiently high power the rarefaction cycle may exceed the attractive forces of the molecules of the liquid and cavitation bubbles will form. Such bubbles grow by a process known as rectified diffusion, i.e. small amounts of vapour (or gas) from the medium enters the bubble during its expansion phase and is not fully expelled during compression. The bubbles, distributed throughout the liquid, grow over the period of a few cycles to an equilibrium size for the particular frequency applied. If the bubbles were only subject to that particular frequency they would remain as oscillating bubbles, however, the acoustic field that influences an individual bubble among the many thousands generated in a cavitating fluid is not uniform. Each bubble will slightly affect the localized field experienced by neighbouring bubbles. Under such circumstances the irregular field will cause the cavitation bubble to become unstable and collapse. It is this collapse that generates the energy for chemical and mechanical effects. For example, in aqueous systems at an ultrasonic frequency of 20 kHz, each cavitation bubble collapse acts as a localized ‘hotspot’ generating temperatures of about 4000 K and pressures in excess of 1000 atmospheres. This bubble collapse, distributed through the medium, has a variety of effects within the system depending upon the type of material involved. 2.1.1.1 Homogeneous liquid-phase systems It is not absolutely correct to describe any system within which cavitation occurs as homogeneous since cavitation bubbles must be present. However, it is logical to refer to systems in the state that they were in before ultrasound is introduced. There are two major zones in which cavitation collapse can influence such systems: in the bulk liquid immediately surrounding the bubble where the rapid collapse of the bubble generates shear forces which can produce mechanical effects and in the bubble itself where any species introduced during its formation will be subjected to extreme conditions of temperature and pressure on collapse, leading to chemical effects.

326 Application of Ultrasound

In order for cavitation to occur it is necessary to pull molecules of the liquid apart to produce a hole (cavity). A pure liquid would require very high power levels to initiate cavitation, too high to be achieved by normal ultrasonic equipment. However, most normal liquids contain some discontinuities, such as gas bubbles or dust motes, which act as weak spots and allow the bubbles to form. Ultrasonic degassing is normally performed before cleaning equipment is used because it increases the efficiency of cavitation by removing air bubbles which absorb acoustic energy and dampen sonication. After degassing continued sonication will provide continuous and more powerful cavitation that will have other effects. The cavitation bubble does not contain a vacuum; during the process of growth in an acoustic field vapour from the liquid medium or dissolved volatile reagents will have entered the bubble. On collapse, these vapours will be subjected to extremely large increases in temperature and pressure, resulting in molecular fragmentation. In the case of water the extreme conditions are sufficient to cause rupture of the O-H bond, leading to the production of small quantities of oxygen gas and hydrogen peroxide. 2.1.1.2 Solid–liquid systems Unlike cavitation bubble collapse in the bulk liquid, collapse of a cavitation bubble on or near to a surface is asymmetrical because the surface provides resistance to liquid flow from that side. The result is an inrush of liquid predominantly from the side of the bubble remote from the surface, resulting in a powerful liquid jet being formed, targeted at the surface. The effect is equivalent to high pressure jetting and is the reason that ultrasound is used for cleaning. This effect can also increase mass and heat transfer to the surface by disruption of the interfacial boundary layers. Acoustic cavitation can also produce dramatic effects on powders suspended in a liquid. Surface imperfections or trapped gas can act as the nuclei for cavitation bubble formation on the surface of a particle and subsequent surface collapse can then lead to shock waves which break the particle apart. Cavitation bubble collapse in the liquid phase near to a particle can force it into rapid motion. Under these circumstances the general dispersive effect is accompanied by interparticle collisions that can lead to erosion, surface cleaning and wetting of the particles and particle size reduction. 2.1.1.3 Liquid–liquid systems The general mechanical effects of cavitation at or close to a liquid–liquid interface lead to very effective emulsification/homogenization. 2.1.2 Power ultrasound in gases

There are two difficulties that pertain to the use of ultrasound in gaseous systems. First, there is a greater attenuation (power loss) in the transmission of sound through air compared with that through liquid. Secondly, the transfer of acoustic energy generated in air into a food material is inefficient due to the mismatch between acoustic impedances of gases and solids or liquids. Significant attempts have been made to alleviate these problems by developing very powerful sources of airborne ultrasound that can achieve more efficient energy transmission to the material (Gallego et al.,

Fundamentals of ultrasound 327

Stepped radiating plate

Piezoelectric ceramics

Mechanical amplifier

Figure 13.2

Sandwich transducer

Dish emitter for airborne ultrasound.

1994). The system is based on the use of a stepped-plate transducer to generate airborne ultrasound (Gallego et al., 1978) and incorporates an extended titanium stepped-grooved radiating plate (350 mm in diameter) which allows the ultrasonic energy to focus (Figure 13.2). Sound pressure levels (SPL) of about 165 dB (ca 3 W/cm2) have been recorded at a distance of about 330 mm from the centre of the plate, when a maximum power of 150 W is applied to the transducer. Using this type of device, airborne ultrasound has been used in the precipitation of airborne powders, drying and defoaming.

2.2 Ultrasonic processing equipment 2.2.1 Laboratory scale

Whatever food processing application is to be studied or developed, the essential requirement is a source of ultrasound. The two most common pieces of laboratory equipment used for processing liquids are an ultrasonic cleaning bath, which is inexpensive and is commonly used to sonicate liquid samples in vessels immersed in the bath (Figure 13.3), or the more powerful probe system, which introduces vibrations directly into the sample (Figure 13.4) (Mason, 1999). 2.2.2 Large scale

There are essentially two types of large scale plant: batch and flow types. The results from successful small-scale experiments can be adapted for large scale work providing that information is available on power input and volume treated. Several scale-up designs are available for food processing which can be broadly divided into batch and flow systems. A general review of large scale processors has been published by

328 Application of Ultrasound

Reaction mixture in conical flask Water with detergent

Optional heater

Stainless steel tank Transducers bonded to base Figure 13.3

Ultrasonic bath.

Generator

Casing containing transducer element

Upper fixed horn (booster) Screw fitting at null point Detachable horn

Replaceable tip

Figure 13.4

Ultrasonic probe system.

Mason and Peters (2002). Several large scale applications using airborne ultrasound will be discussed later in this chapter. 2.2.2.1 Batch systems Batch systems will generally be based upon the ultrasonic cleaning bath using the whole bath as the reactor. Examples can be found in cleaning and decontamination of

Ultrasound as a food preservation tool 329

Region of controlled formation and collapse of cavitation bubbles

Flow from pump

Figure 13.5

Liquid whistle.

equipment, e.g. in the cleaning of chicken shackles to avoid cross-contamination (Quartly-Watson, 1998). 2.2.2.2 Flow systems One of the oldest devices used to achieve emulsification through cavitation is the liquid whistle. Process material is forced under pressure generated by a powerful pump through an orifice from which it emerges and expands into a mixing chamber (Figure 13.5). With no moving parts, other than a pump, the system is rugged and durable (Moser et al., 2001). The systems that are particularly suitable for general usage in the food industry are resonating tube reactors. Essentially the liquid to be processed is passed through a pipe with ultrasonically vibrating walls. In this way the sound energy generated from transducers bonded to the outside of the tube is transferred directly into the flowing liquid. Generally, commercial tube reactors are constructed of stainless steel and the choices for pipe cross-section are rectangular, pentagonal, hexagonal and circular. An alternative arrangement is via the coaxial insertion of a radially emitting bar into the pipe containing the flowing liquid; this would require minimal change to existing pipework. One such system consists of a hollow tube sealed at one end and driven at the other by a standard piezo transducer. Another concept involves a cylindrical bar of titanium with opposing piezoelectric transducers attached at each end. The design of both inserts is such that the ultrasonic energy is emitted radially at half wavelength distances along their lengths.

3 Ultrasound as a food preservation tool Food preservation can be defined as the extension of shelf-life of raw materials or prepared foods beyond their ‘natural’ (i.e. without human intervention) decay times. This extension can be considered to be a competition between different physical, chemical

330 Application of Ultrasound

or biochemical processes. It is also depends upon the growth and development of different microbial populations, which can be sometimes beneficial or, more commonly, detrimental for maintaining desirable food properties.

3.1 Inactivation of microorganisms The food industry has generally concentrated on inactivating or killing microorganisms and enzymes as a means of preservation by using a number of physical methods, mostly involving heat, with enormous success. However, while heat can aid food preservation it can also cause some deterioration, e.g. the loss of some nutrients and reduction in the organoleptic properties of the material. Thus the scientific community has been searching for alternative methods to preserve foods using different strategies or physical principles. The attempt to use ultrasound for food preservation is not new but it has experienced a relative revival in the last 10 years (Mason et al., 2003). Initial investigations of ultrasound were devoted to its effects on the most important food alteration agent – microbial populations. The destruction of microorganisms by power ultrasound was reported in the 1920s when the work of Harvey and Loomis (1929) was first published. Their work examined the reduction in light emission from a seawater suspension of rodshaped Bacillus fisheri caused by sonication at 375 kHz under temperature-controlled conditions. Maintaining the temperature during sonication at 19°C prevented regrowth and all the bacteria appeared to be dead when viewed under a microscope. They attributed microbial death to cell disruption caused by cavitation. Other authors (Lepeschkin and Goldman, 1952; Kinsloe et al., 1954) pointed out that microbial inactivation due to ultrasound could be also achieved in the absence of cavitation, which led them to suggest that other inactivation mechanisms could play a significant role. Subsequently, investigations of the effects of ultrasound on different microbial species revealed very different sensitivities, which were related to the shape and size of the microorganisms. With some exceptions, it is generally accepted that bigger cells are more sensitive than smaller ones and that coccal forms more resistant than rod-shaped bacteria. Further, Gram-positive are more resistant than Gram-negative and aerobic are also more resistant than anaerobic bacteria. The physiological state of the cells also plays a role, with younger cells more sensitive than older ones and spores much more resistant than vegetative cells (Paci, 1953; Jacobs and Thornley, 1954; Davies, 1959; Ahmed and Russell, 1975). However, comparisons between the sensitivities of different species or even within the same species is very difficult mostly due to the different types of equipment used for sonication and the conditions used, especially the control of temperature. In the 1970s and 1980s, a research group led by Burgos explored the effects of ultrasound on sporulated and vegetative forms of Bacillus spp. (Burgos et al., 1972). In 1987, the first report of synergy between ultrasound and heat in the inactivation of bacteria was published by the same group and, interestingly, there was a reduction in the effectiveness of ultrasound at elevated temperatures (Ordoñez et al., 1987). In 1989, Burgos suggested that this efficiency loss could be due to the elevation of vapour pressure in the sonicated medium that would impair or at least diminish the

Ultrasound as a food preservation tool 331

intensity of cavitational collapse (Garcia et al., 1989). Burgos suggested in that paper, that this problem could be avoided by increasing the applied pressure of the sonicated medium and patented the process in 1993 (Sala et al., 1993) that was termed manothermosonication or MTS (reviewed by Burgos, 1999). The enhanced killing of microorganisms when ultrasound is combined with heat or pressure is generally ascribed to a greater mechanical disruption of cells. Very interesting results have been obtained by the group of Sala (Sala et al., 1995; Mañas et al., 2000) in the inactivation of emerging pathogenic microorganisms such as Listeria monocytogenes, a number of strains of Salmonella spp., Escherichia coli, or Staphylococcus aureus which are increasingly found in outbreaks of food poisoning from mildly treated and/or refrigerated foods. Ultrasonic treatment at ambient temperature was not very effective on L. monocytogenes giving a decimal reduction time of 4.3 min. By using manosonication, the D-value of the ultrasonic treatment was reduced to 1.5 min. Temperatures up to 50°C did not have any significant effect on inactivation, but at higher temperatures, a considerable enhancing effect was noted (Pagan et al., 1999). Similar results have been obtained with Salmonella (Mañas et al., 2000). One of the most remarkable features of manosonication and manothermosonication is that the factors that make microorganisms more resistant to heat treatment do not seem to affect their resistance to ultrasound. This makes inactivation by ultrasound very interesting for food preservation as its efficacy would be much less dependent on treatment and real food conditions than current heat treatments. The other advantage is that damage to cells caused by heat can be reversible but, in contrast to this, experimental results show that damage caused by manosonication is irreversible (Pagan et al., 1999). Ultrasound has also been used in combination with other non-thermal technologies (San Martin et al., 2001) such as magnetic fields and high pressure together with lisozyme treatments in model systems using pulses of 20 kHz ultrasound to reduce contamination by E. coli ATCC 11775. Inactivation of Saccharomyces with a combination of heat and ultrasound has been found to be almost independent of pH (Guerrero et al., 2001). Continuous ultrasonic treatment systems have been shown to produce similar results to batch treatments (Villamiel and de Jong, 2000a). Another continuous system working in combination with steam injection has been shown to afford between 1.5- and 4-fold higher inactivation rates of E. coli and Lactobacillus acidophilus in several liquid foods such as milk or fruit juices (Zenker et al., 2003). All of the above refers to the preservation of liquid systems, but microbial decontamination of some solid foods can also be achieved by ultrasonic irradiation. Decontamination of minimally processed fruit and vegetables (lettuce, cucumber, carrots, parsley and others) has been attempted (Seymour et al., 2002) using ultrasound to remove microorganisms from the surface of the vegetable pieces which were subsequently killed by the use of chemical sanitizers, generally chlorine. It was suggested that the low increase in cleaning efficiency did not justify the extra cost of the process. A similar approach by Scouten and Beuchat (2002) to decontaminate alfalfa seeds inoculated with Salmonella or E. coli O157 showed that the combined treatments of ultrasound and Ca(OH)2 could be an alternative to chlorine treatments to avoid contamination

332 Application of Ultrasound

in the sprouts. One to 1.5 log reductions of Salmonella contaminating poultry surfaces have been achieved by using ultrasonic irradiation alone but up to 4 log reductions were achieved by combining the same irradiation intensities with minimal (0.5 ppm) chlorine concentrations (Lillard, 1994). However, Sams and Feria (1991) reported no decrease in total aerobic counts from broiler drumsticks sonicated with and without heat.

3.2 Inactivation of enzymes An excellent review was published some years ago of ultrasound inactivation or denaturation of enzymes and proteins (El’Piner, 1964), nevertheless, the study of enzyme inactivation by ultrasound has received less attention than microbial inactivation. As is the case of microbial species, enzymes are found to show a huge variety of sensitivities toward ultrasonic irradiation but, for the same reasons as given above, comparisons between different work from different authors are difficult. In 1994 a research group headed by Burgos initiated the study of the application of MTS treatments to model enzymes relevant to the food industry (lipoxygenase, peroxidase and polyphenol oxidase) in model buffer systems (Lopez et al., 1994). MTS treatments proved to be much more efficient than heat treatment for inactivating these enzymes, especially those which are more thermally labile (lipoxygenase and polyphenol oxidase). MTS inactivated peroxidase by splitting its prosthetic heme group, the same inactivation mechanism as heat (Lopez and Burgos, 1995a), whereas lipoxygenase seemed to be inactivated by a free radical mediated mechanism (Lopez and Burgos, 1995b). This group extended their studies to other harmful food enzymes. Proteases and lipases from psychrotrophic Pseudomonas (Vercet et al., 1997), the limiting factor for UHT (ultra heat treated) milk shelf-life, were found to be inactivated up to ten times faster by MTS treatments. Thermostable pectin methylesterase from oranges affects the cloudiness in citrus juices and whose inactivation is therefore mandatory in the citric juice industry, is inactivated almost 500 fold faster by MTS treatments than by heat treatment at an identical temperature. This is probably due to the impairment of substrate (pectin) enzyme stabilization by ultrasound (Vercet et al., 1999). Pectic enzymes of tomatoes, pectin methylesterase and the two endopolygalacturonase isozymes are also inactivated by MTS treatments with much higher efficiency, both in model systems (Lopez et al., 1998) and in tomato juice (Vercet et al., 2002a). General trends arose from all these enzyme inactivation studies; thermolabile enzymes are more sensitive to ultrasound than those which are heat resistant. The stabilization mechanisms operating against heat inactivation do not protect against MTS treatments (unlike that which occurs to microbial populations) and small enzymes seem to be more resistant than bigger ones (Vercet et al., 2001a). The use of ultrasound at ambient pressure has also been successfully used to inactivate food relevant enzymes. Peroxidase was inactivated by combinations of heat and ultrasound at neutral (Gennaro et al., 1999) or low pH (Yoon-Ku et al., 2000) and lipoxygenase has been shown to be inactivated at low sonication intensities (Thakur and Nelson, 1997). Villamiel and de Jong (2000b) report no effect of ultrasound on endogenous milk enzymes (alkaline phosphatase, g-glutamyltranspeptidase and lactoperoxidase) at room temperature but synergistic inactivation at 60–75°C.

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4 Ultrasound as a processing aid 4.1 Mixing and homogenization There are a large number of industrial processes that employ power ultrasound as a means of mixing materials (Canselier et al., 2002). Although the ultrasound can be supplied through bath or horn systems, there is another method of achieving the mixing via mechanical means. The device used for this purpose is often called the liquid whistle and it was developed many years ago for liquid processing, particularly for homogenization. As the name suggests, it operates on the whistle principle in that ultrasonic vibrations are generated via the flow of a liquid. The required operating pressure and throughput is determined by the use of different sized orifices or jets and the velocity can be changed to achieve the necessary particle size or degree of dispersion. A typical emulsion base for soups, sauces or gravies would consist of water, milk powder, edible oil and fat together with flour or starch as thickening agent. These materials are premixed together and, after passing through the homogenizer, produce a fine particle size emulsion with a smooth texture. Ketchup is processed in a similar way to produce a smooth product with increased viscosity and improved taste as a result of the complete dispersion of any clumps of tomato pulp. Often the ratedetermining step of a particular process involves an energy interchange through a liquid–solid interface. In the food industry one such process is the extraction of components from vegetable material. A comparison has been made of the oil-in-water emulsions produced by mechanical agitation (Uitra-Turrax, 10 000 rpm, P ⫽ 170 W) or power ultrasound (ultrasound horn, 20 kHz, 130 W) using the same model system: water/kerosene/polyethoxylated (20 EO) sorbitan monostearate. With ultrasound, the drop size is much smaller than that given by mechanical agitation under the same conditions, which makes insonated emulsions more stable. In addition, for a given drop size, less surfactant is required. These two methods were also used in a process for the encapsulation of liquid cheese aroma (20 per cent) in different carbohydrate matrices by spray-drying (Mongenot et al., 2000). In terms of encapsulation efficiency, the best system of cheese aroma encapsulation was obtained using ultrasound for the emulsification step, which gave a lower microcapsule size and a higher aroma retention than when an Ultra-Turrax mixer was used. The effect of position of the ultrasound source from the interface on emulsion quality has been studied using ultrasonic bath and horn (Mujumdar et al., 1997). Large variation in the emulsion properties with small changes in the position of ultrasound source were observed and this offered a possible explanation for discrepancies in the results of heterogeneous liquid phase systems reported in the literature. A continuous ultrasound emulsification process has been compared with other continuous mechanical emulsifying devices (Behrend et al., 2000). Continuous phase viscosity was varied by means of water-soluble stabilizers (o/w systems) and different oils (w/o systems). At constant energy density, droplet size decreases when adding stabilizers, whereas the viscosity of the oil in w/o emulsions has no effect. Qualitative investigations of the local distribution of cavitation have shown very small penetration

334 Application of Ultrasound

depths of cavitation into the liquid which emphasizes the need for improvement of apparatus design to optimize ultrasonic emulsification processes.

4.2 Foam formation and destruction Foam is a dispersion of gas in a liquid in which the distances between the gas bubbles are very small. In fact, foam can be considered as the agglomeration of gas bubbles separated by a very thin liquid film. In a foam system the volume ratio of gas to liquid is very great and the bulk density approaches that of a gas. The generation of a foam may result from the:

• aeration and agitation of liquids (if the gas is already dissolved in the liquid) • vaporization of the liquid phase • action of microbiological or chemical agents that under certain reaction conditions will release a gas. The various types of foam can be classified into different categories depending on their characteristics (Ghildyal et al., 1988). Among different foam-characterizing factors, stability is the most essential one as this defines its natural or forced rupture (Viesturs et al., 1982). Natural collapse of foams takes place because of liquid escape from foams (or syneresis). This process means liquid drains from the upper layers of the foam to the lower ones, while the capillary pressure gradient along the height of the foam column increases, thus preventing running out. Other relevant factors affecting the stability of foams are pH, molecular surface electrical charge, temperature, viscosity and surface tension. Foams are frequently produced in technical situations as unwanted side effects and, in general, they cause difficulties in process control and in equipment handling. Therefore, a great effort has been made either to prevent foam formation or to control it once it has been formed, for example in bioreactors or when gases are released under conditions of sudden pressure relief in a chemical reactor. In the food industry, submerged fermentation processes represent a good example of where foam formation is disadvantageous because it adversely affects productivity, causes fermenter contamination and hinders downstream processing among another effects (Ghildyal et al., 1988; Sandor and Stein, 1993; Freeman et al., 1997). In view of the adverse effects, it is important to control the foam in the reactors. Nowadays, there are several conventional foam control methods employing water sprays, chemical defoamers or mechanical foam breakers, although a combination of chemical and mechanical methods has been found to be more effective in the control of foam. Chemical methods use antifoam agents or defoamers. These products are usually surface-active agents which are unable to produce a stable foam by themselves. They are very effective but sometimes cause adverse effects by contaminating the process. The use of antifoams also tends to be largely based on a ‘hit and miss’ approach both with regard to the type of antifoam and the amount added. Furthermore, the addition of antifoams may also be limited by legislative problems relating to the production of food and pharmaceutical products.

Ultrasound as a processing aid 335

Mechanical methods are more widely used mainly to overcome the disadvantages related to the use of antifoam agents. The collapse of bubbles is produced by any mechanical shock (rapid pressure changes, shear force, compressive force, impact force, suction and centrifugal forces). The use of ultrasonic energy can be considered as a mechanical method based on the propagation of high intensity ultrasonic waves and represents a clean method of breaking foams without contact with the liquid. The breaking and destruction of foams by ultrasound based defoamers is assumed to be a combination of: 1 partial vacuum on the foam bubble surface produced by high acoustic pressure 2 impingement of radiation pressure on the bubble surface 3 resonance of the foam bubbles which create interstitial friction causing bubble coalescence 4 cavitation 5 atomization from the liquid film surface 6 acoustic streaming (Boucher and Weiner, 1963; Gallego, 1999). The potential use of ultrasound for foam breaking was first introduced during the 1950s and 1960s by using various types of acoustic defoamers. Most liquids with viscosities up to 500 cp can be acoustically defoamed (Chendke and Fogler, 1975). The majority of them were based on aerodynamic acoustic sources of various types such as the Hartmann whistle, the rotatory siren, etc. The main disadvantages that these systems present are related to noise problems (they usually operate in the hearing frequency range), but they also require high air generation capacity, control and sterilization of the air-flow and involve high energy consumption. A new concept for a high-intensity ultrasonic defoamer has been developed (Rodríguez et al., 1985) based on the use of a stepped-plate transducer to generate airborne ultrasound (see Figure 13.2) (Gallego et al., 1978) and incorporates an extended titanium stepped-grooved radiating plate (350 mm in diameter) which allows the ultrasonic energy to focus. Sound pressure levels (SPL) of about 165 dB (艐3 W/cm2) were measured at a distance of about 330 mm from the centre of the plate, when a maximum power of 150 W is applied to the transducer. As an example of the potential use of this system, the airborne ultrasound has been successfully applied to the control of excess foam produced during the filling operation of bottles and cans on high-speed canning lines before capping. The focal area covered by the ultrasonic beam is about 3 cm2. Two focused transducers working at 20 kHz were used in parallel to improve this effect in order to cover the can surface. The overflow of foam was also controlled with bottles in a champagne company with powers lower than 100 W due to the great volume ratio of gas to liquid. Other experiments were carried out in milk and beer factories presenting promising results. Details of a more powerful second generation ultrasonic defoamer with a power capacity of 400 W and a radiating plate of 480 mm in diameter was published 2 years later (Rodríguez et al., 1987). The system was applied to defoaming in a beer cylindroconical fermenter 6 m in diameter located in a brewery company in London. This more powerful system allowed an increase in the SPL in the focal area (12 cm2) at up to 170 dB (ca 10 W/cm2) (Gallego, 1998). The pilot trials demonstrated that the ultrasound was

336 Application of Ultrasound

able to break older foam much more effectively than freshly formed foam. There may be several reasons for this: 1 the older foam at the surface would be less stable due to drainage and coalescence 2 the lower liquid content of the less dense foam would cause the foam to reflect less of the ultrasound 3 as the foam was destroyed liquid passed downwards into the lower levels of foam which may have made it more resistant to the ultrasound; any large bubbles formed at the broth surface might make their way to the top of the foam. Nakamura et al. (1996) developed a device to control the large amount of foam generated at the time of filling bottles in a food machine plant. In this work, carried out at a small scale, the mechanism and energy efficiency of various physical and mechanical defoaming technologies were compared. The tests were carried out in a small cell within which the foam was generated and a rectangular (585 ⫻ 93 mm) flat vibrating plate made in aluminium was used in the experiments to generate ultrasonic waves. The experimental results obtained showed that ultrasonic defoaming is the most effective method when compared with laser and infrared techniques. Recently, a more versatile and powerful ultrasonic defoamer system has been developed and tested to break and control foam growth in big reactors in accordance with a procedure patented by Gallego et al. (2002). The application of the system in the dissipation of foam in reactors has been successfully used in the control of foam in industrial fermenters. More recently, this device has been improved by using several airborne focusing ultrasonic emitters, with an electronically controlled rotation system. Most of the bubbles break almost immediately under the influence of the acoustic intensity beam. From the point of view of manufacture, it is a powerful and compact device, without airflow and therefore it does not interfere with the process being treated and it can be easily sterilized.

4.3 Precipitation of airborne powders Having suspended airborne particles in most gases is generally regarded as undesirable and a method of removing airborne particles (solid or liquid) is by means of coagulation. One of the most promising methods to agglomerate and precipitate airborne particles is by using high intensity sonic or ultrasonic vibrations (Table 13.2). It is a well-known fact that the application of a high intensity acoustic field on an aerosol originates a coagulation process of the suspended particles. This phenomenon was reported by Patterson and Cawood (1931) when they observed the rapid particle agglomeration in a standing wave sound tube. Shortly afterwards independent acoustic agglomeration experiments were carried out by Brant and Hiedemann (1936) in Germany, Andrade (1936) in the UK, and St Clair in the USA (1949). They found that the microscopically small particles, both liquid and solid, in the aerosol, which were normally light enough to remain suspended for a considerable time, were agglomerated by the ultrasonic vibrations, the bigger particles then settling more rapidly under the influence of gravity and causing the aerosol to precipitate out.

Ultrasound as a processing aid 337

Although, acoustic agglomeration has been widely studied, the development of this process into an industrial application has been slow. This is probably because of the lack of suitable high-intensity, high efficiency sound sources and appropriate full-scale agglomeration chambers. Methods of obtaining high-intensity levels have involved several types of acoustic sources (Hartmann whistles, St Clair emitter, sirens and stepped plate transducers). The majority of these generators have efficiencies of about 25 per cent and poor directivity in air. Nevertheless, Gallego et al. (1978) and Rodríguez et al. (1987) showed that vibrating stepped plate transducers have very high efficiencies, up to 80 per cent, and can be applied to an industrial process where control and precipitation of fine particles is required. Magill et al. (1992) carried out investigations to remove submicron particles (mean particle diameter of 0.8 m) from a gas stream, using a combined acoustic module and electrostatic precipitator (ESP). Stepped plate transducers with power capacities up to 0.5 kW were used in a pilot-scale plant. The aerosol flow rate ranged from 150 to 1500 m3/h with resulting flow velocities of 0.17–1.7 m/s inside the acoustic chamber and 0.33–3.3 m/s in the ESP. In the experiments, the total particle number concentration was 2 ⫻ 106/cm3 and mass loading of about 1.35 g/m3. The separation efficiency of 0.8 ␮m particles was increased from 87 to 92 per cent when ultrasonic energy was applied at 20 kHz (transducer operating at 400 W). Gallego et al. (1996, 1999a) developed a pilot scale acoustic preconditioning system for fumes of a 0.5 MWt fluidized bed coal combustor. In these tests the objective was to agglomerate not only micron-sized particles but also finer particulates in the submicron range. Fly ash-laden fumes generated by the combustor were let into a 3.6 m long horizontal acoustic agglomeration chamber. The residence time of the aerosol in the acoustic chamber was in the order of 2 s. Four stepped-plate, high-intensity macrosonic transducers were used to achieve average sound pressure levels of about 151 dB at 10 kHz and 152 dB at 20 kHz and peak values of 165 dB, in the entire volume of the chamber. Optical and scanning mobility particle sizers were employed along with cascade impactors to measure a wide range of particles. Using ultrasound (four transducers at 400 W), the reduction in micron-sized particles was up to 70 per cent in terms of the initial number of particles. Reductions of about 30 per cent were obtained in the submicron size distribution. The system can be applied to any industrial process where agglomeration and precipitation of airborne particles is required. The preconditioning procedure is equally useful in combination with other filtering or removal systems such as cyclone filters, bag houses, ceramic filters, etc. Another important benefit is its applicability to high-pressure and high-temperature environments. More recently, Riera et al. (2002a) have investigated the influence of humidity on the ultrasonic agglomeration and precipitation of submicron particles in diesel exhaust by using a linear array of four high-power 20 kHz stepped-plate transducers. At flow rates of 900 Nm3/h, there was a small reduction (25 per cent) in the number concentration of particles at the outlet of the chamber but at a humidity of 0.06 kgwater/kggas the reduction increased to 56 per cent. A general enhancement of the acoustic particle agglomeration was also found with higher levels of initial humidity.

338 Application of Ultrasound

4.4 Filtration and drying 4.4.1 Filtration

The requirement to remove suspensions of solids from liquids is common to many industries. This separation can be either for the production of solids-free liquid or to isolate the solid from its mother liquors. Conventionally, membranes of various sorts have been employed for these processes ranging from the simple filter pad through semipermeable osmotic type membranes to those which are used on a size-exclusion principle for the purification of polymeric materials. Unfortunately, the conventional methodologies often lead to ‘clogged’ filters and, as a consequence, there will always be the need either to replace filters or stop the operation and clean them on a regular basis. The application of ultrasound enables the filtration system to operate more efficiently and for much longer periods without maintenance through two specific effects. First, sonication will cause agglomeration of fine particles and, secondly, will supply sufficient vibrational energy to the system to keep the particles partly suspended and therefore leave more free ‘channels’ for solvent elution. This so-called acoustic filtration has been studied for many years and in many systems achieving, for example, an 18-fold increase in filtration rate of motor oil through a sandstone filter (Fairbanks and Chen, 1971). There have been a number of developments in acoustic filtration and separation processes (Tarleton and Wakeman, 1997). One such is the application of an electrical potential across the slurry mixture while acoustic filtration is performed (Senapati, 1991). The filter itself is made the cathode while the anode, on the top of the slurry, functions as a source of attraction for the predominantly negatively charged particulate material. An example of its application can be found in the dewatering of coal slurry (50 per cent moisture content). Conventional filtration reduces the moisture to 40 per cent, using ultrasound this was improved to 25 per cent and using electroacoustic filtration further improved to 15 per cent. The potential for this process is clearly enormous when applied to a continuous belt drying process in the deliquoring of such extremes as sewage sludge or fruit pulps. 4.4.2 Drying

The reduction of moisture is one of the oldest techniques for food preservation. There are two basic methods to remove the moisture in a solid material: mechanical and thermal. Mechanical drying is based on the application of pressure or centrifugal forces to the material, whereas thermal drying uses heat to evaporate the liquid. The first method acts on the moisture weakly attached to the material and the second one can provide a more complete drying effect on the product. The use of ultrasonic energy in drying is very promising because it can act without affecting the main characteristics and quality of the products. In particular, power ultrasound is an especially attractive means of drying heat-sensitive foods because they can be dried more rapidly and at a lower temperature than in the conventional hot-air driers. Some applications of high-power acoustics in food drying are summarized in Table 13.3 where different authors reported studies of acoustic drying of a number of

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Table 13.2 Experimental data relating to particle precipitation Airborne particle

Frequency (kHz)

Intensity (dB)

Reference

Paraffin oil Carbon black smoke Coal fly-ash Soot; black soot glycerine; glycol fog aerosol Coal fly-ash Fly-ash calcined limestone

10 20.4 1.42–2.5 20 21 1.42–2.5 44

138; 148; 151 159.5 144 and 160 133–149 140–160 144 and 160 160

Brant and Hiedemann (1936) Gallego et al. (1999a) Tiwary (1985) Magill et al. (1991, 1992)

Glycol fog aerosol; soot Glass spheres; quartz particles Fly-ash laden fumes Fly-ash laden; diesel exhausts

10 and 20 0–0.9 10 and 20 10 and 20

140–165 140–165

Song (1990) Hoffmann (1993), Hoffmann et al. (1993) Caperán et al. (1995) Hoffmann and Koopmann (1997) Gallego et al. (1996) Riera et al. (2002a)

materials using airborne radiation, ultrasonic vibration in contact with food, products immersed in hypertonic solutions, in sugar solutions and in salt brine. In all cases high-intensity acoustic waves accelerate the drying process of food solid materials. Although acoustic drying has been known for more than five decades, its application and development has been very slow due to technical problems in the design of efficient and powerful airborne acoustic generators. Boucher dried gelatin, yeast cake and granulated sugar using a multi-whistle device operating in the frequency range of 10–33 kHz (Boucher, 1961). In these experiments the intensity level is considered the main factor governing the evaporation rate. Boucher suggests a minimum intensity level of about 145 dB for industrial purposes. A clear benefit in the final moisture content of about 50–75 per cent less was obtained with ultrasound compared with conventional drying processing. In order to increase the benefit of ultrasound on food drying, ultrasonic vibrations have been applied in direct contact with the samples. Gallego et al. (1999b) applied direct contact ultrasound together with a static pressure at 22°C in the drying of carrots. Results obtained with carrot slices of 2, 4 and 8 mm in thickness and 14 mm in diameter showed that the drying effect was remarkably improved. A final moisture content of about 1 per cent was obtained. Riera et al. (2002b) also obtained encouraging results applying the same technique to the drying of apples, carrots and mushrooms. Osmotic dehydration is widely used for partial removal of water from food materials by immersion in a hypertonic solution. However, one of the main difficulties when applying this technique is the usually slow kinetics of the process. A classical way to increase the rates of mass transfer is the application of a mechanical agitation system; another possibility is the use of power ultrasound. Simal et al. (1998) reported the application of ultrasound with cubes of apples in a hypertonic solution of sucrose (70°Brix) at four different temperatures (40, 50, 60 and 70°C) and showed that the rates of mass transfer increase with the use of ultrasound at 40 kHz. They found an increase of water loss (14–17 per cent) and a sugar gain rate (23 per cent at 40°C; 11 per cent at 70°C) when ultrasound was applied. Therefore,

340 Application of Ultrasound

osmotic dehydration can be carried out at a lower solution temperature to obtain higher water loss and solute gain rates, while preserving the heat-sensitive nutritive components, flavour and colour. Experiments carried out by Sanchez et al. (1999) obtained similar results with cheese immersed in saturated NaCl. Water loss increased by 11 per cent and NaCl was increased by 5 per cent compared with normal brining agitation. Cárcel et al. (2002) studied the mass transfer processes of apple slices immersed in a solution of sucrose (30°Brix) at 30°C when different intensities were applied (from 3.6 to 11.5 W/cm2) at 20 kHz with a horn. The authors (Cárcel et al., 2002) detected an intensity threshold of about 9 W/cm2. Above this intensity the water loss and the solute gain were clearly higher compared with experiments carried out with agitated brine. These experiments were repeated for brining pork loin slices with and without ultrasound at several intensities (from 15 to 76 W/cm2) at 2°C. The results for NaCl gain behaved in a similar way to those that observed for solute gain in apples treated in sugar solution. Above the corresponding intensity threshold, the increase of NaCl gain was proportional to the ultrasonic intensity applied and much higher than in experiments with mechanical agitation of the brine.

4.5 Extraction Power ultrasound has been shown to be a promising and innovative method to assist the extraction of valuable compounds from vegetables and food products (Mason et al., 1996; Mason, 1998; Vinatoru, 2001; Valachovic et al., 2001). It is particularly useful in combination with conventional solvent extraction and a range of examples is given in Table 13.3. The beneficial effects of ultrasound derive from its mechanical effects on the process by increasing the penetration of the solvent into the product and enhancing the mass transfer process to and from interfaces. It is supposed that those benefits are related to the enhancement of diffusion of cellular contents through the disruption of the cell walls produced by acoustical cavitation (Chendke and Fogler, 1975). On collapse, bubbles are capable of producing shock waves. Oscillatory particle motion produced by high-intensity ultrasonic waves can also induce secondary flows, known as acoustic streaming. Moreover, cavitation produces microjets at the surface of the food material that may introduce the liquid into the solid. This effect can increase mass transfer in both directions from the liquid to the solid or in reverse. Therefore, cavitation induced cell disruption and dispersion of suspended solids coupled with enhanced mass transfer rates due to acoustic streaming are believed responsible for the improved extraction (Toma et al., 2001). Herbs provide a source of raw materials for the pharmaceutical, cosmetics and food industries and, more recently, in agriculture for pest control and the effect of ultrasound on various extraction procedures has been reviewed (Vinatoru, 2001). During distillation ultrasound will produce more rapidly boiling centres, but no collapsing bubbles, hence the use of ultrasound during distillation does not produce any significant effect. However, ultrasound can be successfully employed to enhance extraction when a low boiling point solvent is used and the temperature of the extraction mixture is kept below its boiling point.

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Table 13.3 Application of acoustic energy in the drying of food Material

Assisted drying procedure

Reference

Gelatin; yeast cake; granulated sugar Grated cheese; orange crystals; gelatine beds; rice grains Potato cylinders

Airborne radiation in solid–gas system Airborne radiation in solid–gas system Airborne radiation in solid–gas system Airborne radiation in solid–gas system Hypertonic solution of sucrose in solid–liquid system Saturated NaCl brine in solid–liquid system Airborne radiation in solid–gas system Airborne radiation and direct contact in solid–gas system Airborne radiation and direct contact in solid–gas system 30°Brix solution of sucrose in solid–liquid system Saturated NaCl brine in solid–liquid system Ultrasonic vibration in direct contact in solid–gas system

Brun and Boucher (1957) Boucher (1961) Soloff (1964)

Rice Apple cubes Cheese cylinders and parallelepipeds; curd Onions Carrots Carrots; apples; mushrooms Apple slices Pork loin slices Apples; potatoes

Bartolome et al. (1969) Muralidhara et al. (1985) Simal et al. (1998) Sánchez et al. (1999) Da Motta and Palau (1999) Gallego et al. (1999b) Riera et al. (2002b) Cárcel et al. (2002) Cárcel et al. (2003) de la Fuente et al. (2003)

A combined ultrasound and microwave-assisted extraction method of essential oil from caraway seeds has been proposed by Chemat et al. (2003). A microwave-ultrasonic (MW-US) reactor was designed for atmospheric pressure extraction of biological and chemical products. Its application has been shown by extraction of carvone and limonene from caraway seeds. The system basically consists of a microwave oven cavity unit operating at 2.45 GHz with a power of 300 W, an open vessel reactor operating at atmospheric pressure with a volume capacity of 20–150 ml and an ultrasonic horn-type transducer working at 20 kHz. The samples were caraway seeds prepared by grinding with liquid nitrogen in a roller mill. Samples were directly introduced into the extraction reactor and conventional solid–liquid extraction was also performed for comparison. The analysis of the extracts was carried out by gas chromatography and the structure of the specimen analysed by scanning electron microscopy (SEM). A significant improvement in extraction was obtained using simultaneous ultrasound and microwave-assisted extraction. Supercritical fluid extraction (SFE) with CO2 is a non-conventional technique that can offer very good yields. This technique is suitable for fragrance extraction, giving better yields and good quality essential oil. Nevertheless, fixed bed SFE of oil from solid matrix is slow even when solute-free solvent is re-circulated and therefore improvements in mass transfer are required. The use of power ultrasound represents a potentially efficient way of enhancing mass transfer processes. This is due to the

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effects produced by compressions and decompressions, as well as by radiation pressure, streaming, etc. In addition, this is probably the unique practical way to produce agitation in SFE because the use of mechanical stirrers is not possible. Riera et al. (2002c, 2004) examined the effect of ultrasound (20 kHz and 50 W) on the particulate almond oil extraction kinetics using supercritical CO2. As a consequence of the trials (at 280 bar and 55°C) at the end of the extraction time (8 h 30 min) the yield of the oil was significantly increased (20 per cent) when SFE was assisted by ultrasound. Alternatively, mass transfer was speeded up to such an extent that yields comparable to those obtained by SFE alone could be achieved in about 30 per cent shorter time when using ultrasound.

5 Ultrasound effects on food properties The primary goal of food preservation technologies is to extend food shelf-life, mostly by preventing enzymatic deleterious reactions and microbial spoilage. However, on the other hand, they need also to preserve all those attributes that make food a pleasant and nutritious material to be eaten, i.e. flavour, colour and texture. This is especially true in First World consumer societies where interests lie much more in the pleasure of eating than in the Third World where the primary goal of eating is survival. Ultrasonic irradiation concentrates quite large amounts of energy into very small volumes (hot spots) and this has the potential to change food properties in unexpected ways. There are, however, very few reports of these effects either because they are insignificant or perhaps because the experimental findings do not always show the expected (good) results.

5.1 Effects of ultrasound on dairy products Milk is one of the most studied raw materials in relation to the ultrasonic irradiation of foods. Villamiel and de Jong (2000b) report effects on sonication at ambient pressure on several of its constituents. Milk fat globules are finely homogenized, individual caseins seem not to be affected, although the authors do not give any indication about their multimeric structures, the micelles. Serum proteins ␣-lactalbumin and ␤-lactoglobulin, on the other hand, are denatured more extensively when ultrasound is combined with heat than with these two treatments performed separately. Similar results were obtained in fat globule homogenization when applying continuous manothermosonication. This also results in a slight change in milk colour according to instrumental measurements, but this is probably just a consequence of different light reflection (Vercet et al., 2002b). Milk is also a good source of thiamine and riboflavin. The former is stable to light and oxidation but it is the least heat stable vitamin. Meanwhile, riboflavin is thermostable but it is rather sensitive to oxidation and degradation by light. Another parameter to take into account is the Maillard reaction because, in milk, lactose usually reacts with amino groups of lysine to form an Amadori product. This reaction is the first step of a complicated group of reactions

Ultrasound effects on food properties 343

that eventually lead to browning of milk and milk products. However, the ultimate result is not only the changes in colour but also the loss of the bioavailability of lysine (an essential aminoacid). There is no significant loss of riboflavin and thiamine in MTS treated milk compared with samples treated only with heat (under otherwise identical conditions). However, there was a dramatic change in the kinetics of browning when ultrasound was applied with and without pressure, even at the relatively low temperature of 92°C. Although the kinetics was very different, it proved impossible to identify the precise changes induced by ultrasound in the browning pathways (Vercet et al., 2001b). Another problem arose with the modification of milk flavour induced by MTS treatment which had an extreme cooked flavour (Vercet and Lopez Buesa, unpublished observation) very similar to that of UHT milk just after thermal treatment. The cooked flavour of UHT milk is due to exposure of sulphydril volatile groups derived from proteins and disappears after a short storage (a few days) period. Strong flavour changes have also been found in sonicated sunflower oil: they were due to the apparition of volatile derivatives of fatty acids such as hexanal and hept-2-enal (Chemat et al., 2004). Using MTS-treated milk for yoghurt production, better textural properties have been found than in control milks homogenized with standard methods (Vercet et al., 2002c). No flavour defect could be observed in these yoghurts (Vercet and Lopez Buesa, unpublished observation). A similar finding has been reported by Wu et al. (2001) using milk sonicated at ambient pressure. These authors (Wu et al., 2001) attribute the effect to more effective milk homogenization, but work by Vercet et al. (2002c) showed that control yoghurts were even more finely homogenized than MTS yoghurts. The change was attributed to protein modification by ultrasound which agrees also with the cooked flavour derived from proteins in MTS treated milk and with the fact that MTS-treated milk does not coagulate in cheese making (milk coagulation for cheese production is mostly a protein dependent phenomenon) (Vercet and Lopez Buesa, unpublished observation).

5.2 Effects of ultrasound on juices The textural properties of tomato juices after manothermosonication treatments compared with controls have been thoroughly studied (Vercet et al., 2002a). MTS resulted in higher consistency and initial apparent viscosities. Tomato juices are, from a rheological point of view, pseudogels, whose flow properties depend on the interaction or entanglement of cell particles (mostly cell walls), soluble pectin concentration and the chemical properties of the latter. MTS treatments of pure pectin solutions yielded molecules with lower apparent viscosities due to a size reduction (Lopez Buesa, unpublished observation). A similar result was observed by Seshadri et al. (2003). It is difficult to predict what might be expected from a modification of pectin properties in gels, or pseudogel derived from pectin. Longer molecules show higher resistance to flow but shorter ones can interact in a different way with suspended particles leading also to increased resistance to flow. An untrained panel detected some flavour differences in MTS-treated tomato juice but these were not considered detrimental by the panellists, just different. Instrumental

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colour measurements of tomato treated juice also revealed some differences from control samples. This is most probably due to the disappearance of almost 70 per cent of the initial lycopene, the main carotene responsible for tomato red colour (Lopez Buesa, unpublished results). However, no appreciable change was observed in the tomato juice content of another readily oxidable and important molecule, ascorbic acid. Zenker et al. (2003) also found that sonication had no effect on the ascorbic acid content in milk and orange juice. This is somewhat surprising taking into account that MTS treatments are able to produce substantial amounts of hydroxyl radicals (Vercet et al., 1998). The explanation could lie in a competition (different reaction rates) between hydroxyl groups and all the other oxidizable substrates available (i.e. proteins or sugars). A comparison study has been made of the effect of the heating and sonication of orange juice in terms of: 1 ascorbic acid, the most important vitamin in orange juice which is quite sensitive to oxidation 2 carotenoid content which is important for both colour and as a nutrient (provitamin A and functional molecules) and 3 non-enzymatic browning that, in this case, is related to sugars and ascorbic acid degradation. Indeed, browning intermediates derived probably from sugar degradation were found only in MTS treated orange juice. Also a 10 per cent decrease was found in carotenoid content. Only a slight decrease in ascorbic acid was found in MTS treated juice (Vercet et al., 2001b).

5.3 Effects of ultrasound on egg products Liquid eggs have been also submitted to MTS treatments without any noticeable change in functional properties (Mañas, 1999). However, there was a detectable diminution of the gelling properties of isolated ovalbumin submitted to MTS treatments (a 50 per cent decrease in storage modulus of ovalbumin gels). The gelling properties of manosonicated ovalbumin samples recovered partially after 24 hours cold storage which points to protein unfolding and refolding processes occurring during and after MTS treatments, respectively (Sánchez et al., 2002).

6 Conclusions The effectiveness of ultrasound as a food processing tool has been proven in the laboratory and there are a number of examples of scale-up. In most cases the frequency used has been that which is available commercially, i.e. 20 or 40 kHz and this has proved quite satisfactory. In such cases the variable parameters are temperature, treatment time and acoustic power. Little attention has been paid to the use of different frequencies except in a few cases. One such is the use of ultrasound in food preservation

References 345

using the bactericidal action of sonication combined with other techniques such as heat, ultraviolet light and the use of a biocide. The results presented in this chapter should provide a starting point for more comprehensive research and development leading to the introduction of power ultrasound into a wider range of industrially significant food processing operations.

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Irradiation of Foods Monique Lacroix INRS-Institut Armand-Frappier, Canadian Irradiation Center, Université du Québec, Laval City, Québec, Canada

In spite of the technologies developed during the last decade, the level of food loss is still high and is reported in many countries. According to the United Nations, more than 30 per cent of the mortality rate world-wide is caused by alimentary diseases. The desire of most countries to make food safer for consumption requires better food preservation and production techniques. In this regard, irradiation is an interesting alternative to be considered. Some agricultural products are important commodities in international trade. The trade of these products is often seriously hampered by infestation of several species of insects and mites. The presence of parasites, some microorganisms, yeast and moulds are also the source of problems, sometimes directly or indirectly through toxin formation in the food products. Irradiation alone or combined with others processes can contribute to ensuring food safety to healthy and compromised consumers (pregnant mothers, immunocompromised AIDS patients, people on medication and ageing persons), satisfying quarantine requirements and controlling severe losses during transportation and commercialization. The use of irradiation for decontamination of foods is a promising technology that could be applied to the end product. This technology also has the advantage that it can be applied to fresh, frozen or cooked products. It is a physical, safe, environmentally clean and efficient technology. This chapter reviews the application of irradiation alone or in combination with other treatments for preserving some fruit and vegetables, fish, poultry and meat.

1 Introduction 1.1 Importance of food-borne illness World-wide alimentary self-sufficiency and security are main objectives to reach in order to protect human health, reduce alimentary losses/waste and suppress hunger and malnutrition. Unfavourable climatic conditions, such as very hot and humid temperatures, the absence of a continuous chain of protection during stocking operations, conditioning, transportation and marketing of foods, contribute to the increase in alimentary losses (Satin, 1996a). During the last decade different food treatments like heating, freezing, smoking, drying and modified atmosphere storage have been developed alone or in combination in order to improve the efficiency of the treatments. In spite of the development of food technologies in order to avoid contamination, food loss is still high and can reach more than 40 per cent in fruit and vegetables and the loss is higher for fish and meat products since the preservation time is more restricted. In developing countries losses tend to be rather high. Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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Storage losses alone have been estimated from a low of 10 per cent in cereal grains to a high of 75 per cent in the more vulnerable pulses (peas and beans). With some crops, significant insect damage can occur even before storage and resultant losses may run up to 60 per cent (Satin, 1996a). The cost to treat food-borne disease due to meat and meat product contamination is estimated in Canada to be $500 million/year. The estimated cost of food losses for meat, poultry and fish is over $200 million/year (Anon, 1995). Moreover, society has to cope with diseases resulting from the development of pathogenic microorganisms that may be found naturally in foods and also by the production of toxic substances such as verotoxin-producing bacteria like E. coli O157:H7 and aflatoxin produced by moulds. Microbial contamination is responsible for a variety of alimentary diseases such as toxoplasmosis, salmonellosis, campylobacteriosis, listeriosis, trichinellosis, cholera and many more. Campylobacter, Listeria, Shigella, E. coli and Salmonella are the most important bacteria responsible for food-borne illness in Canada. According to Statistic Canada, the number of food-borne illnesses is estimated to be more than 630 000 cases/year for Salmonella, 100 000 cases/year for Staphylococcus aureus, 19 000 cases/year for Shigella, 2800 cases/year for Listeria monocytogenes, 16 000 cases/year for Campylobacter jejuni and 13 000 cases/year for E. coli O157: H7. The costs associate with 10 Salmonella incidents in catering establishments in the USA and Canada ranged from US$57 000 to US$700 000 and the direct cost only of five Salmonella incidents in manufactured foods ranged from US$36 000 to US$62 000 (Socket, 1991). In Canada, 50 per cent of the mortality related to alimentary diseases is due to poultry consumption. According to Health and Welfare Canada, the annual costs to treat foodborne illness are estimated at $1 billion in Canada and from $5 to $86 billion in the USA (Anon, 1994). New inspection systems based on hazard analysis and critical control point (HACCP) systems have permitted a reduction in the number of Salmonella and Campylobacter cases in the USA (Anon, 2000). However, food contamination is still an enormous public health problem. In the USA, 73 480 cases are diagnosed from E. coli O157:H7; 2518 cases from listeriosis and 2 453 926 from Campylobacter jejuni (Etzel, 2001). More than 76 million/year people are affected by food-borne pathogens, which result in 325 000 hospitalizations and 5000 deaths each year in the USA (Mead et al., 1999). It has been demonstrated that many fresh-cut products serve as vehicles for foodborne pathogenic bacteria. In 2000, more than 500 people were ill and one child died from E. coli O157:H7 after consumption of beef contaminated salad bar watermelon (Zhuang et al., 2003). Salmonella was also found in pre-cut watermelon in the USA (Blostein, 1993). Shigella sonnei contamination was associated with shredded lettuce and Listeria monocytogenes was linked with coleslaw consumption (Hurst, 1995). Listeria monocytogenes was found in over 50 per cent of red bell pepper production of Boskovich Farms Fresh Cut Division (Anon, 2001a). Fresh Products Northwest also recalled its Crunch Pak Fresh Sliced Apple packages due to a probable contamination with Listeria monocytogenes (Anon, 2001b). The Salmonella outbreaks in Victoria, Australia, in 1997 cost the Australian small goods industry more than $400 million. A well-known company has paid over $200 000 in fines and spent $3 million

Introduction 355

on food safety research as well as $1.2 million to settle a civil law suit over Listeriacontaminated hot dogs and deli meats by Bil Mar Foods in 1998, even though tests of Bil Mar food products conducted by the US Department of Agriculture during 1998 were negative for Listeria monocytogenes (Zhuang et al., 2003). Food-borne parasites and viruses are also responsible for food-borne diseases. Raw vegetables are particularly potent carriers of parasites when unsterilized manure is used as a natural fertilizer. Giardia lamblia is found throughout nature and causes infection of the small intestine resulting in diarrhoea, cramps and weight loss. This is the most commonly reported parasite in the world in both developing and developed countries. This parasite can persist for many months and cause entamoebiasis. Toxoplasmosis is a serious disease that can attack nervous and muscular tissue. Cysts of this parasite have been found in pork and lamb. Ingestion of undercooked meat from these animals can cause toxoplasmosis. Over 50 per cent of the American population is Toxoplasma seropositive. Trichinella spiralis is another parasite especially found in pork. Ingestion of under cooked meat can liberate the larvae in the intestine and then the parasite can move into the circulatory system and to the muscles provoking inflammation. Anisakis is found in raw fish and is responsible for severe incidents in Japan and Korea. Raw herring is also eaten in the Netherlands and Scandinavia and is also a source of contamination (Satin, 1996b). Viruses are also agents of food-borne disease. Hepatitis A and E viruses, small roundstructured viruses, rotavirus, Norwalk-like caliciviruses, astroviruses and group A rotavirus are also frequent food-borne viruses. Food, water and person-to-person are all vehicles of transmission (Richards, 2001). These viruses are spread by the faecal–oral route and are a major cause of morbidity and mortality world-wide. Foods may be contaminated at any time pre- or post-harvest and many outbreaks are associated with foods handled by infected restaurant workers, improper irrigation or fertilization practices, or by spoilage at any stage of handling (Richards, 2001). In Shanghai, in 1988, it was estimated that more than 16 000 people were infected by hepatits A virus after consumption of raw shell fish (Jianxiang et al., 1988; Wang et al., 1988) and the transmission was then spread to over 300 000 cases of hepatitis by person-to-person transmission. Rotavirus causes an estimation of 2.7–3.9 million illnesses, 49 000–50 000 hospitalizations and about 30 deaths per year in USA but an estimated 800 000 deaths per year world-wide (Anon, 1998; Parashar et al., 1998; Mead et al., 1999). Sanitation during food processing, distribution and food handling is not necessarily under proper conditions and contributes to the increase of the contamination level. The importation of food where sanitation conditions are not necessary under control conditions is also a source of contamination. All of these parameters could be responsible for widespread illness and suffering and the cost to the company concerned can be huge (Moltimore and Wallace, 1998). The most susceptible people to food illness are an ageing population, pregnant mothers, individuals with compromised immune function or antibiotic resistant pathogens, children and people on medication. The time has to come to find ways to decrease losses in food products, increase their preservation time and assure their innocuousness. This in turn, will ensure the

356 Irradiation of Foods

quality of food sold to the population at large and it will also enable the expansion of distribution on a world-wide scale. Irradiation technology is the answer to all these expectations, as it creates the possibility of putting food on the market that is ‘glowing with freshness’ and free from pathogens. Irradiation technology does not supplant sanitary food requirements but it should reduce the risk of disease. According to the Centers for Disease Control and Prevention, based on the assumption of irradiation of 50 per cent of poultry, ground beef, pork and processed meats, could result in a 25 per cent reduction in the annual morbidity and mortality related to the five most important bacteria responsible for food-borne illness and food irradiation on a large scale basis will prevent 900 000 cases of illness, 8500 hospitalizations, over 6000 catastrophic illnesses and 350 deaths each year (Mead et al., 1999). This illness prevention would permit a saving in health care cost and loss of productivity of several billion dollars.

2 Fundamentals of food irradiation 2.1 Definition Irradiated foods are those that have been treated with ionizing irradiation. This process is a physical treatment that consists of exposing foods to the direct action of electronic, electromagnetic rays to assure the innocuity of foods and to prolong the shelflife. The ionizing irradiation that is being discussed is part of the electromagnetic spectrum with radio waves at one end and the high energy X-rays and gamma-rays at the other. In the middle are the visible light rays, with infra-red and ultraviolet rays on either side. Between the radio and infrared rays are the microwaves which are becoming common in every household. Since all these are part of the electromagnetic spectrum, they will also have several things in common. They are all waves with a characteristic wavelength, frequency and a certain amount of associated energy. The higher the wavelength, the smaller is the associated energy. Radio waves, with considerably long wavelengths of 30 cm to 3 km have very limited energy associated with them. Microwaves, although relatively low energy waves, can cause molecular vibrations in materials like food which contain moisture and fat, resulting in very rapid heating. The X- and gamma-rays on the other hand are very short wavelength radiations that have very high associated energy levels. When made to bombard against materials, they can knock off an electron from an atom or molecule causing ionization. For this reason, these are often called ionizing irradiation. When food irradiation is discussed, it is mainly with respect to ionizing irradiation (Lacroix et al., 2002). The irradiation sources that are permitted for use in food processing are gammarays produced from the radioisotopes cobalt-60 (1.17 and 1.33 MeV) and caesium137 (0.662 MeV). Gamma-rays and X-rays transfer energy in a number of ways, each involving the liberation of fast electrons that then lose energy in electronic interactions. The main source of gamma irradiation is cobalt-60 which is a radioactive isotope produced from cobalt-59. Caesium-137, which is a spent fuel from nuclear reactors, also

Fundamentals of food irradiation 357

produces gamma-rays. Another source is beta-rays which are a stream of electrons (maximum energy 10 MeV). Since the associated energy levels of these rays are too low to be of any practical value in terms of irradiation preservation, they need to be accelerated (in cyclotrons, linear accelerators etc.) to make them acquire the required energy. Careful precautions should be taken to ensure that all electrons have enough energy. If the acquired energy is too high, induced radioactivity in foods could occur upon irradiation (Lacroix et al., 2002).

2.2 Doses of irradiation The treatment received by the food product is characterized by the irradiation dose, which is the quantity of energy absorbed by the food while it is exposed to the irradiation field. The international unit of measurement is the Gray (Gy). One Gray represents one joule of energy absorbed per kilogram of irradiated product. One Gy is equivalent to 100 rad (radiation absorbed dose). The technique for measuring the dose is known as dosimetry. The three main purposes of dosimetry in food irradiation are: 1 to develop the proper dose for the food commodity under research 2 to get data for commissioning the food product through the regulatory agency and 3 to establish the quality control procedure in the food production plant. In general, experimental dosimetry is preferred over calculation methods. Dosimeters are placed within the food product being irradiated to measure the distribution of the absorbed energy and to determine the maximum and the minimum dose absorbed by the food. The measurement of the absorbed dose must be accomplished as accurately as possible to establish correct procedures for food preservation and quality control in irradiation processing. Primary or reference dosimeters (e.g. calorimeter) are used to calculate the amount of energy absorbed or to measure the dose directly. Secondary or routine dosimeters give measurements that vary with the amount of energy absorbed. The dose is estimated from a calibration curve in which measurements obtained from a secondary dosimeter are plotted against the measurements made under similar conditions by the primary dosimeter (Lacroix et al., 2002). Once secondary dosimeters are properly calibrated, they can be used to estimate the dose absorbed by the food. Of course, the secondary dosimeter has to be selected with absorption properties that match the food. For example, Ceric-cerous (MDS Nordion International Inc.) is an aqueous chemical dosimeter. Ceric-cerous dosimeters are mainly used for research purposes. Upon irradiation, ceric ions are reduced to cereous ions. The range covers most food requirements (low dose: 0.6–10 kGy, high dose: 5–50 kGy). Aqueous solutions are kept in glass ampoules that are breakable and their size is relatively large. It is usually used in the development of the technology. The Fricke dosimeter is widely used for calibration and is one of the most useful reference dosimeters. Aqueous solutions of Fe2
ions are converted into Fe3
ions and measured by an ultraviolet spectrophotometer to determine the concentration of Fe3
at 305 nm. The range is 0.02–0.4 kGy. Radio chromic film (Far West Technology) is one of the most commonly used dosimeters.

358 Irradiation of Foods

Table 14.1 Applications of irradiation treatment Doses

Effects

1 kGy

Inhibition of germination Disinfestation Ripening delay Shelf-life extension Elimination of pathogens Sterilization

1–10 kGy 10–50 kGy

For routine dosimetry during processing of fruits, FWT 60 radiochromic film (Far West Technology) is normally used. The dosimeter is usable throughout a dose range from 0.1 to 100 kGy in food processing. The desired dose is achieved by the time of exposure and by the location of the product relative to the source. The amount of energy absorbed by the food will also depend upon the mass, bulk density and thickness of the food. For each kind of food, a specific dose has to be delivered to achieve a desired result. If the dose is less than appropriate, the intended preservation effect may not be achieved. If the dose is excessive, the food may be damaged and unacceptable for consumption. Low doses are used to inhibit sprouting of certain crops such as potatoes and onions, to disinfect fruits and vegetables from insects and parasites and to delay physiological processes (e.g. ripening) of fruits (Table 14.1). A delay in post-harvest ripening can occur only in a climacteric fruit which ripens normally after harvest. To eliminate spoilage microorganisms and to extend the shelf-life of foods, medium doses are necessary. High doses are used to decontaminate herbs, spices and food ingredients or for shelf-stable foods (Table 14.1).

3 Wholesomeness of irradiated foods 3.1 Government regulations In 1983, the Codex Alimentarius Commission accepted that foods irradiated up to 10 kGy were safe and therefore toxicological testing was no longer necessary. In 1988, the Agriculture Organization of the United Nations (FAO), World Health Organization (WHO), International Atomic Energy Agency (IAEA) and the International Trade Centre-UNCTAD GATT jointly organized the international conference on the acceptance, control of and trade in irradiated food in Geneva, Switzerland (IAEA, 1989). The use of food irradiation was endorsed by government designated experts from 57 countries. The WHO, FAO and IAEA, through the International Consultative Group on Food Irradiation (ICGFI), convened a study group in Geneva in September 1997 in order to study the data available on safety and wholesomeness of irradiated foods at doses ⬎10 kGy. This study, based on extensive scientific evidence, has confirmed that foods could be treated at any dose without any detrimental effect on their wholesomeness

Wholesomeness of irradiated foods 359

Table 14.2 Status of food irradiation regulations in the USA Product

Dose of irradiation (kGy)

Pork Spices Vegetables, fruit Poultry Meat Frozen meat

1 since 1985 30 since 1986 1 since 1986 1.5–3 since 1992 1.5–3 since 1992 7 since 1997

(WHO, 1999). The study group concluded that high dose irradiation, conducted in accordance with good manufacturing and irradiation practices, could be applied to several types of foods to improve their hygienic quality, to make them shelf-stable and to produce special products (WHO, 1999). Now, countries around the world have brought their regulations in line with the Codex General Standard of Irradiated Foods and have cleared many foods for irradiation. South Africa has been irradiating and successfully marketing irradiated spices and others foods like pre-cooked, irradiation sterilized entrees (chicken curry, country sausage, beef steak with gravy) intent for military and sport use (Molins, 2001b). South Africa also produces the greatest variety of irradiated fruits including mangoes, papayas, bananas, litchis, tomatoes and strawberries. France operates an electron beam for the control of Salmonella in mechanically deboned chicken meat and a gamma source for the control of Salmonella in frogs legs (Molins, 2001b). Thailand irradiates nahn (raw pork sausage) for microbiological safety. In the USA, in 1986, the Food and Drug Administration issued a regulation (CFR title 21 part 179) that permits irradiation of fruits for insect disinfestation, delaying ripening, growth and maturation inhibition. Frozen beef patties, fresh meat and poultry and spices could be irradiated to assure the innocuity and pork to eliminate Trichinella (Table 14.2). Not only are specific applications to foods specified in this regulation but also the type and sources of irradiation: gamma rays from cobalt-60 or caesium-137, accelerated electrons from a machine source not to exceed 10 MeV or X-rays from a machine source not to exceed 5 MeV. Labelling is required; the international recognized logo (Figure 14.1) must also appear on the package and either of the following statements, ‘treated with irradiation’ or ‘treated by irradiation’ should be displayed. If irradiated fruits are shipped to a manufacturer for further processing, a label ‘do not irradiate again’ must be displayed. Anyone may irradiate fruits in compliance with this regulation without further permission from the Food and Drug Administration (FDA) in the USA and from Health and Welfare in Canada. This regulation is a full authorization. There are no FDA licensing procedures for plant facilities. In both countries, plants must comply with current regulations for good manufacturing practices for the production, handling and storage of foods. Moreover, industrial activities must conform to laws designed to protect workers’ safety and the environment. In the

360 Irradiation of Foods

Figure 14.1

International recognized logo for irradiated foods.

USA, the use of a radioisotope such as cobalt-60 or caesium-137 as a source of irradiation, requires a licence issued by the United States Nuclear Regulation Commission (USNRC). In the case of electron accelerators and X-ray machines, concerns regarding safety arise only when the machine is turned on. Thus, manufacturers of machine sources of irradiation are not required to obtain a licence from the USNRC but they must submit a report to the FDA’s Center for Devices and Radiological Control for Health and Safety Act. While this imposes no requirement on food processors, a processor should ensure that the equipment has been reported by the manufacturer. It has to be pointed out that some states require registration or licensing for facilities using machine-generated irradiation. In Canada, products must be irradiated in plants especially constructed for this purpose. These plants must possess a licence issued by the Atomic Energy Control Board (AECB), which inspects regularly to verify safety and compliance. Irradiated food products must be also approved in advance by Health Canada. In the current legislation, irradiation treatment has been permissible to inhibit germination of potatoes and onions since 1960 and wheat, flour and wholewheat flour irradiated to prevent insect infestation during storage since 1969. Since 1984 whole or ground spices and dehydrated seasonings have been allowed to be irradiated to reduce the initial microbial charge (Jategaonkar and Marcotte, 1993) (Table 14.3). To get the permission to irradiate other foods, processors must first prepare a submission to Health Canada according to the pre-clearance requirements for proposed irradiated food. Details, including the purpose of the proposed irradiation, the irradiation dose required, the chemical, physical, microbial and nutritional effects, the details of other processes to be applied before and after irradiation and recommended conditions of storage and shipment, must be specified. Then, the petition will be evaluated in terms of whether it addresses and satisfies the pre-clearance requirements. Health Canada has proposed to recommend amendments to the present regulation in order to permit the application of the food irradiation process for chicken, ground beef, shrimps, prawns and mangoes. This proposal is under evaluation by the Canadian government. If accepted, the petition will follow due regulatory process and these food products will be put on the

Wholesomeness of irradiated foods 361

Table 14.3 Status of food irradiation regulations in Canada Product

Dose of irradiation (kGy)

Potatoes Onions Wheat and flour Spices and dehydrated seasoning preparation

0.15 since 1960 0.15 ince 1965 0.75 since 1969 10 since 1984

clearance list for irradiation in the Canadian Food and Drug Regulations. If the irradiated food is sold in the market place, it must also comply with labelling requirements. Identification of the irradiated food product or food ingredient, using the international symbol, is required if it makes up more than 10 per cent of the product content. A written statement that the food has been irradiated must also be either written on the package or displayed next to the irradiated food. Although many attempts have been made to internationalize regulation of this technology in the world, most countries continue to approve its use on a case-by-case basis. Today, at least 36 countries have collectively approved irradiation of more than 50 different foods. As an example, according to the estimate of FAO-IAEA Division of Nuclear Techniques in Food and Agriculture, more than 100 000 tons of spices alone were irradiated in 1998 all over the world (Molins, 2001a).

3.2 Public acceptance Historically, the food industry had great difficulty in influencing public opinion regarding new food processing technologies. According to Young (2003), the public resisted the use of canned food products for about 50 years after the introduction of the canning process. The development of foods processed by novel and emerging technologies introduce different challenges: the consumer perception, the acceptation of the novel technology, the purchase behaviour and the retention of the physical and the sensorial quality as well as the nutritional value of the treated foods without interfering with the assurance of food innocuity (Cardello, 2003). Due to a misunderstanding of the technology, most of the time irradiation is associated with the nuclear establishment and the lack of awareness about benefits to society, arguments have routinely and successfully been sought to postpone the introduction of this technology. However, faced with new facts and developments in health, environmental, international trade, quarantine, legislative issues etc., the perception about the potential benefits that food irradiation offers in solving some of the most pressing problems in these areas is rapidly changing attitudes everywhere (Molins, 2001a). Education of the public to adopt health practices and the education about irradiation could permit an acceptance of the public face to this new technology (Anon, 1993). Experience in some countries has shown that consumers provided with sufficient information can be convinced of the advantages offered by irradiated foods (Diehl, 1990). In Florida, the public is educated for example through brochures. Efforts are

362 Irradiation of Foods

also made to educate health professionals on the need for the use of irradiated foods through seminars and training programmes. According to Allen (1999), the positive attitude of consumers to accept irradiated foods is related to the efficiency of this technology to improve the food safety. In general, these consumers are willing to pay more for safe foods and the purchase level increase is related to the level of education (Maciorowski et al., 1999). When measured in the marketplace using real products, consumer acceptance is invariably positive. A variety of irradiated products has been sold successfully in the Chicago area since the late 1990s (Molins, 2001a). Progress in the commercial use of food irradiation has been slow, but there have been positive signs along the way (Pszczola, 1997). In 1992, irradiated strawberries were sold at an independent produce and grocery store in Florida (Marcotte, 1992) and one in the Chicago area (Pszczola, 1992). The latter store also sold a variety of other irradiated produce including grapefruits, oranges, onions, tomatoes, mushrooms and blackberries (Pszczola, 1997). In 1993, selected stores in the USA sold irradiated poultry (Pszczola, 1993). California is one of the states which has started a project to allow irradiation of fresh produce that is in demand for the food service industry (Pszczola, 1997). These examples confirm that consumers are willing to buy irradiated foods especially when the product is identified. Also consumers are interested in a process that eliminates harmful microbes and reduce the risk of food-borne disease. Marketing tests have demonstrated that if consumers are first educated about what irradiation is and why it is done, approximately 80 per cent will buy the product (Young, 2003). On occasions, stores in the USA have been selling irradiated exotic fruit such as papaya, mango and rambuttan. Plans are also currently underway for the building of a food irradiation facility in the state of Washington for irradiation of fresh fruit and vegetables as well as poultry, meat and seafood. The quantity of spices treated by irradiation has continued to increase each year. In California, irradiation of dried vegetables seasoning increased by 20 per cent in 1993 and 20 per cent in 1994. For this technology to be successful, the application should be fulfilling a real need. The irradiation process should either be the only solution to a specific problem or possess real advantages over existing technologies. The cost should be comparable to other food processes. The trend in the practical application of irradiation for fruits is likely to increase in the coming years in view of the prohibition or restriction of fumigants used for insect disinfestation. Following the US ban on ethylene dibromide, many producers in several countries employ hot water dips to disinfest fruits such as mangoes and papayas but this technology destroys the quality of these fruits. Methyl bromide, another fumigant used to prevent the migration of microbes and pests around the world, is still used in the USA to disinfest grapes, pineapples and dried fruits. This toxic gas has been related to the depletion of the ozone layer. Irradiation is the only technology available that could replace the utilization of methyl bromide as a fumigant. Because irradiation can be applied to fruits in a ‘fresh-like’ state and because it can kill microbial contaminants and sterilize or kill adult insects as well as larvae and eggs, it is an alternative process of considerable interest. The increase in consumption of exotic fruits originating from developing countries and the demand for safe, nutritious and convenient

Biological effects of irradiation 363

foods in industrialized countries will likely contribute to the application of this technology on a broader scale. In developing countries, post-harvest food losses can be enormous. A low dose application of irradiation (up to 1 kGy) to fruits at post-harvest offers a unique opportunity to eliminate insect infestation. Developing countries will likely benefit from this technology by reducing their losses and the ability to offer an increase in the supply of certain produce. Consumers will benefit from greater price stability due to the availability of many commodities including tropical fruits throughout the year.

4 Biological effects of irradiation 4.1 Effects on microorganisms One of the most important properties of irradiation is inactivation of microorganisms, especially pathogens. Moreover, this technology can be efficient in improving the shelflife by reducing the microorganism level, destroying parasites and insects, delaying the ripening of fruits, inhibiting the germination of onions and garlic and, under certain conditions, deactivating viruses. The biological effects of irradiation create damage in the genetic material of the cell causing a lesion of the DNA or breaking both strands of DNA (Dickson, 2001). This damage prevents multiplication, randomly inhibits cell functions, resulting in the death of the cell. The sensitivity of microorganisms to irradiation is based on the size of their DNA, the rate at which they can repair damaged DNA and other factors. The size of the DNA ‘target’ in the microorganism is one of the most important factors. Parasites and insect pests, which have large amounts of DNA, are rapidly killed by extremely low doses of irradiation with D-values (dose necessary to reduce by one log the content of microorganism) of 0.1 kGy or less. It takes more irradiation to kill bacteria, because they have smaller DNA with D-values in the range of 0.3–0.7 kGy. Some bacteria can form a dense hardy spore, which means they enter a compact and inert hibernation state. Spore-forming bacteria are generally the most resistant to irradiation with D-values in the order of 2.8 kGy. According to Dickson (2001), it is in part due to their low moisture content (10 per cent wet basis) compared to as much as 70 per cent in vegetative bacteria. The prion particles associated with bovine spongiform encephalopathy do not have nucleic acid and they are not inactivated by irradiation except at high doses (Young, 2003). Indirect action of irradiation on water produces radiolytic products through the reaction of the hydroxyl (OH• ) radical. An increase in the bacterial resistance of irradiation is observed under freezing conditions or in reduced moisture content. The food components also affect the bacterial resistance (Quinn et al., 1967; Huhtanen et al., 1989). Bacterial resistance to irradiation is also related to the efficiency of its DNA repair mechanism, on the environmental conditions and the nature of the food where bacteria are located. Table 14.4 presents D10 values for selected pathogenic bacteria.

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Table 14.4 D10 values of selected pathogenic bacteria Bacterium

Medium

Conditions

D10 value (kGy)

Reference

Listeria monocytogenes

Chicken Chicken Ground beef

2–4°C 12°C 12°C

0.77 0.49 0.5–0.9

Huhtanen et al. (1989) Patterson (1989) El-Shenawy et al. (1989)

Staphylococcus aureus

Buffer Saline Poultry Meat

pH 7 20–25°C 10°C 10°C

0.17 0.4 0.42 0.86

Erdman et al. (1961) Ahn et al. (1964) Patterson (1988) Erdman et al. (1961)

Campylobacter jejuni

Broth Ground turkey

0–5°C 0–5°C

0.27 0.19

Lambert and Maxcy (1984) Lambert and Maxcy (1984)

Escherichia coli O157:H7

Ground beef Ground beef

⫺17°C 2–5°C

0.307 0.241

Clavero et al. (1994) Clavero et al. (1994)

Salmonella typhimurium

Roast beef Ground beef Deboned chicken Gravy

3°C 20°C ⫺40°C vacuum 3°C

0.567 0.55 0.497 0.416

Grant and Patterson (1992) Tarkowski et al. (1984) Thayer et al. (1990) Grant and Patterson (1992)

Shigella dysenteriae

Oysters Crabmeat

– –

0.40 0.35

Quinn et al. (1967) Quinn et al. (1967)

Adapted from Dickson (2001).

4.2 Effects on parasites and insects Food-borne parasites are a great problem especially in developing countries but can be found in developed countries. The most important parasites present in foods are Entamoeba histolytica, Giardia lamblia and Toxoplasma gondii. Entamoeba histolytica is the cause of dysentery which can be fatal if the parasite migrates to the liver, lung or brain (Satin, 1996b). The traffic of people and goods between countries creates risks of pest contamination from country to country. According to Satin (1996a), when pests move to new areas they can flourish and become unmanageable. One of the best examples is the Mediterranean fruit fly, which is perhaps the most feared pest in many countries (Satin, 1996a). Damage from Mediterranean flies can result in losses of 80–100 per cent of the crop. Quarantine treatments can prevent the entry of small flies. Quarantine treatments could be assured through chemical fumigation like ethylene dibromide, but this compound is now banned in many countries. Cold treatment could also be used, which consists of a storage of 15 days at 0.5–2.0°C. However, this method is expensive and a large variety of fruit and vegetables such as clementine, papaya and mango are not tolerant to cold storage. Hot dip treatment could be used on unripe fruit but the flavour and aroma are not comparable to the fresh fruit (Satin, 1996a). Irradiation at low doses is an effective treatment in order to control insect infestation. Unlike fumigation irradiation does not leave any chemical residue. Also, this treatment has no negative effect on consumers and the environment. Insect pests are

Biological effects of irradiation 365

Table 14.5 Absorbed doses needed to achieve quarantine security Pest group

Objective

Dose (kGy)

Bruchid weevils Aphids, whiteflies Tephritid flies Scarab beetles Curculionid weevils Noctuidae and Tortricidae Pyralidae and Tortricidae Tetranychid mites Stored product beetles Acarid mites Stored product moths Root-knot nematodes

Sterilized adult Sterilized adult Prevent adult emergence from third instar Sterilized adult Sterilized adult Prevent adult emergence from third instar Sterilized late pupa Sterilized adult Sterilized adult Sterilized adult Sterilized adult Sterilized adult

0.07–0.1 0.05–0.1 0.05–0.25 0.05–0.15 0.1–0.2 0.1–0.3 0.2–0.3 0.3 0.05–0.4 0.5 0.1–1 4.0

Adapted from Gupta (2001).

easily controlled by irradiation treatment. Irradiation could be used to sterilize or to kill the pest. In quarantine applications, the important requirement is prevention of survivors arriving at the shipping destination which has the capability of causing infestation (Urbain, 1986). Table 14.5 shows doses needed to achieve quarantine security against several groups of pests and shows that, depending on the pest group, the dose necessary to control the pest varies from 0.05 to 4 kGy. According to Gupta (2001), the irradiation efficiency is determined based on the prevention of adult emergence when only eggs and larvae are present or of sterility when pupae or adults are present. Also, the irradiation doses necessary to kill or sterilize fruit flies vary from 0.15 to 0.25 kGy. The eggs of P. interpunctella treated with 0.2 kGy do not hatch. If only eggs or larvae were present a dose of 0.2 kGy was sufficient. In dried apricots and figs infested with Corcyra cephalonica and Cadra cautella, or dried dates and raisins infested with Tribolium castaneum, an irradiation dose of 0.25 kGy is needed to be combined with storage at low temperatures (10–20°C) to ensure disinfestation for one year (Wahid et al., 1989). Adult forms may maintain activity for a large number of days after irradiation with doses as large as 1 kGy and present problems for quarantine requirements (Urbain, 1986). Several researchers have tried to reduce the irradiation disinfestation dose by adding another treatment which would predispose insects to damage when irradiated. One of the more promising areas of research to increase the efficacy of irradiation disinfestation is the use of temperature variations before, during, or after the irradiation treatment (Tilton and Brower, 1983). Changes in temperature apparently modify the effects of irradiation primarily by affecting the metabolic state of the insect (Tilton and Brower, 1983). Moreover, the response to irradiation of adult granary weevils was modified by the temperature before, during, or after the treatment. Pendlebury (1966) reported that high temperatures (30°C) before irradiation sensitized the insects to irradiation, but the same high

366 Irradiation of Foods

temperature during treatment did not increase the rate of adult mortality. Prolonged exposure to high temperature after irradiation also accelerated death at all doses investigated. One hundred per cent mortality was reached in 14 and 56 days at 30 and 15°C, respectively. Apparently, the temperature modified the lethal response to irradiation through its effects on metabolic rate, speed of the cell cycle and repair of irradiation damage, but the degree of sterilization induced by irradiation was not affected by the temperature. According to a report from Pakistan, insect control can also be achieved by a combination of irradiation and cold storage (Wahid et al., 1989). It has been observed that the infestation rate was higher in apricots and figs than dates and raisins. Initially, all samples were apparently free of any insect infestation. However, insect attacks increased consistently in irradiated and unirradiated samples, especially during room temperature storage. Ionizing irradiation has been proposed for insect disinfestation at doses of 0.15–0.75 kGy. Most insects are sterilized at these doses. However, it was found that 1 kGy may not sterilize some moth species (Tilton and Burditt, 1983). Doses of 0.25 and 0.5 kGy decreased insect infestation, but in all cases after 12 months of storage at room temperature, insect damage reached 100 per cent. The irradiation dose of 0.25 kGy coupled with lower temperature completely blocked the development of all types of insects during a storage period of one year. Irradiation treatment had no adverse effect on the appearance or taste of any sample. It was observed that packaging of dried fruits in amber bottles or black/silver polyethylene was better for protecting the colour of dried fruits. These results indicated that the packaging of dry fruits in coloured packages was better for protecting the quality of fruits. Specifically, dates are important commodities for international commercial trading. One of the main factors affecting the quality of dates is insects. These pests damage the date fruit externally and internally and limit the export capability of these nutritive fruits. Before shipping, dry dates are usually disinfested using methyl bromide, a fumigant which is not so easy to handle and requires certain precautions. Ahmed et al. (1981) carried out an experiment with low irradiation doses (0.35, 0.7 and 1.05 kGy) combined with heat (40°C) or cold treatment (25°C) in comparison with fumigation and irradiation alone (0.7 kGy) for insect disinfestation of dates. This study demonstrated that a treatment with irradiation alone (0.7 kGy) resulted in a complete kill of insects only after a long storage period that exceeded one month. However, a high degree of killing was achieved within a short period when unirradiated products were held at 40°C and irradiated at the lowest dose of irradiation (0.35 kGy). Therefore, a combination treatment of low doses of gamma irradiation and heat would be advantageous if a short storage period of time is required to cause complete killing of insects in dates, instead of using somewhat high doses which might render irradiation disinfestations of dried dates prohibitive. In fresh fruit and vegetables, certain levels of irradiation may sometimes cause softening and increases in tissue permeability. However, when used at the right dose level, this treatment can slow down the ripening rate and improve the shelf-life (Lacroix et al., 1993). It also allows fruit to be picked in a riper condition rendering the fruit more desirable for taste and texture (Satin, 1996a).

Biological effects of irradiation 367

4.3 Effects on viruses Viruses reproduce only by parasitizing a living cell. When they occur in food they do not generally multiply. However, they can persist in an infectious state for a long period (Urbain, 1986). It is believed that just a few virus particles may be sufficient to elicit infection. Viral contaminants may persist in the environment, on food surfaces or within foods for extended periods (Deng and Cliver, 1995). One of the more common of the viral pathogens is the Norwalk virus. The Centers for Disease Control and Prevention recently estimated the annual incidence of Norwalklike viral illness in the USA at 23 million (Mead et al., 1999). Viruses are the smallest pathogens that have nucleic acid and they are, in general, more resistant to the fragmentation by irradiation at doses approved for foods with a D10 values (dose necessary to eliminate by one log the content of microorganism) ⬎10 kGy (Young, 2003). The irradiation doses necessary to inactivate virus increases inversely with virus size and they are relatively high (20–100 kGy). However, according to Mallett et al. (1991), the level of irradiation required to eliminate 90 per cent of the hepatitis A virus, poliovirus and rotavirus in shellfish is 3.1 kGy but 6.8 kGy is required to inactivate coxsackievirus type B2 from ground beef (Sullivan et al., 1973). Bidawid et al. (2000) have observed that a dose of 2.7 and 3.0 kGy would be required to achieve ⬎90 per cent kill in a hepatitis A population on fruit and vegetables. However, in many cases, 90 per cent inactivation may not be sufficient to eliminate the threat of illness, since only a few virus particles may be sufficient to elicit illness. A combination with other treatments like mild hot treatment, ozone, microwave, or additives could be considered to improve the efficiency of the treatment.

4.4 Effect on ripening delay The principal interest in the use of irradiation to alter the biological processes of fruit is to delay ripening and or senescence in order to improve the shelf-life. A ripening delay can be done only with climacteric fruits but delayed senescence can be achieved with both climacteric and non-climacteric fruit (Urbain, 1986). The influence of irradiation on the ripening delay depends on the delay between harvest and irradiation, the physiological state of the fruit and the sensitivity of the fruit (Thomas, 1986). According to Boag et al. (1990), irradiation increased fruit respiration immediately after the treatment, but delayed the time to attain the climacteric respiratory peak and reduced the magnitude of this peak rate.

4.5 Sprouting inhibition Sprouting of tubers and bulbs during storage is one of the major factors contributing to qualitative and quantitative deterioration during storage. Onions, garlic and shallots are also extensively produced in the world. Sprouting inhibition by irradiation is one of the most effective technologies and it could be achieved at doses from 0.05 to 0.15 kGy. This treatment can replace chemical spraying with maleic hydrazide before

368 Irradiation of Foods

harvesting or the cold storage after harvesting (Thomas, 2001). Developing countries produce more than the third of the world’s potatoes. Losses of crop and tuber could be very high in developing countries due in part to the lack of cold storage facilities. In India, cold storage is mostly used for potato and onion storage. If these products were irradiated, the cold storage might be used for the storage of foods with higher values (Satin, 1996a).

5 Irradiation of foods 5.1 Irradiation of fresh fruit and vegetables All fruit and vegetables are perishable due to physiological changes, post-harvest fungal diseases, other pathological breakdown and insect infestation. A good preservation technique for fresh fruit and vegetables must be efficient as a post-harvest treatment. It should retain the qualities and nutrients of the commodity in a fresh-like state. It should have a proven capacity for controlling insect larvae and eggs if insect infestation is a problem. It should also have a synergistic effect on the commodity if and when combined with other preservation techniques (Moy and Nagai, 1985). Disinfestation and shelf-life extension have been extensively studied and have a great deal of potential and promise, especially for tropical fruit. Ionizing irradiation is a promising technology to maintain the quality of fresh fruits and vegetables because it has the potential to control both spoilage and pathogenic microbes (Hines, 2000). This procedure offers a physical means for pasteurization without changing the fresh state of these commodities (Farkas et al., 1997). The use of irradiation at a pasteurization dose (2–5 kGy) could control post-harvest spoilage and diseases that affect fruit and vegetables without impairing their sensory qualities (Moy and Nagai, 1985). The use of irradiation at a pasteurization dose can produce quality changes such as softening of the product. Therefore, this technology should be used in combination with other treatments. The more promising application is the use of irradiation with heat. Using the synergy, a lower dose could be used to achieve pasteurization. According to Buckley et al. (1969), non-germinating spores were markedly resistant to single applications of heat (hot water at 39–46°C) or irradiation (0.5–2 kGy), a strong inactivation effect (1 per cent survival) was obtained when irradiation plus heat (1.25 kGy
46°C, 5 min) was applied in sequence. The interaction was less pronounced with treatments in the reverse sequence. With germinating spores (6 h incubation) an even greater synergistic effect (0.1 per cent survival) resulted from heating (39°C for 5 min) followed by irradiation (1.25 kGy). Thus the maximum synergy was obtained in non-germinating spores with an irradiation-heat sequence, whereas in germinating spores a maximum synergy was obtained with a heat-irradiation sequence. With both non-germinating and germinating spores, heating and irradiation had a stronger interaction than heating and chilling (Moy, 1983). Tropical fruits are frequently wasted due to problems of deterioration during handling, transport and storage.

Irradiation of foods 369

Extension of storage or shelf-life through control of post-harvest rots is the most difficult of the three main purposes (disinsectization, shelf-life extension, ripening delay) for irradiation of fruit and vegetables. In most instances, the doses of irradiation required to inactivate pathogens would result in tissue damage (Heather, 1986). A widely reported exception is the strawberry. This fruit has been shown to be tolerant to irradiation and it is possible to treat strawberries at doses which eliminate the causes of fruit rots. The minimum effective absorbed dose required is in the range of 1.5–2 kGy, sometimes as high as 3 kGy (Dennison and Ahmed, 1971). Provided that they were subsequently kept cool, high quality can be maintained for more than 14 days.

5.2 Irradiation of fish, meat and poultry During processing and storage, fish, meat and meat products are exposed to several internal and environmental conditions that increase food safety risks. A major cause of such detrimental effect is the proliferation of many pathogenic bacteria such as Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Clostridium botulinum, Vibrio parahaemolyticus, Shigella and Cl. perfringens (Farkas, 1998; Samelis et al., 2001), spoilage microorganisms like Pseudomonas, Acinetobacter, Lactobacillus sake, L. curvatus (Lacroix and Ouattara, 2000) or parasites like Trichinella spiralis, Toxoplasma gondii, Opisthorchis viverrini, Cysticercus cellulosae, Anisakis and various taenias (Kotula, 1983; Loaharanu, 1996; Farkas, 1998). The dose required to inactivate parasites is normally very low and can be achieved at doses of 0.15–0.6 kGy (Farkas, 1987). An irradiation dose of 0.4 kGy can assure pork trichina-free (Kasprzak et al., 1993). However, doses that would kill the larvae of Anisakis in salted herring were reported to be high (⬎6–10 kGy) (Van Mameren and Houwing, 1968). Shelf-life extension and elimination of pathogenic microorganisms such as Salmonella, Yersinia and Campylobacter require a dose of 2.5–5 kGy (Molins, 2001b). Normally Gram-negative bacteria are more sensitive to irradiation than Grampositive bacteria. Hasting and Holzapfel (1987) have confirmed that lactic acid bacteria (Gram-positive) survive irradiation in minced beef better than other spoilage types. Ouattara et al. (2002) have observed that Enterobacteriaceae and other Gram-negative bacteria (total coliforms and Pseudomonas spp.) exhibited a greater sensitivity toward the irradiation treatment than the Gram-positive bacteria tested (Brochothrix thermosphacta, Staphylococcus aureus and lactic acid bacteria). According to their study, lactic acid bacteria appeared to be the most resistant organisms tested, followed by Brochothrix thermosphacta, while presumptive Staphylococcus aureus showed a sensitivity comparable to those of the Gram-negative bacteria, i.e. Enterobacteriaceae, total coliforms and Pseudomonas spp. in ground beef.

5.3 Use of combined treatments The use of different preservation techniques in combination can be beneficial for the elimination of pathogenic bacteria due to the synergistic or additive effect of the

370 Irradiation of Foods

treatments. It also allows a less extreme use of a single treatment and can permit protection of the sensorial quality of the foods (Patterson, 2001). Irradiation in combination with modified atmosphere packaging (MAP) and/or an edible coating could be beneficial to maintain the innocuity and prolong the shelf-life of fruit and vegetables (Hagenmaier and Baker, 1998; Vachon et al., 2002). Combination of irradiation and other food processing techniques such as MAP, refrigeration, freezing and cooking have great potential for improving the quality and the safety of fresh and processed fish, meats and poultry (Molins, 2001b). Irradiation treatment can reduce the numbers of pathogenic bacteria and the level of normal flora while MAP will suppress the growth of the survivors during subsequent storage. Irradiation under MAP could also act synergistically in the killing of bacteria due to the fact that some bacteria are more sensitive to irradiation under MAP conditions (Patterson, 1988; Chiasson et al., 2004). The use of irradiation in combination with heat has synergistic effects for the destruction of vegetative bacteria (Farkas, 1990) and bacterial spores (Gombas and Gomez, 1978). It has also been postulated that when irradiation is performed in the absence of air, an enhanced killing is observed during heat treatment. According to Kim and Thayer (1996), irradiation induced DNA damage while heat induced membrane destabilization. It has also been demonstrated that prepared foods irradiated at doses ranging from 12 to 40 kGy combined with cooking, vacuum packaging and freezing (⫺40°C), can produce meals with a shelf-life of over 11 months when stored at 4°C after the treatment (Desmonts et al., 1998). The use of chemical preservatives in combination with irradiation, especially natural preservatives or natural active edible coating, represents a challenge for scientists to develop new food preservation methods to improve the shelf-life without affecting the sensorial quality. Some studies available have demonstrated synergistic effects to reduce the content of microorganisms in fruit (Vachon et al., 2002), vegetables (Lafortune and Lacroix, 2004), fish (Ouattara et al., 2001), meat (Giroux et al., 2001; Ouattara et al., 2002) and poultry (Mahrour et al., 2003a). It has also been demonstrated that the active compounds present in natural antimicrobials can improve the radiosensitivity of bacteria (Mahrour et al., 2003a; Chiasson et al., 2004). There is also an increased interest in the use of high hydrostatic pressure. Like irradiation, this technology is a cold treatment used to reduce the vegetative bacteria content. Spores are resistant to both treatments. However, when used in combination, a reduction in spore number was observed (Wills, 1975). High pressure induces spore germination and germinated cells are more sensitive to irradiation (Gould and Jones, 1989). It could be anticipated that these emerging food processes would be beneficial to assuring food safety.

5.3.1 Application on fruit and vegetables

The use of irradiation as a sole treatment for post-harvest disease control seems to be limited to only a few commodities, because the doses required for effective control of fungal spoilage frequently result in undesirable changes such as tissue or skin damage, changes in flavour or texture or by affecting the appearance and normal ripening

Irradiation of foods 371

of the fruit (Lacroix and Vachon, 1999). The tolerance to irradiation varies among species and varieties and is influenced by the ripeness at the time of treatment (Kader, 1986; Oufedjikh et al., 2000). Several investigations have demonstrated the usefulness of mild heat treatment prior to low dose irradiation in extending the shelf-life of certain fresh fruits without affecting their normal quality. However, the time sequence of the application of the combination partners may also play an important role (Farkas, 1990). In the case of fungi, the heat treatment preceding irradiation usually results in a greater antimicrobial effect of all combinations (Padwal-Desai et al., 1973; PadwalDesai, 1974). With bacterial spores, the reverse order of treatment (i.e. pre-irradiation followed by heating) was found to be more synergistic, while the effect of heating followed by irradiation seemed to be additive or only slightly more than additive (Farkas and Roberts, 1976). Unfortunately, few fruits can sustain treatments at such high doses. The heat treatment employed is mostly a hot water dip. With fresh fruit and vegetables, it is important to establish the exact parameters such as proper ripening stage, proper pre-treatment, post-treatment and transport conditions yielding optimum results (Langerak, 1982). In South Africa, the combination of hot water dipping at 55°C for 5 min for mangoes, 50°C for 10 min for papayas and waxing and irradiation (0.75 kGy) combined with low temperature storage and shipment (7–11°C) have been shown to be particularly effective in controlling fungal and insect attack and in delaying senescence (Thomas, 1977). Transportation trials from South Africa to Europe demonstrated that combination-treated mangoes and papayas may be transported long distances with much lower losses in quality than untreated lots (Brodick and Thomas, 1978). The combined treatment of mild heat and low-dose irradiation also offers possibilities for delayed ripening and reduction of microbial spoilage of tomatoes (Langerak, 1979), mangoes (Gagnon et al., 1993; Lacroix et al., 1993) and other fruit and vegetables (Langerak, 1979). The causal fungus of anthracnose was sensitive to hot water and dipping for 5 minutes at 55°C (Barkai-Golan et al., 1993) was sufficient to control this disease. On the other hand, irradiation processing targets nuclear DNA and also inactivates the microorganisms (Dickson, 2001). Besides the possibility of microbial reduction, heat treatment can extend the shelf-life of fruit and vegetables by inhibition of phenylalanine ammonia lyase (PAL) activity and by inducing heat shock proteins, resulting in the reduction of phenolic compound accumulation and tissue browning (Saltveit, 2000). Lowering the dose rate of irradiation following storage under MAP conditions was also effective in protecting the colour, by the protection of the cellular membrane, of mushrooms and in assuring a shelf-life improvement (Beaulieu et al., 1999). Another promising way of increasing the effectiveness of irradiation, in the control of food-borne microorganisms without adversely affecting organoleptic qualities of foods, is to combine it with MAP or with an edible coating. A combination of irradiation and MAP treatment played a role in retarding the growth of bacteria during the storage period of mini-peeled carrots. MAP can also prevent the whitening development during storage of minimally processed vegetables (Lafortune et al., 2005; Lafortune and Lacroix, 2004). The alternative combination of an edible coating and irradiation treatment was

372 Irradiation of Foods

used to maintain the quality of fresh strawberries. A treatment of 1.5 kGy was applied to strawberries coated with a cross-linked edible film (Lacroix and Vachon, 1999; Vachon et al., 2002). These results showed that combined treatments were effective in reducing water losses and mould growth. A shelf-life extension over 15 days during storage at 4°C was observed. Coating with edible film could also be efficient in delaying browning by acting as oxygen barriers or scavengers (Le Tien et al., 2001). 5.3.2 Application on poultry, meat and fish

Irradiation of various foods at doses necessary to improve the shelf-life and assure the innocuity could affect the sensorial properties depending on the food treated. The use of combined treatment could permit a reduction of the dose necessary to eliminate the pathogen and also can reduce the growth rate of survival microorganisms during storage. Combined low-dosage irradiation (3 kGy) with a modified atmosphere widely extended the shelf-life of fresh foods stored under refrigeration (Raso and BarbosaCanovas, 2003). A combination of irradiation and heat has been proposed for the required lethality while preserving food quality, thereby reducing the detrimental effects of heat or irradiation alone on food (Raso and Barbosa-Canovas, 2003). Irradiation sensitizes vegetative cells and bacterial spores to a subsequent heat treatment. For example, the D70 value of a strain of Listeria monocytogenes decreased from 22.4 to 5.5 s after a pre-irradiation treatment (0.8 kGy) (Grant and Patterson, 1994). A synergistic effect on the inactivation of vegetative bacteria and bacterial spores can be achieved with thermo-irradiation. For example, Pallas and Hamdy (1976) observed that the D value (dose inactivating 90 per cent of the population) of Staphylococcus aureus decreased from 0.098 to 0.053 kGy, when the irradiation temperature increased from 35 to 45°C. A greater inactivation of Salmonella enteritidis (Schaffner et al., 1989) and Vibrio vulnificus (Ama et al., 1994) was also reported by thermo-irradiation of whole egg and fresh fish respectively. Packaging atmosphere can also influence the sensitivity of foods during irradiation treatment (Nam et al., 2001). Extended shelf-life of fresh meat is possible by irradiation under a modified atmosphere. Ehioba et al. (1987) showed that irradiation at 1 kGy extended the shelf life of vacuum-packed ground pork by 40–70 per cent. High hydrostatic pressure (HHP) in combination with irradiation has also been studied and showed interesting results especially for spore-forming bacteria. Clouston and Wills (1969) have observed that the resistance of Bacillus pumilus spores to irradiation could decrease after a pre-treatment at 500 atm. A one-half D value could be obtained for Clostridium sporogenes in meat by pressurization at 680 MPa at 80°C before irradiation at 4.1 kGy (Crawford et al., 1996). Consumer demand for fresh, natural and minimally processed products has resulted in an increase in the risk of food contamination. Generally, low dose irradiation reduces total bacterial numbers by more than 90 per cent and the shelf-life of meat is substantially increased (Dempster, 1985; Dogbevi et al., 1999; Lacroix et al., 2000). For example, organoleptic shelf-life of fresh chicken could be extended from 6–10 days to 12–20 days when irradiated at 2.5 kGy (Lacroix et al., 1991). However, it is known that irradiation of poultry with over 2.5 kGy is required for the elimination of Salmonella (Katta et al., 1991).

Irradiation of foods 373

Minimally processed red meats may be irradiated to doses up to 5 kGy for fresh refrigerated products or to 7 kGy for frozen products (Sommers, 2003). However, these commercial applications can be limited by oxidation of fatty acids and some amino acids, resulting in off-flavour formation (Lacroix et al., 1991; Giroux and Lacroix, 1998). Lipid oxidation affects the sensorial characteristics resulting in unacceptable odours and flavours and reduction of shelf-life. A pink colour is also developed in irradiated pork and poultry (Lynch et al., 1991; Nanke et al., 1998; Nam and Ahn, 2002). Sulphur volatiles produced by radiolytic degradation of sulphur amino acids are responsible for the irradiation off-odour (Jo and Ahn, 2000) and the complex of heme pigment and radiolytic carbon monoxide are responsible for the pink colour of the meat (Nam and Ahn, 2002). The use of natural antioxidants and antimicrobial compounds can increase the radiosensitivity of bacteria and, at the same time, protect the sensorial quality of foods. According to Nam and Ahn (2003), the use of gallate and alpha tocopherol with double packaging was effective in reducing the red colour of cooked irradiated turkey breast after 10 days of storage. Bio-preservatives such as organic acids, fatty acids, plant and herb extracts and essential oils from spices have been investigated for possible applications as food preservatives. Trends in food irradiation technology consist of developing combined treatments using heat, headspace gas, antimicrobials, etc. in order to reduce the irradiation doses required to kill pathogenic bacteria and/or reduce the overall microbial load (Lacroix and Ouattara, 2000). Some natural compounds have functional properties like antimicrobial and antioxidant properties. Lacroix et al. (1997) and Mahrour et al. (2003b) showed that the natural antioxidants, rosemary and thyme, have the potential to control oxidation of unsaturated fatty acids, particularly those derived from phospholipids in chicken. Mahrour et al. (2003a) reduced the irradiation dose required for complete elimination of Salmonella on fresh poultry by combining gamma irradiation with marinating in natural plant extracts (Table 14.6). The marinade also had an additive effect with irradiation to reduce the bacterial growth and control the proliferation during storage at 4°C (Figure 14.2). Similarly, Farkas and Andrassy (1993) reported that the combination of gamma irradiation at 2 kGy with ascorbic acid or glucono-delta-lactone produced a significant reduction in aerobic viable cell counts and Enterobacteriaceae in vacuum

Table 14.6 Salmonella isolation during storage of poultry Dose (kGy)

Day 1

Day 3

Day 6

Day 9

Day 12

Day 15

0

Air Vacuum Marinated

3
/5 2
/5 1
/5

4
/5 3
/5 1
/5

4
/5 5
/5 1
/5

5
/5 5
/5 1
/5

3

Air Vacuum Marinated

1
/5 1
/5 0

2
/5 2
/5 0

4
/5 2
/5 0

3
/5 4
/5 0

4
/5 4
/5 2
/5

5
/5 4
/5 3
/5

5

Air Vacuum Marinated

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 1
/5 0

12 11 10 9 8 7 6 5 4 3 2 1 0

A

Log cfu/g

Log cfu/g

374 Irradiation of Foods

0

2

4

6

8

10

12

14

16

12 11 10 9 8 7 6 5 4 3 2 1 0

V

0

2

4

Log cfu/g

Time (days) 12 11 10 9 8 7 6 5 4 3 2 1 0

M

0

0 kGy

2

4

6

8

10

6

8

10

12

14

16

Time (days)

12

14

3 kGy

5 kGy

16

Time (days) Figure 14.2 Counts of bacterial population (APCs) in poultry irradiated under air (A), under vacuum (V) or marinated and irradiated under air (M).

packaged chilled meat products. Giroux et al. (2001) showed a significant (P  0.05) additional antimicrobial effect between irradiation at 1–4 kGy and the addition of ascorbic acid. The dose of irradiation needed to eliminate pathogenic bacteria in meat products can produce detrimental effects on the sensory quality (Lacroix et al., 1997). Since radiation-degradation of sensory quality is dose dependent (Sudarmadji and Urbain, 1972), reduction of the dose of irradiation can be useful to improve indirectly the sensory quality of the treated product (Borsa et al., 2004). The use of combined treatments could be useful in order to reduce the dose of irradiation required for a level of microbial kill and to protect the sensorial quality of meat (Mahrour et al., 2003a). The active constituents of thyme that have a wide spectrum of antimicrobial effectiveness are thymol and carvacrol (Aktug and Karapinar, 1987). These compounds inhibit a range of food spoilage microbes, including E. coli, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus and Lactobacillus plantarum at a much lower concentration than herb extracts (Collins and Charles, 1987; Davidson, 1997). The antimicrobial

Irradiation of foods 375

6

6 Control Carvacrol with tetrasodium pyrophosphate

5

5

3

3

2

2

1

1

0 0.0 (a)

4 Log cfu/g

Log cfu/g

4

Control Carvacrol with tetrasodium pyrophosphate

0 0.1

0.2

0.3

0.4

0.5

Irradiation dose (kGy)

0.6

0.7

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

(b)

Irradiation dose (kGy)

Figure 14.3 Radiosensitization of (a) E. coli and (b) S. typhi in ground beef packed under MAP in the presence of carvacrol and tetrasodium pyrophosphate.

properties of thymol and carvacrol are associated with their lipophilic character, leading to accumulation in membranes and to subsequent membrane-associated events such as energy depletion (Sikkema et al., 1995). Also, the mechanism by which microorganisms are inhibited by phenolic compounds involves a sensitization of the phospholipid bilayer and leakage of vital intracellular constituents (Kim et al., 1995) or impairment of bacterial enzymes (Tassou et al., 1996). It was demonstrated by Helander et al. (1998) and by Ultee et al. (1999) that the addition of carvacrol or thymol is responsible for outer membrane disintegration and disruption of the intracellular ATP (the energy level), making it nearly impossible for the cell to make the necessary repairs to the damage caused by radiation. The cytoplasmic membrane disruption altered the cell gradient, leading to the impairment of the essential process in the bacterial cell and to cell death. The addition of these compounds helps increase the lethal effect of radiation. Chiasson et al. (2004) have also observed that Salmonella typhi and E. coli are more sensitive to radiation in the presence of these compounds. The primary mechanism of microbial inhibition by radiation is the breakage of chemical bonds within the DNA molecules or alteration of membrane permeability and other cellular functions (Murano, 1995). This facilitates the contact between antimicrobial molecules and cell membranes and increases their inhibitory effects. Chiasson et al. (2004) have demonstrated that the addition of carvacrol or thymol to ground beef before irradiation treatment, increases the relative sensitivity of E. coli and Salmonella typhi by 2.2 times. It is also established that the radiation sensitivity of a bacterium will vary depending of the packaging atmosphere (Lopez-Gonzalez et al., 1999). Chiasson et al. (2004) have observed that in the presence of thymol and carvacrol, E. coli was more affected under air conditions but Salmonella typhi was more affected under MAP conditions (60 per cent O2; 30 per cent CO2; 10 per cent N2). Under MAP and in presence of carvacrol and thymol, irradiation was able to improve the radiosensitivity of E. coli by 2.7 times and Salmonella typhi by 9.9 times (Figure 14.3) (Chiasson et al., 2004).

376 Irradiation of Foods

14

14

12

Log cfu/g

10

a

a

8

b

bb

10

b c

b

6

d

a

c

b

c

c

2 0

1

Figure 14.4

a

8

d

b b

0 (a)

a

4

aab a

aa

b b

b b

4 2

a

Control Base EO09 EO18

12

a

a a a

aa

6

a

Log cfu/g

Control Base EO09 EO18

3

7

9

Storage time (days)

14

21

1 (b)

3

7

9

14

21

Storage time (days)

Counts of bacterial population (APCs) in (a) unirradiated and (b) irradiated coated shrimp during storage at 4°C.

Research activities on the selection of natural antimicrobial and antioxidant compounds also include the controlled-release of active molecules in food systems and control of pathogenic microorganisms using gamma-irradiation and combined treatments (Lacroix et al., 1997; Giroux and Lacroix, 1998; Lacroix and Ouattara, 2000; Oussalah et al., 2004). Edible coating or biodegradable packaging are new technologies that have been introduced in food processing in order to obtain products with longer a shelf-life. Several applications for meat, poultry and seafoods have been reviewed by Gennadios et al. (1997) with particular emphasis on the reduction of lipid oxidation, weight and moisture loss, microbial content and volatile flavour loss. The coating can serve as a carrier for antimicrobial compounds in order to maintain high concentrations of preservatives on the surface of foods. A study to evaluate the combined effect of low-dose gamma irradiation and antimicrobial coating on the shelf-life of pre-cooked shrimps was conducted in our laboratory (Ouattara et al., 2001). Antimicrobial coatings were obtained by incorporating various concentrations (EO09: 0.9 per cent and EO18: 1.8 per cent) of thyme oil and trans-cinnamaldehyde in coating formulations prepared from soy protein isolates. The coating was applied to the surface of the shrimps before irradiation treatment. Results of counts of bacterial population showed that in unirradiated samples no significant difference was found between the control (uncoated samples) and samples coated with the base solution (without antimicrobial compounds). When antimicrobial compounds were incorporated in the base solution (EO09 or EO18 coating), bacterial counts decreased significantly (P 0.05) as compared to uncoated controls. Without irradiation, the shelf-life was established at 7 days for uncoated shrimps, 8 days for samples coated with the base solution and 12 days for samples coated with both coatings containing the antimicrobial compounds (Figure 14.4a). However, with gamma irradiation (3 kGy), the shelf-life was 12 days for uncoated samples, 17 days for samples coated with the base coat solution, 20 days for samples coated with EO09 and more than 21 days for samples coated with EO18 coating (Figure 14.4b) (Ouattara et al., 2001). These results also agreed with Giroux and Lacroix (1998), where the use of natural antimicrobials in combination with low dose irradiation was able to extend the

Conclusions 377

shelf-life of food products, without any detrimental effects on biochemical and nutritional characteristics. The appearance of the shrimps, as well as their odour and taste, was not affected by the treatment. Control of sensorial characteristics and of the microbial quality of meat and meat products could also be done by direct incorporation of antioxidant and antimicrobial compounds in the products and/or immobilization of the active molecules in natural polymer matrices. Studies on biodegradable packaging showed that incorporation of ascorbic acid and/or application of antimicrobial films on meat resulted in a decrease of bacterial growth and stabilized the microbial growth over one week of storage of sliced fresh beef (Giroux et al., 2001; Ouattara et al., 2002). These results showed that ascorbic acid and a cross-linked film coating by irradiation containing spices powders reduced lipid oxidation and sulphidryl (ˆSH) radical production during post-irradiation storage. More recently, cross-linked milk protein-based films by irradiation containing 1 per cent (w/v) oregano, 1 per cent (w/v) pimento or 1 per cent oregano/pimento (1:1) essential oils mix were applied to beef muscle slices to control the growth of pathogenic bacteria and increase the shelf-life during storage (Oussalah et al., 2004). The incorporation of essential oils into cross-linked protein-based films applied to muscle helps to reduce microbial load, increase antioxidative activity and allows a progressive release of antimicrobial compounds during storage. These promising films of cross-linking by irradiation might find application as a support for immobilization of antimicrobial or antioxidant compounds. Moreover, the cross-linked films could be used as a support for controlled released of these compounds from the film to the food. The combination of irradiation with coating is a promising path that should be developed in the future (Lacroix and Ouattara, 2000). Consumer demand for convenient, freshly-prepared restaurant-quality meals has also led to major growth in the home meal replacement market in the last decade (Dwyer, 1999). This kind of product is generally precooked, refrigerated and reheated prior to consumption by the consumer (Neff, 1997). This process is not so efficient in the reduction of the level of bacteria and cross-contamination is a major problem (Foley et al., 2001). Irradiation of prepared foods at a dose of 2.5 kGy would destroy all pathogens (Grant and Patterson, 1992). However, Foley et al. (2001) have demonstrated that a dose of 5–7 kGy applied on prepared meals has advantages in inhibiting indigenous microbial flora, even in cases of post-processing re-contamination, and thus delaying spoilage and causes no change in the sensory attributes of the product. Moreover, this combined process can eliminate the presence of Listeria, a bacterium commonly found in prepared foods.

6 Conclusions Irradiation processing is a safe and effective process to control food-borne disease. This process has the advantage that it can be applied under fresh conditions and on packed foods. The development of combined treatments with irradiation is attractive because it will enhance product safety and offer a higher quality product. It will also provide the

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opportunity to introduce new food products to the food market. However, more research is needed to evaluate the mechanism of action of each treatment on the most important pathogens found in each type of food in order to optimize the treatment.

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Thayer DW, Boyd G, Muller WS, Lipson CA, Hayne WC, Baer SH (1990) Radiation resistance of Salmonella. Journal of Industrial Microbiology, 59, 1030–1034. Thomas AC (1977) Radiation preservation of sub-tropical fruits. Food Irradiation Newsletter, 1, 19. Thomas P (1986) Radiation preservation of foods of plants origin: Temperate fruits: Pome fruits, stone fruits and berries. Critical Reviews in Food Science and Nutrition, 24, 357– 400. Thomas P (2001) Irradiation of tuber and bulb crops. In Food Irradiation, Principles and Applications (Molins R, ed.). New York: Wiley-Interscience, John Wiley and Sons Inc., pp. 241–271. Tilton EW, Brower JH (1983) Radiation effects on arthropods. In Preservation of Food by Ionizing Radiation, Vol.II (Josephson ES, Peterson MS, eds). Boca Raton: CRC Press Inc., pp.269–307. Tilton EW, Burditt AK Jr (1983) Insect disinfestation of grain and fruit. In Preservation of Food by Ionizing Radiation, Vol. III (Josephson ES, Peterson MS, eds). Boca Raton: CRC Press, Inc., 215–229. Ultee A, Letsand EPW, Smid EJ (1999) Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Applied and Environmental Microbiology, 65, 4606–4610. Urbain WM (ed.) (1986) Biological effect of ionizing radiation. In Food Irradiation. Toronto: Food Science and Technology, Academic Press, pp. 83–117. Vachon C, D’Aprano G, Letendre M, Lacroix M (2002) Effect of edible coating process and irradiation treatment of strawberry Fragaria spp. On storage keeping quality. Journal of Food Science, 68, 608–612. Van Mameren J, Houwing H (1968) Effect of irradiation on Anisakis larvae in salted herring. In Elimination of Harmful Organisms from Food and Feed by Irradiation. Vienna: International Atomic Energy Agency (IAEA), pp. 73–80. Wahid M, Sattar A, Khan I (1989) Effect of combination methods on insect disinfestation and quality of dry fruits. Journal of Food Processing and Preservation, 13 (1), 79–85. Wang, JX, Tang, YW, Qian WH, Guo YF, Xu JX, Xu Zy (1988) Serorpidemiological survey of viral hepatitis A during an epidemic in Shanghai. Acta Academiae Shanghai, 15, 374–379. Wills PA (1975) Inactivation of B. pumilus spores by combination hydrostatic pressure radiation treatment of medical products. In Radiosterilization of Medical Products. Vienna: International Atomic Energy Agency (IAEA), pp. 45–61. World Health Organization (WHO) (1999) High dose irradiation: wholesomeness of food irradiated with doses above 10 kGy. Report of a Joint Food and Agriculture Organization (FAO), International Atomic Energy Agency (IAEA), World Health Organization (WHO) Study Group. Young AL (2003) Food irradiation. Environmental Science & Pollution Research, 10, 82–88. Zhuang H, Margaret Barth M, Hankinson TR (2003) Microbial safety, quality, and sensory aspects of fresh-cut fruits and vegetables. In Microbial Safety of Minimally Processed Foods (Novak JS, Sapers GM, Juneja VK, eds). Boca Raton: CRC Press, pp. 255–278.

New Chemical and Biochemical Hurdles Jakob Søltoft-Jensen and Flemming Hansen Danish Meat Research Institute, Roskilde, Denmark

The use of new chemical and biochemical compounds as antimicrobial hurdles in food is reviewed. Organic acids, chitin/chitosan, enzymes, lactoferrin, nisin, reuterin, plant-derived antimicrobials, ozone and electrolysed oxidizing water are all considered. Among the organic acids, benzoic, lactic, acetic and fumaric acids and parabens show the greatest and most well documented effects against microorganisms. A number of plant-derived antimicrobials have demonstrated good antimicrobial effects and are, at the same time, widely accepted by the consumer as being ‘more natural’ than organic acids. Among the newest antimicrobials, reuterin seems very promising. Generally, the hurdles must be considered in combination in a multivariable way, taking their synergistic effects into account. Challenges like application route, distribution in the food, impact on flavour and consumer acceptance are also discussed. These challenges are to be addressed next, in order to take the emerging hurdles closer to a commercial breakthrough.

1 Introduction Additives are used in foods to preserve, flavour, colour, texturize or to make the food more nutritious. Antimicrobial chemicals and biochemicals for preservation continue to constitute the majority of food additives. Preservatives are used to prevent either chemical or biological deterioration of foods. Chemical deterioration includes browning, oxidation and staling, whereas biological deterioration includes growth of either spoilage organisms or pathogenic bacteria in the foods. This chapter reviews the use of chemicals and biochemicals for biological preservation. The focus will be on new and emerging antimicrobials, leaving out the classic preservatives: sodium chloride, sugar, nitrate/nitrite and wood-smoke. Considering the vastness of the subject, it is not intended to describe every aspect in detail but to focus on promising antimicrobials researched mainly during the last 15 years. These would be organic acids, antimicrobial enzymes, chitin/chitosan, nisin, lactoferrin, plant-derived antimicrobials, ozone, reuterin, electrolysed water and other emerging hurdles that do not belong in any of these categories. The food industry is continuously striving to maintain or increase market share, thus doing its utmost to meet the demands of the consumer of tomorrow. And what the consumer demands today and will demand increasingly in the future is wholesome, Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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tasteful, high quality and no-risk food. To comply with these requirements, food processors are increasingly aware of the fact that no single additive can do the job. Safe foods with a long shelf-life and good eating quality throughout the shelf-life are only possible to achieve by using the hurdle technology. The basic point of hurdle technology is that one single preservative in high concentration is not adequate to prevent the food from deteriorating and at the same time maintaining a good eating quality. Therefore, multiple preservatives used in smaller quantities than if used on their own are called for (Leistner, 1995). Chemical and biochemical preservatives are not the only aspects of the hurdle concept. Factors like temperature and modified atmosphere packaging also play very important roles. However, in this chapter, only the chemical and biochemical preservatives will be dealt with, but it is important to keep in mind that new and promising hurdle combinations can be created only when all the parameters are taken into account in a multivariable way. The fact that food matrices usually are more complex and challenging than laboratory media needs to be borne in mind. Many foods consist of a water phase and a lipid phase. This must be considered when deciding how to apply the hurdles and their distribution in the foods. It is important to remember that using hurdles is a delicate balance between inhibiting growth of spoilage organisms and pathogenic bacteria and at the same time maintaining a satisfactory eating quality of the food. This will be discussed at the end of the chapter.

2 Organic acids Acids and their salts are added to food to control the pH, to prevent oxidation or to serve as preservatives. In this context, focus will be on the antimicrobial effects and the acids will be regarded one by one or in combination as part of a hurdle concept. One might argue that organic acids are not new or emerging hurdles, but the vast amount of literature, particularly from the last ten years, reveals that the application of acids to foods is still developing and that new uses are being found. The use of organic acids in foods is at the same time one of the most common and generally accepted methods of chemical preservation. The preservative mechanism of organic acids is often explained by the antimicrobial effect of the undissociated acid molecule and its energy depletion of the living cell. The undissociated acid enters the living cell and dissociates inside the cell or facilitates proton uptake by the cell. The microorganism tries to maintain its internal pH, extruding the organic acids or protons, thereby using all its energy. In addition, it has been proposed that short chain fatty acids, like sorbic, propionic and benzoic acids influence the membrane either by interfering with the membrane proteins or by changing the membrane fluidity (Eklund, 1989). Other ways of inhibiting growth of microorganisms by the undissociated molecule are by inactivating or affecting metabolic enzymes, protein synthesis systems or the genetic material (Barbosa-Canovas et al., 1997).

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Table 15.1 Antimicrobial activities of organic acids and esters on bacteria, yeasts and moulds, other than those caused by acidification

Acetic acid Propionic acid Lactic acid Sorbic acid Benzoic acid Parabens (p-hydroxybenzoic acid ester) Citric acid Malic acid Fumaric acid Tartaric acid Adipic acid Glucono-delta-lactone

Bacteria

Yeasts

Moulds




*

















  



















  


















 

*


: good antimicrobial effect;

: moderate antimicrobial effect;
: little antimicrobial effect; : no antimicrobial effect. Modified from Barbosa-Canovas et al. (1997); Doores (1993); Eklund (1989).

In this chapter, the following acids and their salts, permitted for food application in a number of countries, will be discussed in some detail: acetic, propionic, lactic, sorbic, benzoic, citric, malic, fumaric, tartaric and adipic acids, esters of hydroxybenzoic acids (parabens) plus glucono-delta-lactone (GdL). Benzoate, propionate and sorbate are often used as antimicrobials in a wide variety of foods, whereas lactate and acetate are used often in meat and meat products. Acetic, citric, propionic, malic, fumaric, adipic and tartaric acids are often used as acidulants, thereby preserving the foods directly by lowering the pH. The comparative effectiveness of all these acids on bacteria, yeasts and moulds can be seen in Table 15.1. In addition to the acids mentioned above, emerging antimicrobial fatty acids such as oleic and pentadecadienoic acids, lauric acid-monoglyceride (monolaurin) and phenolic acid will also be considered.

2.1 Acetic acid Acetic acid is a widely used preservative in food, although it has a pungent odour and taste. Therefore it is either used in very low concentrations, or in foods where its flavour and taste is acceptable, e.g. canned fruit and vegetables, mayonnaise, salad dressings, mustard, ketchup and marinades for meat, poultry and fish. During the last decade, acetic acid in low concentrations has become a common hurdle in meat products. It is often produced directly in the foods from lactic acid bacteria, for example in pickles, sauerkraut and fermented milk products. Compared to other organic acids it has a rather high pK (4.75) making it suitable for low acid foods. Acetic acid seems to be a better inhibitor of bacteria than of yeasts and moulds (Eklund, 1989), although it has been found effective against specific bread moulds.

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Acetic acid interacts with the cell membrane to neutralize the electrochemical potential like the other short-chain fatty acids, propionic and lactic acid. It also inhibits amino acid uptake in membrane vesicles and has been found to denature proteins inside the cell (Eklund, 1989). A number of studies illustrate the antimicrobial efficiency of acetate towards the food pathogen Listeria monocytogenes in meat and meat products, but its effect towards a number of other pathogens has also been investigated. Due to its very unpleasant taste and odour, acetate is very often used in combination with other acid salts, such as lactate or citrate. Used as part of a hurdle concept, the effective acetate concentration can be lowered, so that the negative sensory influence is minimized. Juncher et al. (2000) found that 2 per cent lactate and 0.5 per cent acetate could prevent growth of L. monocytogenes in saveloys without seriously impairing the flavour, whereas Blom et al. (1997) found that 2.5 per cent lactate and 0.25 per cent acetate was the optimal combination in vacuum-packed ready-to-eat cooked meat products. Contrary to the situation for acetate, diacetate used alone in small concentrations has been found effective towards pathogens, for instance L. monocytogenes (Schlyter et al., 1993) and psychrotrophic Clostridium (Meyer et al., 2003). A possible explanation for this is that diacetate in fact is a combination of acetate and acetic acid. The acetic acid lowers the pH increasing the amount of undissociated acid. One might argue that the antimicrobial effect of diacetate would be the same as if the acid were applied on its own. Instead of applying acetic acid in the recipe for meat products, spray application or dipping for surface decontamination of fresh meat cuts has also been tested. Gordon Greer and Dilts (1992) found a reduction in bacterial numbers for eight different pathogens and spoilage organisms ranging from 0.5 log cfu (Salmonella typhimurium) to 3.5 log cfu (Brochothrix thermosphacta).

2.2 Propionic acid Propionic acid is not as widely used in the food industry as acetic acid. It has a pungent, disagreeable odour and salts of the acid have cheesy odours. It is used mainly as a mould inhibitor for cheese, bread and cakes, but it is also effective against rope-forming bacteria in bread dough. The pK value is 4.87 and the upper limit for effectiveness is around 5.5. It can function at higher pH levels, but at pH 6.0 only 6.7 per cent remains undissociated. It is formed naturally in cheeses by propionic acid bacteria. The antimicrobial mode of action of propionate is the same as for sorbate and benzoate. As mentioned earlier, the undissociated form of the acids is essential for the antimicrobial activity. These acids are lipophilic and soluble in the cell membrane, where they apparently act as proton ionophores. They facilitate proton transport into the cells, forcing the microorganisms to use more energy pumping out protons to keep their usual internal pH (Jay, 1986). Due to their pungent odour like acetate, propionic acid and propionate must be used in low dosage only in order to maintain the organoleptic quality of the food. Therefore, they are often used in combination with other organic acids or their salts.

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Maca et al. (1997a, b) found in both beef top rounds and patties that a combination of sodium lactate and 0.1 per cent or 0.2 per cent sodium propionate increased the shelf-life by decreasing microbiological growth. The authors also found that the sensory properties were improved, due to a lower level of oxidation during the shelf-life for products with the salts of the organic acids than without. Hu and Shelef (1996) also investigated the effect of propionate and lactate on growth of L. monocytogenes in pork sausages with different contents of fat. They found that both salts were antilisterial, although the antilisterial activity of the salts increased with increasing fat content. This indicates that the concentration of the salts in the water phase, where the bacterial growth occurs, is important.

2.3 Lactic acid Lactic acid has a moderately strong acid, bitter taste. It is used in foods for acidification (pK 3.08), flavour enhancement and microbial inhibition, although it is generally viewed as less effective than other organic acids. However, it is not without antimicrobial effects, as is witnessed for instance by its wide use in meat products in the form of lactate. It is also used in sugar confectionary, dairy products, beer, wine, beverages and bakery products. In nature, lactic acid is one of the most common organic acids, formed both from lactic acid bacteria and in vivo in living tissue. Lactic acid influences the membrane potential of microorganisms in a manner similar to acetic and propionic acids and also depresses the pH of the foods below the growth range (Doores, 1993). The importance of the effects depends on the pH of the food, its buffer capacity and whether lactic acid is added as lactate or in the form of the acid itself. Lactate has been studied extensively for the last 15 years as a meat antimicrobial and its antibacterial effect is widely documented for beef, pork and poultry products. Several reviews are available, one being by Shelef (1994). Lactate is used in meat products both on its own and as part of a hurdle concept, typically together with acetate or propionate. The application level is 2–4 per cent. It is also used as a meat sanitizer for fresh meat or meat cuts. It controls spoilage organisms and a number of Gram-positive and Gram-negative pathogens (Miller and Acuff, 1994; Gordon Greer and Dilts, 1995). In addition to its antimicrobial effects, lactate has also been found to enhance the sensory freshness of meat (Papadopoulos et al., 1991). In the most recent investigations, lactate or lactic acid have been used in combination with the bacteriocin, nisin. Martinez et al. (2002) found that a combination of 1.5 per cent lactic acid and 500 IU/ml nisin could decrease aerobic plate counts, total coliforms and E. coli by up to 2 log-units. The researchers maintain that the acidic conditions created by the lactic acid increase the amount of the undissociated acid and the effectiveness of nisin. Scannell et al. (1997) obtained a similar result when testing lactate and nisin against Staphylococcus aureus and Salmonella kentucky in fresh pork sausages. To prolong the acidification effect from lactic acid, successful tests with poly lactic acid (PLA) have been carried out. PLA releases free lactic acid for an extended period

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of time, thereby maintaining a lower pH during a longer period than when lactic acid is added on its own (Ariyapitipun et al., 1999). Lactic acid has also been found effective against Salmonella and Listeria in other foods, for instance yoghurt and crayfish meat (Rubin et al., 1982; Pothuri et al., 1995).

2.4 Sorbic acid Sorbic acid is mainly used in foods in the forms of calcium, sodium or potassium sorbates. Calcium sorbate is tasteless and flavourless. Sorbates are mainly used as fungistats in products such as cheese, bakery products, fruit juices, beverages and salad dressing. In meat products they can function as antibotulinal agents, reducing the need for nitrite. Sorbates are primarily effective against yeasts and moulds, but can also be used against a range of bacteria, especially catalase-positive cocci, sporeforming bacteria and aerobes. Sorbic acid works best below pH 6.0 and is generally ineffective above pH 6.5. Between pH 4.0 and 6.0 sorbates are generally more effective than benzoates. The pK of sorbic acid is 4.80 (Sofos and Busta, 1993). The effect of sorbates is not only associated with energy depletion of the living microorganism due to accelerated proton pumping out of the cell as for other organic acids, nor is it just based on alteration of the electrochemical membrane potential. A number of specific activities have been discovered (Eklund, 1989). Sorbates inhibit L-alanine and L-cysteine induced germination of developing spores. A number of enzymes are specifically inhibited by sorbates: enolase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, alpha ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and aspartase. Finally, sorbate has been found to interfere with the genetic material, RNA and DNA, in Pseudomonas fluorescens. In cooked bologna, sorbate was found to be more effective than acetate and lactate against Listeria monocytogenes (Sofos et al., 1994) and 0.4 per cent potassium sorbate in combination with smoking was very effective in controlling the microbial quality and extending the shelf-life of fresh catfish (Efiuvwevwere and Ajiboye, 1996). Eklund (1983) determined the minimal inhibitory concentration of sorbic acid for Bacillus subtilis, B. cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans and found that the minimal inhibitory concentration varied between the bacteria and with different levels of pH.

2.5 Benzoic acid Benzoic acid or its sodium salt, benzoate, was the first chemical preservative permitted in foods in the USA. It is still widely used today for a large number of foods. The pK of benzoic acid is rather low (pK 4.20), so its main antimicrobial effect, due to the undissociated acid, will be for high acid foods such as ciders, soft drinks and dressings. It is most suitable for foods with a pH lower than 4.5, but has also found use in margarine, fruit salads, sauerkraut, jams and jellies. Benzoate acts essentially as a mould and yeast inhibitor in high acid foods and the poor activity at pH values above 4.0 limits its use against bacteria. Benzoic acid naturally occurs in cranberries,

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prunes, strawberries, apples and yogurts (Chipley, 1993). In certain foods, benzoate may impart a disagreeable taste described as ‘peppery’ or burning. The antimicrobial effect of benzoic acid has been assumed only to be expressed by the undissociated acid interfering with the permeability of the cell membrane and the proton-motive force (Eklund, 1989). However, as for sorbic and propionic acid, benzoic acid has a certain antimicrobial activity in the dissociated form (Chipley, 1993). Benzoate also specifically inhibits amino acid uptake and certain enzymes within the cell: alpha-ketoglutarate, succinate dehydrogenase, 6-phosphofructo-2-kinase and lipase. In a comparative study, Islam (2002) investigated the effect of dipping turkey frankfurters in 25 per cent solutions of propionate, benzoate, diacetate or sorbate on the growth of Listeria monocytogenes. The organic acids were equally effective in reducing L. monocytogenes when the frankfurters were stored at 4°C for 14 days (reduction around 3–4 log cfu/g) but when stored at 13°C, benzoate and diacetate were more effective than propionate and sorbate. A quite new application method for benzoic acid is active packaging. Weng et al. (1997) treated ionomer films with alkali. The resulting release of benzoic acid inhibited Penicillium and Aspergillus in microbial media.

2.6 Parabens Parabens (esters of p-hydroxybenzoic acid) are used in foods mainly in the form of methyl, propyl and heptyl esters. Parabens are typically used in bakery products, soft drinks, fish products, pickles and salad dressings. The esterification of the carboxyl group of benzoic acid allows the parabens to be undissociated (antimicrobial) in a broader pH spectrum than benzoic acid, from pH 3 to 8 (Davidson, 1993). The parabens are effective against a broad range of microorganisms, including Gram-positive and Gram-negative bacteria, yeasts and moulds. For most types of microorganisms, there is a correlation between effectiveness and the length of the alkyl chain as microbial inhibition increases with increasing alkyl length. The exception to this rule is some Gram-negative bacteria that are resistant to the higher parabens. Like most of the other lipophilic food preservatives, parabens have been reported to interact with several targets in the microbial cell (Eklund, 1989). Contrary to the weak acids, it appears that parabens do not inhibit specific enzymes directly. However, indirectly the free hydroxyl group may be involved in some enzyme interaction. The most common explanation for the antimicrobial mechanism is that the parabens dissolve in or ‘through’ the cell membrane, interfering with membrane-related processes or structures. These are the amino acid uptake and the proton-motive force as shown for sorbate. In the cell, the parabens are also claimed to inhibit the synthesis of protein, RNA and DNA (Eklund, 1989).

2.7 Citric acid Citric acid is the most widely used acid in the food industry. It is a tricarboxylic acid with pK values of 3.14, 4.77 and 6.39 (for each carboxylic group). Citric acid is water

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soluble and enhances the flavour of citrus-based foods. Citric acid has antimicrobial properties due to its acidulation, but it is also used as an antioxidant indirectly by chelating metal ions that catalyses oxidation. Foods preserved with citric acid include tomato juice, ice cream, sherbets, beverages, salad dressings, jams and jellies. Citric acid has also been studied extensively as a nitrite-saving agent in meat products (Jay, 1986). In addition to the antimicrobial effect of citric acid by lowering the pH, studies have indicated that the chelating effect of citric acid also inhibits bacteria (Doores, 1993). By chelating or binding metal ions, the substrate for bacterial growth is diminished in the food, thus influencing growth. Qvist et al. (1995) found that citric acid added to pork sausages at a concentration of 0.125 per cent inhibited Listeria monocytogenes for 35 days when stored at 5°C but not when stored at 10°C. Unfortunately, the acid seemed to reduce the sensory quality of the product. Citric acid was compared to malic, acetic, lactic and hydrochloric acids for their effect on thermal inactivation of Bacillus stearothermophilus and B. coagulans in frankfurter emulsion slurries (Lynch and Potter, 1988). At pH 5.2, the acids had no effect, but at pH 4.6 a greater inactivation rate was obtained with citric, lactic and acetic acids than with malic and hydrochloric acids. The lower the pH, the higher the amount of undissociated acid. In restructured roast beef, Sabah et al. (2003) found an antibacterial effect of 2 per cent or 4.4 per cent citric acid against Clostridium perfringens. Application of 0.21 per cent citric acid reduced Salmonella by 0.7–1.1 log cfu in slices of Gala apples prior to dehydration and storage (Dipersio et al., 2003).

2.8 Malic acid Malic acid is a dicarboxylic acid with pK values of 3.40 and 5.11. Malic acid has a smooth, tart taste that lingers in the mouth without imparting a burst of flavour. Malic acid is highly water soluble. It is inhibitory to yeasts, moulds and bacteria, probably due to its impact on pH (Doores, 1993). It is used in beverages, hard candies, canned tomatoes and fruit pie fillings. A study by Buchanan and Golden (1998) determined how the pH and malic acid concentration interacted to inactivate growth of Listeria monocytogenes in BHI (brain heart infusion). The effect was rather weak and the authors concluded that malic acid is a rather benign organic acid, much less bactericidal than lactic or acetic acids. This supports the theory that the main antimicrobial effect of malic acid is due to a lowering of the pH.

2.9 Fumaric acid Like malic acid, fumaric acid is a dicarboxylic acid with pK values of 3.03 and 4.44. It has a strong acid taste, but a positive flavour effect is that it blends with certain flavours to intensify their aftertaste. Fumaric acid is used in fruit drinks, gelatine desserts, pie fillings, biscuit doughs and wines. It is naturally present in rice, sugar cane, wine, plant leaves, mushrooms and gelatine (Barbosa-Canovas et al., 1997). Like citric acid,

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it also seems to have antimicrobial properties working as a chelator, binding micronutrients essential for bacterial growth. As for malic acid, the antimicrobial mechanism for fumaric acid has only been investigated to a limited extent. One of the main effects is probably due to its lowering of the pH. In ground beef patties, the antimicrobial effect of fumaric acid was compared to the effect of lactic acid on the total number of bacteria (Podolak et al. 1996b). Fumaric acid treatment resulted in greater reduction of microbial growth than lactic acid treatment, for aerobic plate counts, psychrotrophs and faecal coliforms. Increasing the acid concentration from 1 to 5 per cent of course significantly decreased the rate of growth of the microorganisms. However, as the authors correctly conclude, further research is needed on the effect of fumaric acid on sensory properties. The same research group also found that fumaric acid was more bactericidal than lactic and acetic acids on beef surfaces dipped in solutions of the acids. Listeria monocytogenes, Escherichia coli and Salmonella typhimurium were reduced by 1.5 to 2.5 log cfu by 1.5 per cent fumaric acid (Podolak et al., 1995, 1996a).

2.10 Tartaric and adipic acid Tartaric and adipic acids have no antimicrobial properties other than those ascribed to their ability to reduce pH in the foods. Tartaric acid (pK 2.98) has a strong, tart taste and enhances grape-like flavours. It is used in fruit jams, jellies and grape flavoured beverages. Adipic acid (pK 4.43) is used in evaporated milk, puddings, desserts and flavourings.

2.11 GdL GdL (glucono-delta-lactone) is a widely used acidulant in meat products, especially for dry cured sausages. It is used when a slow release of acid is required. In the presence of water it reverts to gluconic acid. The lowering of the pH inhibits growth of bacteria and accelerates the drying (Bertelsen et al., 1995). Both Juncher et al. (2000) and Samelis et al. (2002) found GdL effective in preventing growth of Listeria monocytogenes when used in combination with lactate in emulsion type meat products. Juncher et al. (2000) also found that the lowering of pH by GdL together with lactate significantly improved the oxidative stability of the meat product and resulted in higher a-values (red colour).

2.12 Long chain fatty acids and phenolic acids Long chain fatty acids and phenolic acids such as oleic, chlorogenic, hydroxycinnamic, caffeic, p-coumaric, ferulic and lauric acids are currently being investigated for their antimicrobial activities in food. Stecchini et al. (1996) found that monolaurin (monoglyceride of lauric acid) could reduce the number of Listeria monocytogenes in Italian Stracchino cheese. Oleic acid

396 New Chemical and Biochemical Hurdles

and potassium oleate could reduce the number of bacteria on poultry skin. Aerobic bacteria, enterobacteriaceae, Campylobacter and Listeria monocytogenes were most sensitive; Escherichia coli and Pseudomonas aeruginosa were more resistant, while Enterobacter cloacae, Staphylococcus lentus and Salmonella typhimurium had the greatest resistance (Hinton and Ingram, 2000, 2003). Selected phenolic acids (chlorogenic, hydroxycinnamic, caffeic, p-coumaric and ferulic acids) were screened for their activity against five strains of Listeria monocytogenes using a broth dilution method (Wen et al., 2003). The results showed differences between the acid fractions, varying from effective to ineffective, but also a strong relationship between pH and activity. The authors speculate whether a possible application for phenolic acids could be against L. monocytogenes in processed vegetables. From the marine organism Gorgoniidae, Carballeira et al. (1997) isolated several novel fatty acids. Of these, (5z, 9z)-14-methyl-5,9-pentadecadienoic acid showed antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus and Streptococcus faecalis, but not against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. The implication of these findings for a possible use in foods is still to be investigated. Of the acids described, benzoic, lactic, acetic and fumaric acids and parabens seem most effective against a broad spectrum of microorganisms in different foods. The latest trends point towards use of the organic acids as surface disinfectants instead of applying the acids to the food matrices.

3 Plant-derived antimicrobials Plants, being live organisms, are constantly under attack from fungi, insects, worms and other herbivorous animals. For protection, plants have developed defence strategies, one of them being the synthesis of numerous antimicrobial chemical compounds. These antimicrobial compounds can be either secondary metabolites from the plants’ normal metabolism stored in specialized tissue or vacuoles in the plant (prohibitins, inhibitins), or antimicrobial compounds produced only in defence against an attack, e.g. from fungi (phytoalexins). Examples of preformed or constitutive antibiotics are catechol, carvacrol, thymol and caffeic acid. In many cases inhibitins may be stored as less active or inactive precursors, being enzymatically transformed into active antimicrobials upon damage to the plant tissue. Examples of inactive precursors are alliin (in garlic) and sinigrin (in cabbage) that are converted into the active compounds allicin and allyl isothiocyanate by allinase and myrosinase. In contrast, phytoalexins are only produced after infection of the plant, induced by elicitor molecules from the invading organism. Many vegetables especially from the family leguminosae (e.g. chick peas, peanut, french bean, soya bean) or Solanaceae (potato, green pepper, egg plant) produce such phytoalexins (Walker, 1994). Numerous plants are known to be antimicrobial or to contain antimicrobial compounds which may be exploited in food preservation. Most commonly known is the

Plant-derived antimicrobials 397

Table 15.2 Some herbs/spices and other foods with known antimicrobial activity and their active components Plant

Major active component

Basil, sweet Oregano Rosemary Sage Thyme Olive Allspice Cinnamon Clove Nutmeg Vanilla Cumin Cayenne/chilli Citrus fruits (Orange) Cocoa, coffee, tea

Linalool, methyl chavicol Carvacrol, thymol Camphor, 1,8-cineole, -pinene, Linalool Thujone, 1,8-cineole, borneol, camphor Thymol, carvacrol Oleuropein Eugenol, ␤-caryophyllene Cinnamic aldehyde, eugenol Eugenol Myristicin, -pinene, sabinene Vanilin Cuminaldehyde Capsaicin D-limonene, citral Caffeine

Modified from Davidson and Naidu (2000); Beales (2002).

antimicrobial action of many vegetables, citrus fruits, spices and herbs besides coffee, tea and cocoa (Table 15.2). Although vegetables such as carrot, leek, asparagus, radish, horseradish, broccoli, cauliflower, cabbage, parsley, spinach, garlic and onions are reported to be antimicrobial (Beuchat and Brackett, 1990; Delaquis and Mazza, 1995; Manderfield et al., 1997; Bowles and Juneja, 1998; Kyung and Lee, 2001), the main research has been done on oleoresins or essential oils from spices and herbs. Reports on the very early use of spices and herbs for preservation can be traced back to 1550 BC when the ancient Egyptians used cinnamon, cumin and thyme both for food preservation and for the mummification process. During the last century numerous scientists have reported on the antimicrobial effect of different spices and herbs or their extracts/essential oils and many excellent reviews have been published recently (Walker, 1994; Nychas, 1995; Davidson and Naidu, 2000; Beales, 2002; Suppakul et al., 2003). Although there is a good agreement in general terms, several publications are conflicting, when considering specific spices against specific bacteria. A lot of reasons might be given for this. Oil from different cultivars may be genetically diverse and contain different phenolic compounds or different amounts of the phenolics. Also, geographical origin, the climate of the harvest year and the preparation or extraction method are known to influence the chemical composition of the essential oil (Lis-Balchin et al., 1996; Suppakul et al., 2003). In addition to these factors, the lack of standardized laboratory methods for testing the antimicrobial activity strongly influences the data obtained. However, it is generally recognized that essential oils from herbs and spices such as cinnamon, clove, thyme, sage, rosemary, oregano, basil, allspice, nutmeg, coriander, lemon grass, cumin and bay laurel exert a strong antimicrobial activity, but many other spices and

398 New Chemical and Biochemical Hurdles

Table 15.3 Examples of chemical components in plant essential oils Chemical type Alcohols Aldehydes Esters Ketones Oxides Phenols Terpenes Sesquiterpenes

Citronellol, menthol, terpineol, linalool, nerol, zingerberol, geraniol Cinnamaldehyde, benzaldehyde, anisaldehyde, citral Linalyl acetate, terpinyl acetate, methyl salicylate Menthone, thujone, camphor, carvone Cineole, caryophyllene oxide Eugenol, chavicol, thymol, carvacrol Sabinene, terpinene, myrcene, limonene Humulene, selinene, curcumene, zingiberene, caryophyllene

Modified from Beales (2002).

herbs are also reported to be antimicrobial. It is usually a broad-spectrum activity against Gram-positive and Gram-negative bacteria and fungi, although the majority of the studies shows better activity against Gram-positive than Gram-negative bacteria. The antimicrobial effect of essential oils is mainly being attributed to the content of phenolic compounds, terpenes and aldehydes, but many other components such as alcohols, esters, oxides and ketones (Table 15.3) may contribute to the antimicrobial activity. The most accepted ‘mode of action’ is that terpenes or phenols interfere with the cytoplasmic membrane, resulting in changes in permeability or possible leakage of the cell. Other suggested mechanisms are inhibition of enzyme systems or even denaturing of enzymes (Davidson and Naidu, 2000; Beales, 2002). In addition to having antimicrobial activity, essential oils from many spices and herbs exert an antioxidative effect as well. At present many commercially available preparations of, e.g. rosemary, thyme, sage and oregano, are being prepared and sold as antioxidants for the food industry, but they are also being marketed as natural antimicrobials. Also, many essential oils are added to foods as flavour components and the antimicrobial action is a positive side effect. These multifunctional properties of essential oils should encourage food producers to use them in novel food products. However, the essential oils also suffer from certain drawbacks. Although their antimicrobial activity is well established in laboratory systems, many papers report of a much weaker effect in complex food systems (Tassou and Nychas, 1995; MendozaYepes et al., 1997; Vrinda Menon and Garg, 2001; Vrinda Menon et al., 2002), probably due to interference with other food components. The antimicrobial compounds from spices and herbs are usually lipid soluble and will thus be present in the fat fraction, whereas the bacteria – or at least the multiplication of the bacteria – will be in the water phase of the food. In order to obtain an acceptable antimicrobial activity in such complex food systems, large amounts of the spice or the essential oils may need to be added which, in the majority of cases, will lead to an unacceptable taste, odour or colour. This could theoretically be overcome by removing the components responsible for the strong flavour, but these components are usually the ones responsible for the antimicrobial activity as well. Another solution, illustrated by Mendoza-Yepes

Chitin/chitosan 399

et al. (1997) would be to prepare a blend of different oils in such a way, that no specific flavour would dominate the finished product. However, it may be necessary to develop many different blends of essential oils to cover the entire product range for a certain food manufacturer and this may prove difficult to control in a food plant. Another factor to consider is that the essential oils may show great variations due to different cultivars, geographical origin, climate, etc. (Suppakul et al., 2003) and the antimicrobial activity may thus vary from batch to batch. This lack of consistency could be overcome if the purified components were used instead, but then the ‘natural preservative’ is no longer ‘natural’ and may have to be labelled as a chemical additive. Indeed, many consumers demand ‘chemical-free’ foods and will not accept the use of more chemicals instead of ‘natural preservatives’. It seems that even though extracts/essential oils from many spices and herbs exert strong antimicrobial activity, several problems need to be addressed and solved before they can be widely used for preservation of food products.

4 Chitin/chitosan Chitin is one of the most abundant natural biomolecules in nature. The term ‘chitin’ is derived from the Greek word ‘chiton’ meaning envelope and being the major component of the exoskeleton of invertebrates chitin is easily available as a by-product, e.g. from the shellfish industry. Chitin is a polymer consisting of N-acetyl-D-glucosamine units linked by ␤(1,4) bonds. Chitosan, or more precisely the chitosan family, is a deacetylated form of chitin usually obtained by treatment with strong alkali. The degree of deacetylation usually varies between 60 and 95 per cent. The deacetylation leaves a free amino-group and chitosan thus reacts as a weak base in acidic solutions. Chitin is also degradable by enzymatic action (chitinase, ␤-N-acetylglucosaminidase, lysozyme) resulting in oligomers or monomers. The degree of deacetylation and depolymerization influences the antimicrobial effect of chitosan (Shahidi et al., 1999; Rudrapatnam et al., 2003). Chitosan is soluble in weak organic acids such as lactic acid or acetic acid, whereas chitin is insoluble except in a few organic solvents such as dimethylformamide or hexafluoroisopropanol. Chitosan has been used for many years in different fields as water purification/ clarification, cosmetics (in hair shampoo and conditioners), for pharmaceutical use, for packaging films and in the food industry. Even in the food industry, a broad range of applications for chitin/chitosan exists (Table 15.4). As an antimicrobial agent, chitosan has been studied quite extensively during the past 10 years. Despite this, the exact mechanism of the antimicrobial action is still unknown. One key feature of chitosan is that the amino group in the C-2 position is positively charged at pH values below its pK of 6.3. The positively charged chitosan interacts with the negatively charged microbial cell membrane, eventually leading to the leakage of intercellular constituents (Shahidi et al., 1999; Helander et al., 2001). Other antimicrobial actions might consist of chelating with trace metal ions, binding

400 New Chemical and Biochemical Hurdles

Table 15.4 Food applications of chitin and its derivatives Area of application

Examples

Preservation

Bactericidal and fungicidal agent Measurement of mould contamination in agricultural commodities

Edible film industry

Controlled moisture exchange Controlled release of antimicrobials, antioxidants, nutrients, flavours and drugs Reduction of oxygen partial pressure Controlled rate of respiration Temperature control Controlled enzymatic browning in fruits Reverse osmosis membranes

Additive

Clarification and deacidification of fruits and beverages Natural flavour extender Emulsifying agent Food mimetic Thickening and stabilizing agent Colour stabilization

Nutritional

Dietary fibre Hypocholesterolaemic effect Reduction of lipid absorption Production of single cell protein Antigastritis agent Livestock and fish feed additive Infant feed ingredient

Purification of water/ wastewater

Affinity flocculation Fractionation of agar Recovery of metal ions, pesticides, phenols and PCBs Removal of dyes

Other applications

Enzyme immobilization Encapsulation of nutraceuticals Chromatography Analytical reagents

Modified from Shahidi et al. (1999).

of water and inhibition of various enzymes (Shahidi et al. 1999). Provided that the major antimicrobial action depends on interaction with the cell membrane, it is evident that the size of the molecule (the degree of polymerization) and the net charge (degree of deacetylation, pH) are important parameters in defining the antimicrobial effect of chitosan (No et al., 2002). Also the solubility of the chitosan (degree of deacetylation) is an important parameter for the antimicrobial action. Several researchers describe antimicrobial action of chitosan in food (Darmadji and Izumimoto, 1994; Simpson et al., 1997; Devlieghere et al., 2002; Sagoo et al., 2002). Devlieghere et al. (2002) found that chitosan was inhibitory to a number of bacteria including Gram-negatives (Enterobacter and Pseudomonas) and Grampositives (Bacillus, Listeria monocytogenes, Brochothrix and Lactobacillus). They reported that, in general, Gram-negative bacteria were more sensitive than Grampositive. This difference between Gram-positives and Gram-negatives is not consistent;

Antimicrobial enzymes 401

Simpson et al. (1997) found that Pseudomonas was very resistant to chitosan, whereas Darmadji and Izumimoto (1994) found Pseudomonas and S. aureus to be much more sensitive than E. coli and Micrococcus. In general, bacterial growth is inhibited by adding chitosan at levels of 0.005–0.01 per cent in broth cultures (Darmadji and Izumimoto, 1994; Devlieghere et al., 2002), but much higher levels are required to inhibit growth of microorganisms in real food products. Although Simpson et al. (1997) found a level of 0.01 per cent sufficient to inhibit microbial growth in shrimps stored at low temperatures, it is more frequently found that levels around 0.5–1.0 per cent are necessary to inhibit growth in pork, minced beef and strawberries (Darmadji and Izumimoto, 1994; Devlieghere et al., 2002; Sagoo et al., 2002). Even at this level of chitosan the growth inhibition is not complete, but growth is reduced by 1–3 log cfu/g depending on the storage temperature. High storage temperatures usually result in lower inhibition than low storage temperatures. Also the physical environment in the food product interferes with the antimicrobial effect of chitosan. Devlieghere et al. (2002) found that the antimicrobial effect of chitosan was diminished by the addition of 2 per cent NaCl. The pH in the medium also markedly influenced the inhibitory effect of chitosan, whereas addition of oil did not affect the antimicrobial action. Addition of 10 per cent whey protein at pH 6.0 strongly reduced the effect of chitosan, but this effect was much less pronounced at pH 4.0. It seems that chitosan may have a potential for growth inhibition in foods with low fat and protein contents to which no NaCl is added. Chitosan may thus be an appropriate preservative for fruit and vegetables and some marine foods, but less appropriate for meat and meat products.

5 Antimicrobial enzymes Antimicrobial enzymes are found widespread in nature acting as a defence mechanism for many living organisms against infection from microorganisms. These enzymes can be either hydrolases, which degrade key components of the microbial cell wall, or oxidoreductases, which generate smaller molecules, reactive against the bacteria (Fuglsang et al., 1995). The best known antimicrobial enzymes are lysozyme, lactoperoxidase and glucose oxidase.

5.1 Lysozyme Lysozyme is a natural constituent of blood, milk and eggs in fish and mammals, but can also be found in plants like figs and papaya. Lysozyme from hen egg white consists of 129 amino acids with four disulphide bonds and a molecular weight of 14–15 kDa, although the size and structure may vary depending on the source of origin. The enzyme is stable at pH 3–7 and resists temperatures up to 50°C (Lück and Jager, 1997). The lysozyme used for food applications is primarily derived from hen egg white. Lysozyme hydrolyses the ␤(1-4)-glucosidic linkage of the peptidoglycan present in the bacterial cell wall. Lysozyme is mainly effective against Gram-positive bacteria, as

402 New Chemical and Biochemical Hurdles

they lack the outer membrane. However, the peptidoglycan in some Gram-positive bacteria may be chemically modified and thus resistant or much less sensitive to lysozyme (Fuglsang et al., 1995; Masschalck et al., 2002). The Gram-negative bacteria are generally resistant to lysozyme as an outer membrane consisting of lipopolysaccharide, phospholipid, protein and lipoprotein protects them. However, if the outer membrane is destabilized by other preservatives such as EDTA, an increased effect against Gramnegative bacteria can be obtained. The action of lysozyme on the peptidoglycan layer eventually leads to cell lysis due to pore formation and loss of cytoplasm. Lysozyme has been used as a preservative for a wide range of foods, e.g. cheese, meat, fruit, vegetables and wine (Losso et al., 2000). An important use is in cheese manufacturing, where addition of lysozyme may inhibit germination and growth of the saccharolytic Clostridium butyricum and Clostridium tyrobutyricum, thus preventing ‘late blowing’ in hard cheeses (Hughey and Johnson, 1987). In the same study Hughey and Johnson (1987) found that Clostridium botulinum and Listeria monocytogenes also were inhibited, whereas Cl. perfringens, Bacillus cereus, Staphylococcus aureus and Gram-negative bacteria generally were resistant to lysozyme. An improved antimicrobial effect of lysozyme can be obtained by means of other additives. Wang and Shelef (1992) demonstrated a slightly increased inhibition of Listeria monocytogenes in fresh cod by adding a combination of EDTA and lysozyme. Mehdi Razavi-Rohani and Griffiths (1996) found a markedly increased antimicrobial effect of lysozyme in combination with EDTA, whereas other chelators such as Nacitrate and monoglycerol-citrate had no effect. Proctor and Cunningham (1993) report a synergistic effect of lysozyme and nisin in inhibiting Listeria monocytogenes in hot dogs. Combinations of lysozyme, nisin and EDTA also inhibited Staphylococcus aureus on hot dogs, but no synergism was found if the combinations were added to hamburgers. In contrast, a recent study reports no synergism of lysozyme and nisin but a synergistic effect of lysozyme and EDTA (Gill and Holley, 2003). According to these authors (Gill and Holley, 2003), their study was carried out under different conditions than previous studies and therefore has given rise to the observed discrepancy.

5.2 Glucose oxidase The oxidoreductases exert no antimicrobial effect themselves, but depend on the endproducts formed in the reaction catalysed by the enzyme. The best known oxidoreductases in food preservation are glucose oxidase and lactoperoxidase. The glucose oxidase produced by Aspergillus niger and Penicillium spp. catalyses the following reaction: O2
-D-glucose

:

H2O2
D-glucono-␦-lactone

The D-glucono-␦-lactone then reacts with H2O and forms an equilibrium with D-gluconic acid D-glucono-␦-lactone
H2O M

D-gluconic acid

The antimicrobial action of the glucose oxidase system is due to the H2O2 that has been formed, but the pH lowering effect of the gluconic acid may also influence the

Antimicrobial enzymes 403

inhibition of some microorganisms. The effect of H2O2 may be a short-term effect, as H2O2 is readily inactivated, e.g. by catalase, glutathione or ascorbic acid. Long-term exposure of foods to H2O2 may cause rancidity as a result of lipid oxidation. Glucose oxidase has not been used commercially as an antimicrobial, but as an antioxidant in combination with catalase, owing to the O 2 consumption by the combined reaction of the two enzymes. The O depletion may in turn help prevent growth from strict 2 aerobic microorganisms (Fuglsang et al., 1995).

5.3 Lactoperoxidase The lactoperoxidase system (LP) is secreted from various mammalian glands and is thus found in milk, saliva and other body fluids. Bovine milk contains approximately 20 times more lactoperoxidase activity than human milk. The LP system catalyses the oxidation of thiocyanate (SCN) to hypothiocyanate (OSCN) or to (SCN)2: H2O2
SCN :

OSCN
H2O

or H2O2
2SCN
2H
: (SCN)2
H2O

:

(SCN)2
2H2O

OSCN
SCN
2H


The created OSCN is responsible for the antimicrobial activity of the LP system probably due to reaction with sulphydryl groups on various proteins. The LP system is an important factor contributing to the antimicrobial action in milk. The enzyme is fairly heat resistant and is only partially inactivated by short-time pasteurization at 74°C (Wolfson and Sumner, 1993). Lactoperoxidase is more active at acidic pH with an optimum around pH 5. The LP system has a broad-spectrum antimicrobial effect against both Gram-positive and Gram-negative bacteria, including pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157, Salmonella, Shigella and Campylobacter (Wolfson and Sumner, 1993; Garcia-Garibay et al., 1995; McLay et al., 2002). The LP system has been used to prolong the shelf-life of milk, but the use of lactoperoxidase in other foods, such as for instance minced beef, remains to be documented (McLay et al., 2002).

5.4 Other oxidoreductases Other oxidoreductases such as myeloperoxidase from human leucocytes and haloperoxidases produced by fungi also exert antimicrobial acitivity. Myeloperoxidase seems irrelevant to the food industry, but the haloperoxidases produced by fungi could be of interest for future food applications, as these enzymes can be produced in bulk amounts at low cost. Recently, the antimicrobial activity of a haloperoxidase produced by the fungus Curvularia verruculosa was reported (Hansen et al., 2003).

404 New Chemical and Biochemical Hurdles

6 Nisin The use of lactic acid bacteria in fermented vegetables, meat and dairy products is indeed a very old food processing practice. The resulting increase in shelf-life and food safety is due to different factors: the acidification due to the organic acids, the undissociated acids themselves, competition between lactic acid bacteria and the spoilage and/or pathogenic microflora for nutrients and ‘space’ and specific antimicrobial compounds or metabolites produced by the lactic acid bacteria. Bacteriocins are one such group of antimicrobial compounds produced by lactic acid bacteria. Adding lactic acid bacteria as starter cultures to fermented products is widely accepted by food authorities, but the use of bacteriocins or the bacteriocin-producing bacteria as protective cultures in other foods (biopreservation) is more controversial from a statutory point of view and only a few applications are approved in some countries. A detailed discussion of biopreservation is beyond the scope of this chapter and the reader is referred to specific books on the subject. Bacteriocins can be defined as a group of antimicrobial proteins or peptides produced by bacteria and primarily active against closely related organisms (Holzapfel et al., 1995). A large number of bacteriocins have been described, but only nisin is at present approved for use in some specific foods and will thus be discussed briefly in this chapter. Table 15.5 summarizes the use of nisin in foods and beverages. Nisin is a polypeptide produced by Lactococcus lactis consisting of 34 amino acids in its mature form. The nisin monomer has a MW of 3.4 kDa, but is normally found as the more stable dimer (approx. 7 kDa). Nisin is rather heat stable at acidic pH, withstanding 115°C for 20 minutes without significant loss of activity. Nisin is a relatively broad-spectrum bacteriocin with antimicrobial activity against many Gram-positive bacteria and the endospores formed by many Clostridium and Bacillus spp. The mechanism of nisin against spores is not fully understood, although it is known to be different from the effect on vegetative cells. The primary action of nisin against bacteria is interference with the cytoplasmic membrane resulting in formation of small pores, through which cellular components will diffuse leading to lysis of the cell (Thomas et al., 2000).

Table 15.5 Use of nisin in foods and beverages Food product

Use/main function

Swiss-type cheese Milk Tomato juice Canned foods Sauerkraut Beer Wine

Prevention of ‘late blowing’ caused by Clostridium butyricum Extension of shelf-life Lower heat processing requirements Prevention of ‘flat-sour’ caused by thermophilic Bacillus spp. Optimizing controlled fermentation by improving competitiveness Inhibition of spoilage by lactic acid bacteria Inhibition of spoilage by lactic acid bacteria

Modified from Smid and Gorris (1999).

Lactoferrin 405

Although some authors report an antimicrobial effect against Gram-negative bacteria if nisin is combined with cell wall degrading factors like EDTA, high pressure or heat (Thomas et al., 2000; Branen and Davidson, 2004), nisin is mainly used for inhibiting spore formers and Listeria monocytogenes or the Gram-positive spoilage flora in different products. Nisin has been used in foods like cheese, milk, tomato juice, canned foods, sauerkraut, beer and wine to inhibit other Gram-positive bacteria such as lactic acid bacteria and clostridia, thereby extending the shelf-life and preventing late blowing in cheese (Smid and Gorris, 1999). Excellent reviews on this topic are given by Muriana (1996), Smid and Gorris (1999) and Thomas et al. (2000). Like lysozyme, the effect of nisin may be enhanced if added in combination with other hurdles, e.g. EDTA, lysozyme and lactic acid. Combinations of nisin and EDTA (Tu and Mustapha, 2002; Gill and Holley, 2003; Branen and Davidson, 2004) and of nisin and monolaurin (Mansour and Milliére, 2001; Branen and Davidson, 2004) increase the antimicrobial activity against Gram-positive pathogenic or spoilage bacteria. Synergistic effects of nisin and lysozyme have been described (Thomas et al., 2000; Branen and Davidson, 2004), but this combinatory effect is much less documented. A recent publication also reports a synergistic effect of nisin and lactoperoxidase on fish spoilage flora (Elotmani and Assobhei, 2003).

7 Lactoferrin Lactoferrin is an iron-binding glycoprotein found in mammalian milk, saliva, tears and other secretions and makes up the antimicrobial barrier of milk together with lactoperoxidase and lysozyme. Normal milk contains 2–3 g/l (Naidu, 2000) and due to the enormous production of cow’s milk, lactoferrin can be made available in bulk amounts at relatively low cost. Lactoferrin owes its antimicrobial activity to three different mechanisms. First, as a metal-binding protein, lactoferrin (as other transferrins) binds two Fe3
ions thereby inhibiting growth of bacteria depending on free available iron. The inhibitory action of lactoferrin in laboratory models has been reported by several authors (Naidu, 2000). However, recently Branen and Davidson (2004) studied the effect of 2 g/l lactoferrin in tryptic soy broth (TSB) against Listeria monocytogenes, Salmonella Enteritidis, Pseudomonas fluorescens and several Escherichia coli and failed to find any inhibitory effect of lactoferrin alone. In the meantime, addition of lactoferrin increased the antimicrobial effect of nisin and monolaurin, although the synergism was less pronounced in UHT milk with 2 per cent fat than in TSB. Second, in mammalian organisms lactoferrin prevents binding to or colonization of mucosal cells, which is necessary for certain bacteria to invade the host and thus promote disease. This effect, called detachment or blocking effect, is used in a commercialized product (activated lactoferrin or Activin™). In its activated form Activin™ effectively prevents bacteria from attaching, e.g. to a meat surface or even removes already attached bacteria (Naidu, 2002). The same author (Naidu, 2002) reports strong antimicrobial detachment effect against a broad panel of pathogenic and spoilage bacteria, when used on a meat surface. Whether activated lactoferrin retains

406 New Chemical and Biochemical Hurdles

the antimicrobial action when added into a food instead of being used as a surface treatment is yet to be investigated. The third mechanism is based on an antimicrobial domain of the molecule, rich in basic residues, which interact with the cell wall or membrane of the bacteria. Hydrolysates of lactoferrin (lactoferricins) obtained by action of proteases such as pepsin or acid protease have been shown to exert nine to 25 times stronger antimicrobial activity against Listeria monocytogenes in broth cultures (Tomita et al., 1991). The antibacterial activity of lactoferricin against pathogens was also demonstrated in milk (Murdock and Matthews, 2002) and in laboratory cultures (Wakabayshi et al., 1992). However, this pronounced antimicrobial activity has also been shown to depend strongly on the growth medium (Branen and Davidson, 2000) and to a smaller extent on various salts in the growth medium (Wakabayshi et al., 1992). From the present publications it seems that lactoferrin has a strong potential for surface decontamination of raw meat and possibly fish, fruit and vegetables, although the latter remains to be documented. Adding lactoferrin into foods as a preservative with long-term effect seems more unlikely, based on the present knowledge.

8 Ozone Ozone (O3) is a gaseous compound normally present in the atmosphere and formed as a result of lightning or high-energy UV-radiation. Ozone is a rather unstable molecule having a half-life of about 12 hours in gaseous form, but only 20–25 minutes in aqueous solution. Ozone has been used for decades for potable water treatment in order to disinfect the water and to help remove foul odour and organic/inorganic impurities (Rice, 1999; Muthukumarappan et al., 2000). Although ozone is an efficient, broad-spectrum bactericidal attacking the bacterial cell wall and outer membrane, ozone is also a strong oxidizer and highly reactive with other organic or inorganic compounds. If added to foods, ozone may react with other food components (e.g. lipids, vitamins or other nutrients), leading to unacceptable changes in sensory or nutritional properties. Thus despite its many advantages, the widespread use of ozone as a hurdle in foods is limited due to the high reactivity. However, aqueous ozone may have a good potential for short-time surface treatment (decontamination) of fruit and vegetables and as a disinfectant for process water in food producing plants (Muthukumarappan et al., 2000; Khadre et al., 2001; Garcia et al., 2003).

9 Reuterin Reuterin (␤-hydroxypropionaldehyde) – sometimes referred to as a bacteriocin – is an intermediate metabolite produced by Lactobacillus reuteri during anaerobic metabolism of glycerol. It is a broad range antimicrobial, resistant to heat and proteolytic

Discussion 407

enzymes and active over a broad pH range and easily soluble in water and lipids. These qualities indeed characterize an excellent preservative, but although several papers report good antimicrobial activity of reuterin in various food systems (El-Ziney et al., 2000) this antimicrobial has not yet found widespread use in the food industry, probably due to legislative restraints.

10 Electrolysed water and other concepts A novel antimicrobial system recently developed in Japan is ‘electrolysed oxidizing water’ (EO water). EO water is produced by passing an NaCl solution across an electrically charged membrane resulting in two different fractions. An acidic solution (pH  3) containing hypochlorous acid, free chlorine and other highly reactive chlorine based compounds, and an alkaline solution (pH  11) containing NaOH (Fabrizio and Cutter, 2003). Usually the acidic fraction is used for decontamination due to three different antimicrobial mechanisms: low pH, chlorine or chlorine based compounds and a high oxidation–reduction potential (ORP  1100 mV). The antimicrobial effect of EO water against pathogenic bacteria such as Listeria monocytogenes, Escherichia coli O157 and Salmonella has been clearly demonstrated (Park et al., 2001; Hsu et al., 2004; Fabrizio and Cutter, 2003). Nevertheless, the antimicrobial effect is not significantly different from a laboratory made chlorine solution, containing the equivalent amount of free chlorine, adjusted to same pH (Park et al., 2001). At present many new commercially available antimicrobial agents for decontamination of foods are being marketed. Two examples are ‘calcinated calcium’ and ‘acidified calcium sulphate’. ‘Calcinated calcium’ has been reported to kill Escherichia coli O157, Salmonella and Listeria monocytogenes by surface treatment on tomatoes (Bari et al., 2002), whereas Keeton et al. (2002) reported a strong inhibition of L. monocytogenes on frankfurters when dipped in ‘acidified calcium sulphate’ compared to dipping in saline. These new tailor-made commercial antimicrobial products may be the future preservatives of choice, but they need much more investigation in order to gain approval from food authorities.

11 Discussion As can be seen, a number of opportunities exists for present and future chemical preservation of foods. However a ‘silver bullet’ still has to be discovered. None of the antimicrobials discussed in this chapter will work for all food items and against all relevant microorganisms. The solution to this problem may be found in combinations of several preservatives, making use of the synergistic hurdle concept. The food manufacturer must use different combinations of hurdles for different foods and for any given food the hurdles must be combined and optimized according to new multivariable predictive modelling algorithms.

408 New Chemical and Biochemical Hurdles

In addition, a number of very relevant problems still have to be addressed: application of the hurdles, distribution in the food matrices, influence on flavour and colour and long-term effects of the preservatives especially regarding the possibility that microorganisms may become resistant to the new hurdles. Finally, for the emerging additives to have large-scale commercial success, the consumers have to accept them fully, the food authorities have to approve them for use in foods and the food manufacturers need access to the hurdles in bulk amounts at reasonable costs. The problem of application and distribution is most obvious when moving from laboratory studies into a ‘real life’ food production. Our experience, as well as the reviewed literature, reveals major difficulties in reproducing the results from laboratory tests when moving to real food matrices. In the laboratory, the test matrix is often fluid or semi-solid, has few constituents and is very homogeneous and the hurdles and microorganisms are evenly distributed in the test matrix. However, these conditions are unrealistically uniform and do not represent a food environment. In our work, a number of emerging hurdles have been found which are very effective against unwanted microorganisms in meat when tested on a laboratory scale. However, in pilot plant produced meat products only very few hurdles retained a similar effect. Several explanations can be given for the decrease in the antibacterial activity seen in products produced in a pilot plant. The added hurdles may not be evenly distributed in the food matrix due to their solubility or the hurdles may be found in a food fraction different from where the target microorganism will be situated. Another important factor is that a number of the food constituents may react with the hurdles, thereby decreasing the inhibiting effect against the microorganisms. Finally, the time of application for the hurdle might also prove important. If the chemical hurdle is heat sensitive, adding it prior to heat treatment is useless or, in the best case, provides only a very short-term effect, as the hurdle will be inactivated by the heat treatment. Instead, the hurdle should be added after heating or another hurdle being more heat resistant should be considered. Many commercially available hurdles are coupled to a carrier substance or need to be dissolved in e.g. acid or alcohol before being added to the food. Chitosan, for instance, must be dissolved in weak acids, e.g. acetic acid, but if tested in acid solutions, are the effects seen on the microorganisms caused by chitosan or by the acids? In fact Krøckel et al. (2003) investigated the antimicrobial activity of four different ‘natural’ food additives based on herb extracts. They found surprisingly that the antimicrobial effect of the additives was due to the small amount of benzalkonium chloride present in the additive and not the ‘natural herb’. As benzalkonium chloride is not permitted for use in e.g. meat products, the use of these commercially available additives for such products is questionable. Such factors are important to keep in mind, both when reviewing the literature and when conducting own investigations. When reviewing the literature another matter of great importance is the lack of focus on the flavour influence of the tested hurdles. When testing on laboratory scale, a huge number of hurdles prove to be quite effective. However, when tested in real foods, high levels of the hurdles often need to be added in order to obtain a comparable antimicrobial effect. This will, in many cases, strongly influence the organoleptic quality of the food. In our opinion, if no sensory assessments are made,

Conclusions 409

the results and the recommendations offered in the conclusion are of little practical use for a food manufacturer. Adaptation of microorganisms to the hurdles or development of resistance is the least researched area in dealing with emerging hurdles. In our experience adaptation studies are needed as the last stage in the investigation of new hurdles. We have done experiments on the possible adaptation of L. monocytogenes to lactate and acetate. These tests showed no such tendency as L. monocytogenes grown in lactate and acetate conditions for ten consecutive growth cycles were equally sensitive to lactate as L. monocytogenes organisms with no prior history in a lactate environment (SøltoftJensen, 2000, unpublished data). However, this may be different for other hurdles and needs to be investigated for each hurdle before routine use in food production. Many foods contain a microflora either added as a starter culture or being present as a natural flora. This normal flora can be an important factor or hurdle, when estimating the growth potential of pathogenic bacteria. Thus adding hurdles to prevent deterioration caused by the spoilage flora might indirectly promote growth of pathogenic bacteria by inhibiting the normal flora. This balance between food safety and food quality also needs to be considered when implementing new hurdles for a food product.

12 Conclusions This chapter shows that a number of effective antimicrobial chemicals and biochemicals have emerged, though none of them on their own can replace traditional preservatives like salt, sugar and nitrite. The ‘oldest’ emerging preservatives are the organic acids with a widespread use, a general acceptance and well-documented effects. Of these, benzoic acid and parabens have the greatest overall effect against a broad range of bacteria, yeasts and moulds. Acetic, lactic and fumaric acids are also effective, but only against bacteria. Of the biochemical preservatives, the antimicrobials from herbs and spices or their extracts and oleoresins seem promising. Essential oils, e.g. from cinnamon, clove, rosemary, oregano and thyme, show a great potential with respect to preservative effect, antioxidative effect and consumer acceptance. As the consumers increasingly demand food with zero or very few ‘chemicals’ (clean label food) herbs and spices are good candidates, provided that the adverse effects (e.g. off-flavour, strong odour, discoloration) in the food can be avoided. Nisin and lysozyme are also excellent hurdles for inhibiting Gram-positive bacteria in certain foods. If used in combination with chelators or other new hurdles, their antimicrobial range can be extended to cover inhibition of Gram-negative bacteria as well. Reuterin could be the ‘dark horse’ in this context. It is still in a very early stage of development, but the prospects are good, since it has been shown to be effective against a broad range of microorganisms, is highly water and lipid soluble, is physically robust and is effective over a wide pH range. Lactoferrin in its activated form has been shown to be effective in removing bacteria, e.g. from meat surfaces and, together with for example ozone and acidified

410 New Chemical and Biochemical Hurdles

calciumsulphate, it can be used for decontamination of raw materials rather than being inhibitors in the finished products. Future research should be focused on the effects of hurdles or combination of hurdles in real foods rather than in laboratory models. Also, an evaluation of the adverse effect on sensory qualities as well as the possibility of bacteria developing ‘immunity’ to the hurdles after long-term use must be investigated. That would be the next step towards a commercial breakthrough of the emerging hurdles discussed in this chapter.

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Recent Developments in Microwave Heating Gülüm Sumnu and Serpil Sahin Middle East Technical University, Food Engineering Department, Ankara, Turkey

The use of microwaves for food processing is continuously developing world-wide. Faster heating and high energy efficiency are the major advantages of microwave processing of foods. However, there are still some problems in microwave processes in terms of food quality and non-uniform heating. Research is directed towards developing new formulations, changing oven design and combining microwaves with other heating methods in order to overcome these problems. In this chapter, recent studies about the dielectric properties of foods, process modelling and developments in microwave food processes and product formulations will be summarized.

1 Introduction There has been a significant increase in the number of household microwave ovens in recent years. Nowadays, the microwave oven is widely used for reheating of prepared foods in the food service sector. Microwave heating has also found applications in the food industry including tempering of frozen foods, pre-cooking of bacon, pasteurization of packaged foods and final drying of pasta products. Microwave food processes offer a lot of advantages such as less start up time, faster heating, high energy efficiency, space savings, precise process control, selective heating and foods with high nutritional quality (Decareau and Peterson, 1986). There are differences in the mechanisms of microwave heating and conventional heating. These differences may be beneficial or detrimental depending on different processes. For example, a short processing time is desirable in terms of less nutritive loss in microwave processed foods but undesirable in baked products since biochemical reactions may not be achieved during this short period of time. Non-uniform heating, lack of colour and flavour development, soggy surface, high moisture loss and firm texture are the common problems observed in foods processed in microwave ovens. In order to reduce these problems, new formulations are being developed especially for baked products and microwave food processes are being modelled. Another recent trend is changing the oven design such as phase control heating, a variable frequency oven and combining microwave with other heating methods such as Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

16

420 Recent Developments in Microwave Heating

infrared or hot air to obtain food products with high quality in a shorter time. The difficulty in determination of temperature distribution is another disadvantage of microwave heating. New methods such as magnetic resonance imaging, chemical markers and time-temperature indicator methods are being developed for this purpose. The recent studies about sterilization are towards combining microwaves with ultraviolet (UV), hydrogen peroxide or gamma irradiation. This chapter will provide a general review about improvements achieved in the field of microwave heating in recent years. The dielectric properties of foods are important in understanding the microwave heating characteristics of foods. Therefore, the work presented here first concentrates on the recent studies in the dielectric properties of various foods. Then, advances in the fundamental understanding of microwave heating through heat and mass transfer modelling will be described. Finally, advances in microwave food processes and product formulations will be discussed.

2 Dielectric properties of foods Dielectric properties play an important role in understanding the interaction of microwave energy with materials. Knowledge of the dielectric properties of foods is essential for proper understanding of heating behaviour of foods in microwave ovens. Therefore, research is continuing in the investigation of the dielectric behaviour of foods which will provide insights to the design of microwave food products, processes and equipment. The dielectric properties are the dielectric constant (
) and the dielectric loss factor (
) which are the real and the imaginary parts, respectively of the relative complex permittivity (
r) given by the following equation:
r 
  j


(1)

The dielectric constant is an ability of a material to store microwave energy, while the dielectric loss factor is the ability of a material to dissipate microwave energy into heat. Dielectric properties of foods depend on food composition, temperature and frequency (Calay et al., 1995). The comprehensive reviews on the dielectric properties provide good sources of experimental data for many foods (Nelson and Datta, 2001; Datta et al., 2005). Studies in recent years include the determination of the dielectric properties of fruit and vegetables (Sipahioglu and Barringer, 2003), ham (Sipahioglu et al., 2003a), turkey meat (Sipahioglu et al., 2003b), starch solutions (Piyasena et al., 2003), glucose solutions (Liao et al., 2001, 2003), mashed potatoes (Regier et al., 2001), macaroni (Wang et al., 2003a), various food proteins (Bircan et al., 2001; Bircan and Barringer, 2002; Wang et al., 2003a) and insect pests (Wang et al., 2003b). There are limited dielectric properties data on foods below freezing temperatures. Data obtained for frozen foods and during melting of these foods are important to achieve uniform heating and prevent runaway heating in microwave thawing and tempering. Sipahioglu et al. (2003a) investigated the effects of moisture and ash content on the dielectric properties of ham below and above freezing temperatures (35 to 70°C). Frozen ham samples had low dielectric properties until melting started at 20 to 10°C.

Dielectric properties of foods 421

After melting took place, the loss factor of ham was shown to increase with ash content (Sipahioglu et al., 2003a). The loss factor of a salt solution is affected by the combined effects of two mechanisms, dipole loss and ionic loss. The ionic component of the loss factor increases as the concentration of dissociated ions increases (Mudgett, 1995). This explains the increase in the loss factor of ham with ash content. Similarly, the addition of salt to starch solutions was shown to increase their dielectric loss factor (Piyasena et al., 2003). However, increasing ash content reduced the dielectric constant of the ham sample (Sipahioglu et al., 2003a). This can be explained by the fact that salts are capable of binding water, which decreases the amount of water available for polarization. Ash content was not found to be significant in affecting the dielectric constant of fruits since ash concentration is low in fruits (Sipahioglu and Barringer, 2003). The dielectric loss factor of ham samples with high ash content increased more with temperature (Sipahioglu et al., 2003a). At higher salt concentrations, ionic loss is dominating and the loss factor increases with temperature due to the decreased viscosity of the liquid and increased mobility of the ions. The increase in loss factor with temperature was also observed in turkey meat which contains high amount of ash (Sipahioglu et al., 2003b). Mashed potatoes were used in another study that investigated the effects of temperatures below freezing on dielectric properties (Regier et al., 2001). In this study, in the temperature range of melting, a sharp increase in both the dielectric constant and dielectric loss factor was observed. Dielectric properties data above the boiling point of water are also limited in the literature. Dielectric properties at high temperatures are important for microwave sterilization and pasteurization. Dielectric properties of whey protein gel, liquid whey protein mixture, macaroni noodles and macaroni and cheese mixture were measured over a temperature range of 20–121°C at frequencies 27, 40, 915 and 1800 MHz to cover typical conditions used in commercial pasteurization and sterilization (Wang et al., 2003a). There was a sharp increase in the loss factors of samples with temperatures in the radio frequency (RF) range (27 and 40 MHz) since ionic conductivity, which increases with temperature, is known to play an important role at radiofrequencies. However, both ionic conductivity and dipole rotation play a combination role at microwave frequencies (915 and 1800 MHz) and it is known that the ionic loss factor increases while the dipole loss factor decreases with temperature. Therefore, the increase in loss factor of samples with temperature in the microwave range is mild. As temperature increased, the dielectric constant of all samples, except noodles, increased in the RF range but decreased in the microwave range. The increase in the dielectric constant of cooked macaroni noodles with temperature is due to its low moisture content. For foods having a low moisture content, the dielectric constant increases with temperature due to the dominant bound water relaxation mechanism. Dielectric properties of various fruit and vegetables have been reported in various studies (Nelson, 1983; Tran et al., 1984; Seaman and Seals, 1991; Nelson et al., 1994). Sipahioglu and Barringer (2003) measured the dielectric properties of different fruit and vegetables at 2450 MHz over the temperature range of 5–130°C. As expected,

422 Recent Developments in Microwave Heating

the dielectric constants of all fruit and vegetables, except garlic, decreased with temperature. The dielectric constant of garlic increased up to 55°C and then decreased as the temperature increased. This is due to the fact that garlic contains 30 per cent oligofructosaccharides in the form of inulin which binds water (Van Loo et al., 1995). The dielectric properties of sugar solutions where sugar is an important microwave absorbing food ingredient have been studied (Liao et al., 2001, 2003). The increase in glucose concentration decreased the dielectric constant of sugar solutions since less water was free to respond to the electric field (Liao et al., 2003). The loss factor– concentration relationship depends on temperature. At lower temperatures, the loss factor increased with concentration up to a certain concentration and then decreased. When the temperatures exceeded 40°C, the loss factor increased with concentration for all concentrations. This might be due to the increase in solubility of sugar with increase in temperature. There has been an increasing interest in using RF and microwave energies for postharvest insect control in agricultural commodities as a new method (Tang et al., 2000). Information on the dielectric properties of insect pests is needed in developing thermal treatments for post-harvest insect control based on RF and microwave energy. When the dielectric properties of insects and agricultural commodities were determined, the dielectric properties of nuts were found to be very low compared to that of insects (Wang et al., 2003b). This suggests the possible differential heating of insects in nuts when treated at the same time in an RF system. The dielectric properties of proteins change during denaturation. Moisture is either bound by the protein molecule or released to the system during denaturation, which shows significant changes in dielectric properties. There are various studies showing that the dielectric properties can be used to understand protein denaturation (Bircan et al., 2001; Bircan and Barringer, 2002). In these studies the dielectric properties were found to be as effective as differential scanning calorimetry for the determination of the denaturation temperature. Dough mixing was shown to affect the dielectric properties (Kim and Cornillon, 2001). As mixing time increased, the dielectric constant of wheat dough decreased due to the low amount of mobile water in the sample after mixing. The loss factor also decreased during mixing since mixing resulted in a decrease in the amount and mobility of dissolved ions and water. Dielectric properties are recently being used for quality control of foods. They can be used for monitoring the freshness of fish (Martinsen et al., 2000) and for evaluating frying oil quality (Inoue et al., 2002). The dielectric properties give a rapid, nondestructive sensing of the moisture content of agricultural products (Kuang and Nelson, 1997). Moisture of agricultural products could be determined indirectly by measuring the dielectric properties and by using the relationship of the dielectric constant with moisture content, frequency and bulk density. Dielectric measurements were used to obtain discrimination between bound and free water (Henry et al., 2003). Clerjon et al. (2003) investigated the feasibility of a microwave sensor for water activity measurements and a correlation was determined between water activity and the dielectric properties of animal gelatin gel, which needs further research on real food samples.

Heat and mass transfer in microwave processing 423

3 Heat and mass transfer in microwave processing Microwaves directly interact with food and heat is generated volumetrically. The mechanisms of microwave heating of foods can be categorized into two groups: dipolar rotation and ionic conduction. Some dielectric materials such as water contain permanent dipoles that tend to reorient under the influence of an alternating field thus causing orientation polarization (Metaxas and Meredith, 1983). Heat is generated as a result of the inability of rotating molecules to keep pace with the alternating field. In ionic conduction any charged particles in foods will experience a force alternating at the rate of microwave frequency which will accelerate the particle in one direction and then in the opposite. The accelerated particle collides with an adjacent particle, sets it into more agitated motion and heat is generated (Buffler, 1993). In foods heated by microwaves, time-temperature profiles within the product are caused by internal heat generation due to the absorption of electrical energy from the microwave field and heat is transferred by conduction and convection. The surface temperature of a food heated by microwave energy is cooler than the interior because of the lack of ambient heat and the cooling effect of evaporation. For microwave heating the governing energy equation can be shown below: T Q   2 T

t  Cp

(2)

The relationship of Q to the electric field intensity (E) at that location can be derived from Maxwell’s equations of electromagnetic waves as shown by Metaxas and Meredith (1983) where the magnetic losses of the food material have been ignored: Q  2
0
fE2

(3)

Radiative heat transfer is often neglected in microwave heating except for the cases when the surfaces of packaging material act as susceptors. The generalized surface boundary conditions for microwave heating are shown by (Datta, 1990): k

T  h(T  T ) e (T 4  Ts4 ) mw    
  
 n
 convective heat gain or loss

radiative heat transfer

evaporative heat loss

(4)

When food is heated in a microwave oven, moisture evaporates and moves out of the food material through the surface. The equation to express moisture transport for a porous body can be written as a combination of concentration, pressure and temperature gradients where flow due to the temperature gradient is generally ignored in microwave heating (Datta, 1990): M  m 2M mp 2P m T 2 T t

(5)

A multiphase porous media model that includes spatially varying and intense internal heat generation, evaporation and pressure driven flow was developed to predict moisture

424 Recent Developments in Microwave Heating

50 30 W/g 20 W/g

Over pressure (Pa)

40

10 W/g 30

5 W/g

20

10

0 W/g

0 0

5

10

15

20

25

30

Drying time (min) Figure 16.1 Pressure for microwave spouted bed drying of diced apples at different microwave power levels. (Reprinted from AICHE Journal, 47, Feng H, Tang J, Cavalieri RR, Plumb OA, Heat and mass transfer in microwave drying of porous materials in a spouted bed, 1499–1511, Copyright © (2001) with permission from AICHE.)

transport during intensive microwave heating of wet materials (Ni et al., 1999). During microwave heating of a high moisture food, pressure rose much faster and reached a higher value than during conventional heating (Ni et al., 1999). At higher pressures since enough moisture was pushed to the surface and this surface could not hold any more water, liquid was pumped without any phase change. This phenomenon is called liquid pumping and leads to an excessive moisture loss in microwave heating of a high moisture material. The pressure dropped as the moisture was depleted. Pressure gradient driven flow was also shown to play an important role in moisture migration of diced apples during drying in combined microwave-spouted bed (Feng et al., 2001). Figure 16.1 shows the variation of pressure during microwave drying at different power levels. It can be seen that as power increased, internal vapour pressure increased which could be explained by more internal heat generated at higher powers. The maximum pressure value could be reached within 5 minutes of drying. The higher pressure led to more rapid moisture removal. The decrease in pressure build up when no microwave (0 W/g) was used showed that moisture transfer due to the vapour pressure gradient in hot air drying was significantly lower than in microwave-spouted bed combination drying. When infrared is added to microwave heating, the already complex transport processes can be modified. Infrared can penetrate significantly into the material which can affect surface moisture. Datta and Ni (2002) investigated the effects of infrared and hot air assisted microwave heating on temperature and moisture profiles of foods. For foods with small infrared penetration depth (less than 1 mm) the addition of infrared increased surface temperature which, in turn, increased surface evaporation (Figure 16.2). However, for foods with large infrared penetration depth (greater than 4 mm) that is comparable to microwaves, infrared can increase surface moisture. The behaviour of infrared with large penetration depth was similar to that of

Heat and mass transfer in microwave processing 425

100

4 mm

0.8

No infrared 0.6 0.4

1 mm

80

No infrared 60

40 0.2 0 mm

0.0 0 (a)

0 mm 1 mm 4 mm 8 mm

8 mm Surface temperature

Surface water saturation

1.0

1

2

3

4

Heating time (min)

5

6

20 7

0 (b)

1

2

3

4

5

6

7

Heating time (min)

Figure 16.2 Effect of infrared penetration depth on surface saturation and temperature in infrared assisted microwave heating. (Reprinted from Journal of Food Engineering, 51, Datta AK, Ni H, Infrared and hot-air-assisted microwave heating of foods for control of moisture, 355–364, Copyright © (2002) with permission from Elsevier.)

microwaves. Addition of hot air at different temperatures to microwave heating reduced the surface moisture and increased the surface temperature but not as effectively as infrared heating. The magnitude of surface heat and mass transfer coefficients in a microwave oven is important to control surface temperature of foods. Knowledge of these coefficients may provide possibilities for significant improvements in the design of microwave ovens and heating processes. Verboven et al. (2003) determined the surface heat transfer coefficients in microwave ovens by using computational fluid dynamics to model the process of air flow in an oven cavity. Convective heat transfer coefficients were computed as 1.5–3.8 W/m2°C and 3.3–11.1 W/m2°C for the top and side surfaces, respectively. Radiation heat transfer coefficients were in the same range. Rotation of the food by turntable did not affect the magnitude of the convective heat transfer coefficient but provided uniform heating. The surface convective mass transfer coefficient was computed as 0.0063 m/s. The low value of the convective mass transfer coefficient might show that moisture might not be removed at a rapid rate. This explains a soggy food surface in microwave heated products. The mass transfer characteristics of potato slabs and cylinders dried in convection, microwave and combined convective-microwave conditions were evaluated by McMinn et al. (2003). Biot number increased with temperature and power level in convection and microwave drying, respectively. Combination of convection and microwave heating increased Biot number significantly. Table 16.1 shows the moisture diffusivity and mass transfer coefficient values of potato cylinders during drying at different conditions. As can be seen in Table 16.1, the increase in temperature in convective drying and power in microwave drying increased the magnitude of diffusivity. Process variables and different drying methods also affected mass transfer coefficients.

426 Recent Developments in Microwave Heating

Table 16.1 Moisture diffusivity (D) and mass transfer coefficient (K) values for convective, microwave and combined drying of potato cylinders Experimental conditions Air temperature (°C)

Air velocity (m/s)

Microwave power (W)

40 50 60 60 60 – – – 50 50 50 40 60

1.5 1.5 1.5 2.0 1.0 – – – 1.0 1.5 2.0 1.5 1.5

– – – – – 30 90 650 30 30 30 30 30

D ⴛ 10ⴚ8 (m2/s)

K ⴛ 10ⴚ4 (m/s)

4.88 2.90 4.03 3.60 4.18 7.04 19.99 24.22 4.32 3.15 5.36 5.34 5.17

0.963 0.269 0.234 0.188 0.299 0.251 0.371 0.264 0.086 0.043 0.086 0.141 0.131

Reprinted from Food Research International, 36, McMinn WAM, Khraisheh MAM, Magee TRA, Modelling the mass transfer during convective, microwave and combined microwave-convective drying of solid slabs and cylinders, 977–983, Copyright © (2003) with permission from Elsevier.

4 Microwave processing of foods Microwave processing has been successfully applied on a commercial scale to meat cooking, bacon pre-cooking, tempering of meat, poultry, fish, butter and fruit, drying of snacks and vegetables and pasteurizing ready meals and pasta (Schiffmann, 2001). The development of industrial microwave heating of foods in Europe has recently been reviewed by Bengtsson (2001). Many food processes which became popular in past years such as chicken cooking, potato chip processing, doughnut proofing and frying have been replaced by conventional methods (Buffler, 1993). The reasons for lack of success in microwave processing are high capital cost of the industrial microwave ovens and quality problems in microwave processed foods. Now, with the development of more reliable magnetrons and the invention of ferrite circular to protect magnetrons, microwave equipment has a longer operating life. The cost of the equipment has been recently reduced. The major reason for cost reduction is the increase in the power rating of magnetrons from 25 to 75 kW with few design changes and little cost penalty. The introduction of strong competition between companies is another factor responsible for cost reduction (Edgar and Osepchuk, 2001). In recent years, there has been much research on microwave baking, drying, thawing, pasteurization, sterilization, roasting and blanching. The following sections will summarize what progress has been made in the literature in different microwave food processes.

Microwave processing of foods 427

Table 16.2 Amylose content (%) of cakes baked by different methods Storage time (days)

Microwave baking

Conventional baking

0 1 2 3 4

1.151 1.007 0.814 0.604 0.499

0.341 0.307 0.198 0.118 0.067

Source: Seyhun, 2002.

4.1 Microwave baking The quality problems observed in microwave baked products are firm and tough texture, rapid staling, lack of colour and crust formation and a dry product (Sumnu, 2001). Firm and tough texture are related to microwave induced gluten changes, high amylose leached out during baking and insufficient starch gelatinization. The mechanism of rapid staling of microwave baked products is not yet clear. High amylose leached out during microwave baking compared to conventional baking may be considered as the reason for rapid staling of microwave reheated (Higo and Noguchi, 1987) or baked products (Seyhun, 2002). More amylose leaching from the starch granule was observed in microwave cakes during baking compared to conventional cakes (Table 16.2). The rate constant of amylose leaching was 0.18 and 0.07/min for microwave and conventional baked cakes during storage, respectively. This shows that more amylose was released in microwave cakes not only during baking but also during storage. Long exposure time in the conventional oven ensures the completion of Maillard reactions responsible for browning. In microwave ovens, heating is short and heat is absorbed by the food sample and the air around the product is cold. Therefore, evaporated water molecules from the food system directly come across this cold air around the product and condense. This prevents browning and crisping reactions (Schiffmann, 1994). Relatively large amounts of interior heating create significant internal pressure and concentration gradients which increase the flow of liquid through the food to the boundary in microwave heating (Datta, 1990). Therefore, foods heated in a microwave oven lose more moisture than conventional heating (Datta, 1990; Sumnu et al., 1999; Seyhun et al., 2003). The studies in recent years about microwave baking involve improving the quality of microwave baked products. Gluten content was found to be the significant factor in affecting the firmness of microwave baked breads (Ozmutlu et al., 2001a). Breads formulated with low gluten flour were softer and had higher volume compared to the ones formulated with high gluten flour. In the same study, the increasing fat, emulsifier and dextrose contents were shown to reduce the weight loss of microwave baked cakes. When proofing was performed in the microwave oven, the time of proofing affected the volume and firmness of microwave baked breads (Ozmutlu et al., 2001b). Seyhun et al. (2003) studied the effects of different types of emulsifiers, gums and fat contents on the staling rate of microwave baked cakes. Usage of emulsifiers and gums

428 Recent Developments in Microwave Heating

retarded the staling of microwave baked cakes. Emulsifiers and gums had also synergistic effects. Fat content significantly reduced the variation of firmness and weight loss of microwave baked cakes during storage. The effects of different browning treatments were investigated on achieving the brown colour and crisp crust of microwave baked breads (Sahin et al., 2002). When dough was placed on top of susceptors, desired browning and hardness were obtained on the bottom surfaces of the breads. Susceptors consist of metallized, generally aluminized, biaxially oriented polyester film laminated to paperboard, on top of which or within which the product is placed (Zuckerman and Miltz, 1997). They have the property of absorbing the microwave energy and converting it to heat, which is transferred to the product by conduction and radiation. Combination of microwaves with halogen lamp heating is a recent development in microwave baking. The ‘halogen lamp-microwave’ combination oven combines the browning and crisping advantages of halogen lamp heating with the time saving advantages of microwave heating. This oven is called Advantium™ oven and is produced by General Electrics (Loisville, KT, USA). ‘Halogen lamp-microwave’ combination baking has been recently used in bread baking and it has reduced the conventional baking time of breads by about 75 per cent (Keskin et al., 2004). Specific volume and colour values of breads baked in a ‘halogen lamp-microwave’ combination oven were comparable with that of conventionally baked breads but weight loss and firmness values of those breads were higher (Table 16.3). Breads baked in a microwave oven had the highest specific volume. This can be explained by the significant internal pressure which might result in a puffing effect and high volume. The increase in halogen lamp power reduced specific volume and increased weight loss, firmness and colour values (E) of breads in ‘halogen lamp-microwave’ combination baking. Total colour change (E) was calculated by considering the colour of dough as a reference. The increase in halogen lamp power and processing time increased the E values of bread, meaning that the crust colour became darker (Figure 16.3). There is little research in the area of microwave baking of biscuits. Microwave baked biscuits required a high breaking stress during storage compared to conventionally baked ones (Ahmad et al., 2001). Less breaking was observed in microwave

Table 16.3 Effects of different baking methods on quality parameters of breads Baking methods

Conventional Microwave Halogen lamp Halogen lampmicrowave combination

Quality parameters Weight loss (%)

Specific volume (ml/g)

Firmness (N)

⌬E

4.06 10.80 8.20 17.86

1.60 2.04 1.44 1.57

0.67 2.88 0.80 3.05

47.7 3.0 55.6 25.6

Reprinted from Food Research International, 37, Keskin SO, Sumnu G, Sahin S, Bread baking in halogen lamp-microwave combination oven, 489–495, Copyright © (2004) with permission from Elsevier.

Microwave processing of foods 429

baked biscuits which could be explained by the uniform moisture profiles observed in microwave heating.

4.2 Microwave drying Although the primary objective of food drying is preservation, depending on the drying mechanism, some undesired changes may be observed in the product due to browning reaction, lipid oxidation, nutritional loss and microbial growth. High temperatures or long drying times in conventional air drying may cause serious damage to the colour, flavour, nutrients and rehydration capacity of the dried product. Microwave drying may be an alternative to reduce product degradation. It is suitable for products having a high moisture content like carrot, mushroom and cabbage because of the high dielectric properties of water that can quickly absorb the microwave energy (Prakash et al., 2003). The physical mechanisms involved in microwave drying are different from the mechanisms of conventional drying. The internal heat generated during microwave heating provides a vapour pressure within the product and pumps the moisture to the surface (Turner and Jolly, 1991). Case hardening does not occur in microwave drying because of this moisture pumping effect. Thus, an increased drying rate without increased surface temperature and improved product quality are obtained. However, the progress of microwave drying at the industrial level has been relatively slow due to its high initial capital investment. Non-uniform heating is another problem that hinders the commercial application of microwave drying (Mullin, 1995). The usual means of applying microwaves to a drying process is at the end of the falling rate period, which is referred to as finish drying (Schiffmann, 2001).

50 45

Colour change (E)

40 35 30 25 20 15 10 5 0 2

2.5

3

3.5

4

4.5

5

Baking time (min) Figure 16.3 Colour change (E value) of breads during ‘halogen lamp-microwave’ combination baking at 30 per cent microwave power at different halogen lamp powers (■): 70%, (▲): 60%, (●): 50%, (*): 40%. (Reprinted from Food Research International, 37, Keskin SO, Sumnu G, Sahin S, Bread baking in halogen lamp-microwave combination oven, 489–495, Copyright © (2004) with permission from Elsevier.)

430 Recent Developments in Microwave Heating

Generally, microwave drying of foods or food ingredients with a high moisture content (over 20 per cent moisture) is not economical. Although water has a high dielectric constant and absorbs microwaves easily, it also has a very high specific heat capacity. Therefore, if the bulk of water is high, a considerable amount of microwave energy will be needed to raise significantly the temperature for dehydration (OwusuAnsah, 1991). In general, microwave energy has been combined with hot air to shorten the drying times especially in the falling rate periods. In recent years, there have been a great number of studies about the applications of combined ‘microwave-hot air’ drying to different foods such as orange slices (Diaz et al., 2003), soybeans (Stewart et al., 2003), garlic cloves, (Sharma and Prasad, 2001), berries (Venkatachalapathy and Raghavan, 1999), apple (Feng et al., 2001; Prothon et al., 2001), asparagus spears (Nindo et al., 2003), carrot (Sanga et al., 2001, 2002) and potato (Sanga et al., 2001). Drying curves during combined microwave and air drying of orange slices were modelled by considering two periods, with different kinetic constants which were related with the effective moisture diffusivities (Diaz et al., 2003). These constants increased linearly, whereas the time required to reach critical moisture content decreased with increasing microwave power (Table 16.4). On the other hand, applied microwave power did not affect the rehydration behaviour of orange slices. It is known that the unsaturated fatty acid content of foods reduces significantly during drying due to high air temperatures. Therefore, microwave drying of soybeans, which is an excellent source of unsaturated fatty acids, becomes significant. Microwave assisted drying of soybeans was found to be less destructive to fatty acids and trypsin inhibitor activity than forced convection drying (Stewart et al., 2003). Microwave assisted drying is highly applicable for soybeans to be used for germination purposes but not for food purposes. A combined ‘microwave-hot air’ technique applied to garlic cloves resulted in saving to an extent of about 80–90 per cent of conventional drying time (Sharma and Prasad, 2001). Garlic cloves dried by a combined ‘microwave-hot air’ process were lighter in colour compared to hot air dried ones because of a lower drying temperature and shorter time. The retention of volatile components responsible for flavour strength was also higher in ‘microwave-hot air’ drying.

Table 16.4 Values of kinetic constants (k1 and k2), critical times (tc) and moisture contents obtained for each drying condition Microwave power

k1 (1/min)

k2 (1/min)

a

tc (min)

a

0 0.17 0.36 0.69 0.88

0.0031 0.0120 0.0209 0.0324 0.0453

0.014 0.033 0.038 0.076 0.085

0.605 0.888 0.876 1.005 1.038

653 127 72 44 29

0.14 0.13 0.10 0.11 0.14

a

Xwc (db)

Xwf (db)

Xwc is critical moisture content and Xwf is final moisture content. Reprinted from Innovative Food Science & Emerging Technologies, 4, Diaz GR, Monzo JM, Fito P, Chiralt A, Modelling of dehydration–rehydration of orange slices in combined microwave/air drying, 203–209, Copyright © (2003) with permission from Elsevier.

Microwave processing of foods 431

Microwave drying is a potential production method for some spices like red pepper. Red pepper is traditionally produced by laying the fresh red pepper on plastic sheets and then sun drying. Red pepper is one of the best substrates for aflatoxin production even when it is cooked (Ito et al., 1994). Studies about red pepper drying have increased in recent years but only on conventional drying methods (Shin et al., 2001; Kaymak-Ertekin, 2002). Microwave drying for such a perishable vegetable is a potential application. It is advantageous to use osmotic pre-treatment prior to microwave drying in terms of quality improvement. Qualities of osmotically dehydrated microwave dried berries were found to be comparable with freeze-dried berries (Venkatachalapathy and Raghavan, 1999). In another study, it was shown that osmotic pre-treatment in sucrose solution before ‘microwave-assisted-air’ drying of apple cubes increased the final overall quality of the product and decreased the drying time (Prothon et al., 2001). The effects of microwave drying on the microbiological quality of a traditional product, tarhana dough have been studied recently (Daglioglu et al., 2002). Tarhana is a traditional fermented product in Turkey which is a dry form of yoghurt/cereal mixture. Pathogens are likely to be present on the raw materials or may easily contaminate the product due to the lack of sanitary conditions since it is usually home made. Microwave energy was found to be more effective than conventional methods in destroying the microorganisms in tarhana during drying. Little information is available in the literature on the effects of microwave drying on non-homogeneous material. A coupled heat and mass transfer model for moisture content and temperature distribution in carrots during microwave air drying was developed by considering shrinkage (Sanga et al., 2002). The predicted model showed good agreement with the experimental data. In recent years, the problem of non-uniform heating in microwave drying has been overcome for particulate materials by combining microwave and spouted bed drying (Feng and Tang, 1998; Feng et al., 2001). A heat and mass transfer model was developed for combined microwave and spouted bed drying of apple, a hygroscopic porous material (Feng et al., 2001). Combined microwave and spouted bed drying produced asparagus particles with good rehydration and colour characteristics and was the fastest among the methods where heated air was used (Nindo et al., 2003). Another recent advance in microwave drying to improve food quality and temperature distribution is the use of intermittent microwave drying. In traditional convective drying processes a continuous constant air temperature, humidity and airflow is used for moisture removal. On the other hand, in intermittent drying, drying is achieved with time varying operating conditions (temperature, flow rate and/or operating pressure) (Chua et al., 2003). Sanga et al. (2001) studied the intermittent microwave convection drying of carrot and potato pieces. Considering shrinkage, a diffusion model including temperature and concentration dependent diffusivity was developed which agreed well with the experimental data. Intermittent microwave input to continuous convection drying gave a better colour to the product than continuous microwave input. Chen and Wang (2001) investigated the effect of the intermittent microwave heating patterns on batch fluidized bed drying of porous particles theoretically. The drying time and microwave energy consumption changed with the pattern of microwave heating

432 Recent Developments in Microwave Heating

application. The highest microwave energy consumption but the shortest drying time was obtained in the case of intermittent heating with a rectangular wave pattern. In addition to the beneficial effect of microwave drying to product quality, microwave drying can also be used for the determination of total solid content of food products in a short time. Microwave oven drying has been accepted as a rapid alternative method to the vacuum oven method for determination of total solids in processed tomato products (Chin et al., 1985), tomato pulp (Wang, 1987), flour (Davis and Lai, 1984) and oilseeds (Oomah and Mazza, 1993). Evaporation takes place in the product extremely rapidly since water molecules are selectively heated. This results in a very short analysis time, which limits the exposure of the sample to heat and, therefore, weight loss due to degradation reactions (Reh and Gerber, 2003). This is especially important for products containing sugars like dairy products. Improved repeatability and reproducibility in the microwave oven method for determination of the total solid content in high moisture dairy products (sweetened condensed milk, non-sweetened condensed milk, ice cream premixes, yoghurt and other dairy products) was obtained due to the reduction of weight loss caused by degradation reactions (Reh and Gerber, 2003).

4.3 Microwave thawing and tempering Tempering is defined as bringing the temperature of food up to a few degrees below complete thawing (5 to 2°C). Microwave tempering is the most successful application of microwave heating in the food industry. Tempering systems at 915 MHz in batch and continuous form are used in the USA, Europe, the UK, China, Japan, Korea and Australia (Schiffmann, 2001). The disadvantages of conventional thawing are large space requirements and long time which may result in chemical and biological deterioration of the product. Thawing time should be as short as possible to minimize microbial growth, chemical deterioration, excessive drip loss and dehydration. Microwave thawing is faster than other methods. However, the disadvantage of microwave thawing is that it does not occur uniformly. For example, some parts of meat may cook while the other parts remain frozen during thawing. This phenomenon is known as runaway heating. Therefore, it is necessary to control the heat generated by the microwaves. Nonuniformity in heating arises due to uneven power distributions and the increasing power absorption in liquid regions. Non-uniformity of temperatures in thawing of biological materials inside a microwave oven is affected by the size, shape and dielectric properties of the biological materials and magnitude and frequency of the microwaves (Taher and Farid, 2001). Power cycling or using lower power levels in a continuous manner are the effective ways to minimize non-uniform heating (Chamchong and Datta, 1999a). The fraction of time when the microwaves are off allows the non-uniformity in temperatures to equilibrate and therefore results in more uniform heating in commercial microwave ovens. Since the water in the food material has a number of dissolved components, it freezes or thaws gradually over a range of temperatures resulting in a heterogeneous region of coexisting solid and liquid phases known as the mushy zone (Basak and Ayappa, 2002). This is in contrast to pure material in which phase change occurs at a

Microwave processing of foods 433

single temperature. The method used in modelling of food samples which melt over a range of temperatures are known as enthalpy or effective heat capacity method. A successful model for microwave thawing must also be able to cover these partially thawed regions. In addition to the formation of the mushy region, models for microwave thawing are further complicated due to the penetration of microwaves which results in multiple connected liquid domains. Moreover, dielectric properties vary significantly during thawing and should be considered in the model. There has been progress in mathematical modelling of microwave thawing in recent years. Phase change over a range of temperatures has been included in recent research (Basak and Ayappa, 1997, 2002; Chamchong and Datta, 1999a, b). A heat transfer model has been recently developed to relate the thawing time and the nonuniformity of thawing to power cycling, power level and the surface heat transfer coefficient using apparent specific heat which includes both specific heat and the latent heat during phase change (Chamchong and Datta, 1999a). The microwave flux at the surface and its decay varied with the changes in the power level. Power cycling and using a lower power level in a continuous manner had an almost identical effect on non-uniformity. The decrease in thawing time and increase in non-uniformity of heating with power level were significant before the salty shield developed. The thawing time and the non-uniformity of thawing were related to the shape, size and dielectric properties in microwave thawing of a tylose sample (Chamchong and Datta, 1999b). The increase in sample thickness and volume caused an increase in thawing time linearly. Thawing time decreased as the load aspect ratio decreased (flatter sample). Thawing time reduced quickly in the lower power range and much more slowly in the higher power range for all sample volumes, which could be explained by the rapid development of a thawed region near the surface at higher power levels. The nonuniformity increased for higher salt content since salt, which is a lossy material, decreases the penetration depth and focuses microwave energy near the surface. A theoretical model based on a moving boundary was developed to predict temperature distribution in microwave cyclic thawing of frozen meat samples of different thickness using an effective heat capacity method (Taher and Farid, 2001). Microwave thawing time was found to be less than one-fifth of that necessary in conventional thawing. It was observed that thawing started from the surface and progressed slowly down to the bottom since penetration depth was small. Microwave thawing of 2D frozen cylinders of tylose samples exposed to uniform plane waves from one face was modelled using the effective heat capacity method (Basak and Ayappa, 2002).

4.4 Microwave pasteurization and sterilization The heat generated by microwaves can significantly reduce the time required for commercial pasteurization and sterilization. As a result, application of microwave heating for food pasteurization or sterilization provides better product quality. However, microwave sterilization has some problems such as unpredictable and non-uniform energy distribution and the difficulty in monitoring and predicting the microwave heating pattern during processing. Therefore, progress of microwave sterilization at

434 Recent Developments in Microwave Heating

the industrial level has been relatively slow. It is difficult to assess the adequacy of sterilization in microwave ovens since the position of the coldest spot cannot be defined as in retort sterilization. The ‘time-temperature’ profiles in microwave heating depend on the dielectric properties, size, shape, composition, etc. Accordingly, there can be different transient and spatial temperature profiles within microwave processed foods (Dolande and Datta, 1993). Measuring temperature during microwave heating is more difficult than for conventional heating methods. The temperature profile can be determined by inserting fibre optic probes at different positions in a food material but this method is not practical and affects the microwave field. Another method to measure temperature during microwave heating is infrared thermal imaging. The disadvantages of infrared thermal imaging is that it is a surface only technique and studies are usually limited to model systems that can be easily manipulated (Bows and Joshi, 1992; Mullin and Bows, 1993). The temperature mapping of the non-uniform temperature distributions induced by microwave heating with magnetic resonance imaging (MRI) in both model and real food systems was evaluated by Nott et al. (2000). MRI phase mapping was found to be more advantageous than other MRI temperature mapping methods. Kim and Taub (1993) have developed a method based on the analysis of intrinsically produced compounds (chemical markers) upon thermal processing to assess the sterility of aseptically processed particulate foods. Three different intrinsic chemical markers have been identified in food systems: 2,3-dihydro-3,5-dihydroxy-6-methyl(4H)-pyran-4-one (M-1), 4-hydroxy-5-methyl-3(2H)-furanone (M-2) and 5-hydroxymethylfurfural (M-3) (Kim and Taub, 1993). Chemical markers have also been used to determine the temperature distribution within the food material during microwave sterilization (Kim et al., 1996; Prakash et al., 1997). Formation of compound M-2 in a model system (20 per cent whey protein gel) during microwave heating has been kinetically modelled (Lau et al., 2003). M-2 formation followed first order kinetics and the temperature dependency of rate constants was described by the Arrhenius relationship. Time temperature indicator (TTI) is a new method to monitor thermal processes (Van Loey et al., 1996). TTI should have the same kinetic reaction rate as the target attribute and should be easily prepared, easily recovered and read out. They can be microorganisms, enzymes and chemicals. Deng et al. (2003) used the enzyme peroxidase as TTI to monitor the time temperature profile of food particles during microwave heating. Thermal destruction of bacterial vegetative cells is much more temperature dependent than that of quality attributes. Therefore, high temperature short time (HTST) processes are preferred for liquid foods to minimize thermal damage in food qualities while ensuring food safety. It is difficult to achieve rapid heating in glass bottles using conventional processes. On the other hand, glass bottles do not provide additional resistance to microwave heating since microwaves can directly interact with foods in glass bottles. When pickled asparagus was pasteurized in glass bottles using microwave energy, the process time was reduced by one half compared to water bath heating (Lau and Tang, 2002). It was possible to achieve uniform microwave heating of bottled asparagus by selecting the proper microwave power and using partial

Microwave processing of foods 435

microwave shielding of one-third of the glass bottles. Microwave pasteurization was found to be suitable for inactivation of Escherichia coli in apple juice (Canumir et al., 2002). The inactivation of microorganisms was reported to be mainly due to heat, i.e. there was no non-thermal effect of microwaves on the microorganisms. This result was also obtained by other researchers (Rosenberg and Bögl, 1987; Knutson et al., 1987; Kozempel et al., 1998). Microwave pasteurization of milk by continuous flow has been found to be an efficient way to achieve a product with satisfactory microbial and sensory quality without excessive heat damage (Villamiel et al., 1996). Microwave pasteurization had no adverse effects on milk flavour (Valero et al., 2000). Microwaves have recently been combined with other sterilization methods such as UV light, hydrogen peroxide and gamma irradiation to improve the effectiveness of sterilization on microorganisms. A novel ‘microwave-UV light’ sterilization system has been constructed (Iwaguch et al., 2002). This system was highly effective for sterilization compared to the sterilization system with microwaves alone. This would be due to the generation of active oxygen species under UV light. Active oxygen species easily react with biomolecular substances and induce cell death. Synergistic effects of microwave heating and H2O2 treatment on E. coli destruction were observed in continuous flow microwave heating (Koutchma and Ramaswamy, 2000). The combined effects of gamma irradiation and microwave treatment enhanced the shelf-life of beef products and their safety with little effect on their chemical and sensory qualities (Aziz et al., 2002).

4.5 Microwave roasting In microwave roasting studies contradictory results are seen in the literature. Microwave roasting of sunflower seeds did not cause any significant loss in the amount of tocopherol and polyunsaturated fatty acids in the seeds (Yoshida et al., 2002). There was a minor increase in chemical or physical changes in the oil such as carbonyl value, p-anisidine value and colour development after a long roasting period. Therefore, short exposure to microwaves to retard seed deterioration was found to be technically feasible. However, a change in the fatty acid profiles of peanuts during microwave roasting was reported by Megahed (2001). Microwave roasting was not recommended in this study (Megahed, 2001) due to the formation of oxygenated compounds which could decrease stability and accelerate oil rancidity in roasted peanut kernels. Microwave roasting can be recommended for coffee beans since the antioxidant activity of coffee beans roasted by microwaves were found to be higher than that of conventionally roasted ones (Nebesny and Budryn, 2003). The decrease in caffeine content in microwave treated beans was lower than that in conventional ones.

4.6 Microwave blanching The advantages of microwave blanching over conventional blanching are minimizing the undesirable changes in flavour and texture, minimizing nutrient losses and reducing

436 Recent Developments in Microwave Heating

the amount of waste effluents (Henderson et al., 1975). However, there are some disadvantages of microwave blanching like non-uniform heating and difficulty in controlling the temperature during blanching. Most of the microwave heating applications like domestic microwave ovens use multimode cavity application. The field distribution in such systems is complex. Single mode cavities have a well-defined simple field distribution but their usage in the food industry is limited. The reason for this is that the volume of a food has to be extremely small in order to maintain the resonance. Several monomode microwave cavities have been designed to investigate the thermal inactivation of polyphenol oxidase found in mushrooms for industrial blanching (Sanchez-Hernandez et al., 1999). Polyphenol oxidase was inactivated in a shorter time when microwave blanching was used compared to conventional blanching. Mushrooms blanched by microwaves had a higher antioxidant concentration and less browning. Microwave blanching has shown promising results for improved final product quality by decreasing the process time and reducing the browning rate of current industrial blanching methods. The temperature uniformity of heating can be improved by pulsed microwave heating which is achieved by turning the magnetron on and off in an interrupted sequence. When pulsed microwave blanching was compared with conventional blanching better retention of vitamins was achieved by microwave blanching (Ramesh et al., 2002). This was explained by the avoidance of leaching losses during processing and heat generated by microwaves. It was shown by other researchers that microwave blanching of carrots resulted in higher nutritional quality in terms of higher carotene content, dry matter and sucrose (Kidmose and Martens, 1999).

4.7 Future developments in microwave processing Research is still going on about improving the quality of microwave foods and heating uniformity by changing the oven design. Phase control heating, variable frequency ovens and combining microwave with other heating methods are the novel methods that are being studied. Conventional microwave applicators (multimode resonant applicators) used in domestic and industrial ovens produce one resulting heating pattern. Usage of rotating turntables or mode stirrers is not always sufficient to achieve temperature uniformity. This results in runaway heating during thawing of frozen foods, focusing in spherical and cylindrical objects and overheating of edges (Bows et al., 1999). However, in phase control heating two different heating patterns were found which remained constant during heating (Bows et al., 1999). The use of constructive interference techniques to allow more control of heating is called phase control. In a laboratory instrument, phase control heating was achieved by varying the relative phases of two microwave signals to control the microwave field and the consequent temperature distribution. The heating patterns were simpler and appropriate phase conditions could be combined to achieve more uniform heating. A variable frequency oven is another application that can be used to achieve uniformity. Industrial microwave food processing is based on a single frequency

Nomenclature 437

microwave source. Variable microwave frequency heating can be used to control the heating pattern in a spherical product whose geometry dominates the heating pattern when heated in a fixed frequency applicator (Bows, 1999). One disadvantage of this oven is its high cost. More research is needed to apply this oven to the food industry. Microwave heating has been combined with hot air or infrared heating to reduce quality problems in foods such as lack of crust colour, firmness, excessive moisture migration to the surface and non-uniform heating. As discussed in the microwave baking section, halogen lamp–microwave combination ovens have been developed to reduce the processing time significantly while achieving a high quality product. According to the study of Keskin et al. (2004) it was possible to achieve crust and colour formation in breads baked in this oven. Further research is necessary to investigate the effects of this oven on food processes and the quality of different foods.

5 Conclusions Microwaves directly interact with food and heat is generated volumetrically. In addition to concentration gradients, pressure gradients play an important role in moisture transfer during microwave heating. Short processing time in microwave drying, sterilization and thawing is advantageous to reduce quality losses especially for perishable food products. However, some quality problems are observed in microwave baked products because of insufficient time for some biochemical reactions to occur. Development of new formulations or the use of halogen lamps in combination with microwaves have been shown to improve the quality of baked products. Product quality and heating uniformity in various microwave processes can be improved by changing the oven design such as phase control heating, variable frequency ovens, cycling microwave power method, using continuous microwave power at lower levels or combining microwaves with other heating methods. New methods such as magnetic resonance thermal imaging, intrinsically produced compounds (chemical markers) and time temperature indicator methods, which are used for the determination of temperature distribution within the food material, will be helpful for widespread usage of microwave pasteurization and sterilization systems in the food industry. As well as using microwaves in food processing they have the potential for determining the moisture content of foods. In recent years, it has been shown that dielectric properties, which give an idea about microwave heatability of a product, can also be used for food quality control.

Nomenclature Cp E f h

specific heat capacity (J/kg K) root mean squared (average) value of electric field (V/m) frequency of oven (Hz) convective heat transfer coefficient (W/m2 K)

438 Recent Developments in Microwave Heating

j k M mw n P Q T Ts T t

imaginary unit thermal conductivity (W/mK) moisture content (kg/(kg dry solid)) rate of evaporation (kg/s) direction normal to the boundary pressure (Pa) heat generated per unit volume of material (W/m3) temperature (K) temperature of the susceptor (K) air temperature (K) time (s)

Greek letters  thermal diffusivity (m2/s) m moisture diffusivity (m2/s) p pressure gradient coefficient T temperature gradient coefficient
surface emissivity
0 dielectric constant of free space (8.854  1012 F/m)
 dielectric constant
 dielectric loss factor
r relative complex permittivity

Stefan-Boltzman constant (5.67  108 W/m2 K4)  latent heat of vaporization (J/kg)  Density (kg/m3)

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Turner IW, Jolly P (1991) Combined microwave and convective drying of a porous material. Drying Technology – An International Journal, 9, 1209–1270. Valero E, Villamiel M, Sanz J, Martínez-Castro I (2000) Chemical and sensorial changes in milk pasteurised by microwave and conventional systems during cold storage. Food Chemistry, 70, 77–81. Van Loey A, Hendrickx M, De Cordt S, Haentjens T, Tobback P (1996) Quantitative evaluation of thermal processes using time-temperature integrators. Trends in Food Science and Technology, 7, 16–26. Van Loo J, Coussement P, de Leenheer L, Hoebregs H, Smits G (1995) On the presence of inulin and oligofructose as natural ingredients in Western diet. Critical Reviews in Food Science and Technology, 35, 525–552. Venkatachalapathy K, Raghavan GSV (1999) Combined osmotic and microwave drying of strawberries. Drying Technology, 17, 837–853. Verboven P, Datta AK, Anh NT, Scheerlinck N, Nicolai BM (2003) Computation of airflow effects on heat and mass transfer in a microwave oven. Journal of Food Engineering, 59, 181–190. Villamiel M, López-Fandiño R, Corzo N, Martínez-Castro I, Olano A (1996) Effects of continuous-flow microwave treatment on chemical and microbiological characteristics of milk. Zeitsrift für Lebensmittel Untersuchung und Forschung, 202, 15–18. Wang SL (1987) Microwave-oven drying methods for total solids determination in tomatoes – Collaborative survey. Journal of the Association of Official Analytical Chemists, 70, 758–759. Wang Y, Wig TD, Tang J, Hallberg LM (2003a) Dielectric properties of foods relevant to RF and microwave pasteurization and sterilization. Journal of Food Engineering, 57, 257–268. Wang SJ, Tang JA, Johnson E et al. (2003b) Dielectric properties of fruits and insect pests as related to radio frequency and microwave treatments. Biosystems Engineering, 85, 201–212. Yoshida H, Hirakawa Y, Abe S, Mizushina Y (2002) The content of tocopherols and oxidative quality of oils prepared from sunflower (Helianthus annuus L.) seeds roasted in a microwave oven. European Journal of Lipid Science and Technology, 104, 116–122. Zuckerman H, Miltz J (1997) Prediction of dough browning in the microwave oven from temperatures at the susceptor/product interface. Lebensmittel Wissenschaft und Technologie, 30, 519–524.

Radio-Frequency Processing Valerie Orsat and G S Vijaya Raghavan Bioresource Engineering Department, McGill University, Ste-Anne de Bellevue, Québec, Canada

Radio-frequency (RF) dielectric heating/drying has been used in various industrial applications for many years, especially in wood, textile and some food industry processes. Since dielectric heating transfers energy directly to the product, applications of RF present obvious advantages over other conventional techniques (reduction in processing time and space, improvement in product quality, etc.). The success of an RF heating/drying set-up lies in its design and in the impedance matching between the power generator and the applicator. The quality of the applicator’s design is very important for its efficiency. Attention must be given to the choice of materials (quality of the electric contacts, resistance to corrosion, etc.) and to the set-up as a whole (durability, dielectric behaviour of insulators, proper grounding, etc.). The development of new applications for RF and the design of applicators require sophisticated tools (network analyser) and a considerable amount of expertise for fine tuning. The investment costs are high, from five to ten times higher (per kW) than conventional means of heating. However, when used in combination with conventional methods, RF energy can considerably reduce processing time, improve energy use and provide a quality product with a processing that meets new environmental constraints and new production means.

1 Introduction The market pressure to put out ever-tastier, ever-cheaper low- and no-fat, chemicalfree and safe products is one of the strongest trends in the food industry. Low-fat baked goods, crackers and snacks need to be dried carefully, because the stiffness of their dough makes them especially fragile. To drive out the last 1–2 per cent of moisture, it is sometimes necessary to overheat the crust, which can lead to surface damage, breaking and crumbling. Radio-frequency heating offers a way around that, offering electromagnetic energy of a longer wavelength than microwave, is of greater industrial interest. Radio-frequency heating targets the product, not the air surrounding it. In fact, because the interior of the product gets hot faster than the surface, RF treatment tends to drive the moisture from inside out, equalizing moisture throughout the product and avoiding overheating and dehydration of the surface of the product. Many applications of RF heating, as supplemental heat, have been developed Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

17

446 Radio-Frequency Processing

successfully in the food drying industry for pasta, crackers and snacks (UIE, 1992). Nevertheless, the development of applications in the agricultural and food sectors is still limited and warrants further investigation and most importantly further application developments. In an environmental friendly perspective, more efficient utilization of electric power is required in the food-processing sector. In this respect, dielectric techniques may play an important role in the future. Selection of dielectric energy input and its combination with conventional technologies lead to products which meet, and often improve, the quality requirements of existing products and open the field for new product development (Demeczky, 1985, Jones, 1987). In today’s food market, consumers want healthy, biologically grown, preservativefree, high quality produce. Alternatives being used to answer the preservative-free issue are freezing, sterilizing, drying, refrigeration and distributing a fresh product. Pasteurization can solve some shelf-life problems if a producer has the capability of distributing a refrigerated product (Harlfinger, 1992). Sterilization, on the other hand, can offer greater shelf-stability to foods. In some applications, dielectric sterilization can deliver quality products because electromagnetic waves are able to heat the product three to five times faster than conventional sterilization systems (Wang et al., 2003b). The sterilized product is not temperature abused, so the food has better overall quality attributes than products processed by any other available technology. Microbial and pest reduction by dielectric heating has been studied in a large number of experiments including meat and meat products, poultry, eggs and egg products, fish and shellfish, fruit and vegetable products such as canned fruit, fruit juice and jam, soy milk, sugar beet molasses, pea protein concentrates, ready-cooked meals, milk and its products, puddings, cereals, breads, cakes, pasta, starch and spices (Harlfinger, 1992; Rosenberg and Bögl, 1987). The frequency most used is 2.45 GHz microwave. However, conventional cooking and heating techniques generally yield lower colony counts than microwave treatment. The difference usually is in the order of 1–2 decimal reductions, when both techniques are compared for similar final conditions and temperatures of the product. Some research has also been conducted on the radio-frequency sterilization of food products. Wang et al. (2003b) studied the RF sterilization of a model food and macaroni and cheese. Their findings showed that the RF process (27.12 MHz) produced better quality products and faster, while using less energy than the conventional retort process used to produce shelf-stable foods. The resistance of various bacterial types applies for dielectric treatment as for any other heating technique. For example, moulds and yeasts are more rapidly killed than bacteria.

2 Dielectric heating Dielectric heating lies in the electromagnetic spectrum in the range of frequencies from 300 kHz to 300 GHz (Assenheim et al., 1979). Radio-frequencies (RF) range

Dielectric heating 447

Table 17.1 Dielectric heating frequency ranges 300–3000 kHz 3–30 MHz 30–300 MHz 300–3000 MHz 3–30 GHz 30–300 GHz

Medium frequency (MF) High frequency (HF) Very high frequency (VHF) Ultra high frequency (UHF) Super high frequency (SHF) Extremely high frequency (EHF)

from 300 kHz to 300 MHz and microwaves (MW) range from 300 MHz to 300 GHz (Table 17.1). The difference between radio frequency (RF) and microwave (MW) is principally in the technology. In RF, an electric field is developed between electrodes while in MW, it is a wave being propagated and reflected under the laws of optics. RF works well with large quantities having high ionic conductivity. MW works well with smaller quantities of a dipolar nature. At 27.12 MHz, the wavelength is in the order of 10 m. The skin depth is very small in conducting metals and the transfer of the RF is carried onto the perimeter, thus requiring the use of thin strip connectors rather than cables. RF is often perceived as a mysterious technology since each installation requires individual tuning and specific design characteristics (Anon., 1987). Only certain frequency bands are allowed by law for industrial and scientific use in order to avoid interference with bands used in telecommunications. The frequency bands currently used for industrial applications are 13.56 MHz (central wavelength 22 m), 27.12 MHz (central wavelength 11 m), 40.68 MHz (central wavelength 7.3 m), 915 MHz (central wavelength 32.8 cm), 2450 MHz (central wavelength 12.2 cm), 5800 MHz (central wavelength 5.2 cm) and 24124 GHz (central wavelength 1.24 cm). Many biological materials are colour sensitive to temperature and rigorous temperature control must be maintained to preserve the appearance of the material. Temperature excesses or extended periods of time at tolerable temperatures may change the appearance, taste or quality of the food product. Research on mutual interactions between food products and dielectric processing equipment is still needed to provide a practical basis for process control and minimizing of process energy costs, microbial safety and product quality. Processes for high-moisture solid foods have been less successful in extensive research conducted at 2450 MHz, unless combined with convection heating methods. This results to some extent from low penetration depths that could be increased at frequencies below 2450 MHz, such as 915 and 27.12 MHz.

2.1 Difference between radio frequency and microwaves Which of the four principal frequencies, 13.56, 27.12, 915, 2450 MHz, should be adopted for a particular dielectric heating application? Some recommendations apply: 1 Since the dielectric-properties of the material to be processed vary as a function of frequency along with temperature and moisture content as variables, they should point to a specific frequency range.

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2 For large scale processing applications of materials, radio frequency with its longer wavelength is less prone to standing waves and resulting non-uniform heating. 3 Moisture levelling is more effective at radio frequency for wet planar materials in drying applications. 4 If drying needs to be carried out under vacuum to reduce the boiling point, as could be the case with some temperature-sensitive materials, microwave energy is preferred since the likelihood of arcing is much smaller. Certain frequencies are better suited for loads of given dimensions; at certain frequencies, there is the formation of standing waves where the wave is reflected, reinforcing the original wave. If a set of electrodes were designed so that their length was equal to a half or quarter wavelength at a given frequency, standing waves would be set up along the electrodes and instead of all points on the electrode receiving full voltage during a complete cycle, the voltage would be a maximum at one point and zero at another (Kinn, 1947). Since the heating effect in dielectric heating is proportional to the square of the voltage, this would mean that a large variation in heating would take place across the electrodes and the uniformity of heating inherent in dielectric heating would be absent. The general concept in RF heating is that it yields uniform heating. Investigations by Zhong et al. (2003) indicate that it is not always the case with products having a high dielectric loss where, in such cases, the temperature of the product may show a significant gradient.

2.2 Heating mechanism of RF In a radio-frequency heating system, the RF generator creates an alternating electric field between two electrodes. The material to be heated is placed between the electrodes where the alternating energy causes polarization, where the molecules in the material continuously reorient themselves to face opposite poles (Figure 17.1). When the electric field is alternating at radio frequencies, e.g. 27.12 MHz, the electric field alternates 27 120 000 cycles per second. The friction resulting from the rotational movement of the molecules and the space charge displacement causes the material to heat rapidly throughout its mass. The amount of heat generated in the product is determined by the frequency, the square of the applied voltage, dimensions of the product and the dielectric loss factor of the material, which is essentially a measure of the ease with which the material can be heated by radio waves. The principle of dielectric heating is described by the general expressions for time rate of temperature rise and power dissipation in a dielectric, as follows: dT P ⫽ 0.239 ⫻ 10⫺6 dt cr

(1)

P ⫽ 2␲f E2␧o␧⬙

(2)

Which yields the following equation since, ␧o ⫽ 8.85 H10⫺12 F/m.

Dielectric heating 449

Electrode

⫹

⫹ ⫺ ⫹

⫺

⫹

⫹

⫹

⫺

⫹

⫺ ⫹

⫹

⫹

⫺

⫺ ⫺

⫺ ⫺

Figure 17.1

⫹ ⫺

⫺

Alternating electric field ⫹

⫺ ⫺

⫹

⫺ ⫹ ⫺

⫹

Space charge and dipolar polarization in an alternating electric field at radio frequencies.

P ⫽ 55.61 f E2␧⬙ ⫻ 10⫺12

(3) 3

where dT/dt is expressed in °C/s, P is expressed in W/m , c represents the specific heat of the dielectric material (J/kgAK), ␳ is its density (kg/m3), f is the frequency (Hz), E is the rms value of the dielectric loss factor (V/m), and ␧⬙ is the imaginary part of the complex relative permittivity, ␧* ⫽ ␧⬘ ⫺ j␧⬙. Because the heating rate (dT/dt) depends on the variable P, and on c and ␳, which are fixed for any given substance and condition, the variables that influence the value of P, the power dissipation per unit volume, are important in determining the differential heating of the components of a mixture. If a mixture of materials is subjected to dielectric heating by high-frequency or microwave electric fields, the relative power absorption will depend upon the relative values of E and ␧⬙ for each of the materials in the mixture (Nelson, 1985). Too high a value of ␧⬙ will result in a small skin depth, which annuls the desirable volumetric heating effect. On the other hand, too low an ␧⬙ renders the material practically transparent to the incoming energy. Experience has shown that materials with an effective loss factor in the range 102 ⬍ ␧⬙ ⬍ 2 will be suitable for processing with this form of energy (Metaxas, 1988). Therefore, a knowledge of the dielectric properties is very important when assessing the feasibility of heating a given material. Selective polar or ionic additions to a low-loss host material can enhance its effective loss and render it suitable for dielectric processing. It is sometimes possible to modify a low loss factor material, without significantly altering its other properties, with a small amount of high loss factor additive, such as carbon black added to natural rubber, sodium chloride to urea-formaldehyde glues (UIE, 1992) and immersion in a saline solution for cherries (Ikediala et al., 2002). Trials were conducted by Orsat (1999) to study the effects of increased ionic conductivity on the behaviour of RF heating and the energy coupling at the electrode/material level. Wheat samples of 215 g were stirred with 0, 5 g, 10 g and 25 g of salt and RF treated at 400 W output power for 3 min. The phase and load values of the impedance matching varied significantly

450 Radio-Frequency Processing

4300 4200

Voltage (V)

4100 4000 3900 3800 3700 3600 0

20

40

60

80

100

120

140

Time (s) No salt

5 g of salt

10 g of salt

25 g of salt

Poly. (No salt)

Poly. (5 g of salt)

Poly. (10 g of salt)

Poly. (25 g of salt)

Figure 17.2 Voltage decrease at the electrode with increasing salt content for wheat samples RF treated for 3 min at 400 W incident power (Orsat, 1999).

to adjust to the impedance of the circuit with increasing product temperature over time. The salt addition had a significant effect on the impedance adjustment with an increase in salt content with the most pronounced effect with the highest salt addition. The electrode voltage decreased with time and was proportionally affected by the salt content increase from 0 to 25 g (Figure 17.2). The phase and load values of the matching box are excellent indicators of the dielectric behaviour of the material being heated. The dielectric properties were affected by the increased ionic conductivity which can clearly be noted with the necessary changes in impedance matching with both load modulation and phase changes. Dielectric materials exhibit the property of polarization because their molecular structure has strongly bound electrons unlike that of conductive materials which have free or loosely bound electrons (Metaxas, 1996; Kasevich, 1998). In the case of heating at radio-frequencies there exist principally two mechanisms of polarization, namely dipolar polarization, where polarized molecules are realigned with the alternating field, and space charge polarization, where some charge carriers migrate under the influence of the alternating field as represented in Figure 17.1. Owing to the interaction of the dipole moment with the electric field, a polar substance has a dielectric constant which is larger than that of a non-polar material. The dielectric constant of a polar material is strongly dependent upon various physical parameters such as temperature, pressure and frequency of the applied field (Grant et al., 1978). For a polar substance the relative permittivity ␧⬘ (dielectric constant) decreases with increasing frequency as the motion of molecular dipoles is unable to keep up with the changes in direction of the electric field. Accompanying this fall in permittivity is an absorption of

Material properties 451

energy by the medium from the field. Biological molecules are polar therefore they are likely to respond well to electromagnetic stimulation.

3 Material properties The process of heating through permanent and induced polarization is called dielectric heating. The overall process of heating by microwaves and RF is defined by the dissipation of electrical energy in ‘lossy media’. The polarization effect is a function of the radiation frequency, the dielectric and electric properties of the material, the viscosity of the medium and the size of the polar molecules. Water is the major absorber of electromagnetic waves in foods and, consequently, the higher the moisture content, the better the heating. The organic constituents of foods are dielectrically inert (␧⬘ ⬍ 3 and ␧⬙ ⬍ 0.1) and, compared to aqueous ionic fluids or water, may be considered transparent to electromagnetic waves. Only at very low moisture levels, when the remaining traces of water are bound and unaffected by the rapidly alternating field, do the components of low specific heat become the major factors in heating. In high carbohydrate foods, such as bakery products and alcoholic beverages, the dissolved sugars and alcohol are the main susceptors (Rosenthal, 1992). Foods with phases of diametrically opposed dielectric properties are likely to be heated with drastically different temperature gradients: highly absorbing components suspended in a continuous phase of low absorbance (custard inside a doughnut); low absorbance components suspended in a continuous phase of high absorbance (meat and vegetable pieces in soups); or layered products with alternating phases of low and high absorbance (cheese, tomato sauce and dough layers in pizza). The overall efficiency of heating is affected by the radiation frequency, the food composition, the size of the material, its salt content, its moisture content, its temperature and a few other factors. Foods of a lower density heat faster at a given level of power absorption than do denser foods of similar composition. Air is practically transparent to electromagnetic waves because of its low dielectric constant (␧⬘ ⫽ 1) and therefore its presence in a material reduces the amount of power absorbed. In spite of a low dielectric loss and poor absorption, a compound of a low specific heat can be heated up easily by electromagnetic waves because of the lower amount of calories needed per unit weight to raise the temperature. In the context of foods, this property is most relevant for fats and oils. Although the relative dielectric constants (␧⬘ ⬍ 3) and loss factor (␧⬙ ⬍ 0.1) of oils and fats are much smaller than that of water at 20°C (␧⬘ ⫽ 80), they heat considerably faster since the thermal capacity of 2 kJ/kg°C is less than half that of water (4.2 kJ/kg°C). The thermal conductivity influences the homogeneity of the heating process. Heating has a direct relationship between the amount of energy for heating and size of material subjected to heating. If the size of an individual piece is very large in comparison to the wavelength, superficial heating is favoured, whereas for sizes closer to the wavelength, the temperature may be higher in the centre. The more regular the shape, the more uniform the heating. Thinner parts may be overheated compared to larger parts. This effect may be controlled by reducing

452 Radio-Frequency Processing

the power input and extending the heating time. Although the speed of heating can be easily increased by boosting the electromagnetic power, in practice, this option is treated with caution as an excessive rate will lead to non-uniform temperature profiles. While in conventional heating the limiting factor of the heating rate is the thermal conductivity of the material, with dielectric heating, the heterogeneous dielectric properties are the determinant. Each processing operation, such as cooking, baking, drying, pasteurizing, etc. requires specific optimized heating gradients to enable the desired physicochemical changes to occur properly. Speed and evenness of heating are influenced by the composition and mass of food as well as by features of the heating unit. Since the heating during dielectric cooking could be uneven, the presence of relatively cool regions might account for the survival of bacteria even when very high temperatures are recorded in other parts of a food. The permittivity is found as follows (Risman, 1991): D ⫽ e abs ⫽ eo e ⫽ eo (e⬘⫺ je⬙) E

(4)

where, ␧o is the absolute permittivity of vacuum, also called the electric constant (␧o ⫽ 8.854188 ⫻10⫺12 F/m). It would be difficult to separate the various power absorption mechanisms by macroscopic measurements at a given frequency, hence the imaginary part ␧⬙ generally includes the conductivity contribution ␴/␻␧o, where ␻ is the angular frequency (␻ ⫽ 2␲f ) and ␧⬙abs ⫽ ␧⬙ ⫹ ␴/␻␧o. The dielectric constant ␧⬘ varies significantly both with temperature and frequency for many typical workload substances and in many microwave and dielectric heating applications, it is actually necessary to account for that change during processing. The ratio ␧⬙/␧⬘ is often called the loss tangent in dielectric and microwave heating. The terms recommended are dielectric dissipation factor or loss tangent for ␧⬙/␧⬘ ⫽ tan ␦. The permittivity is a measure of a material’s ability to store electrical energy and the loss factor is a measure of its ability to dissipate electrical energy. The loss tangent tan ␦ ⫽ ␧⬙/␧⬘ is related to the material’s ability to be penetrated by an electrical field and to dissipate electrical energy as heat (Mudgett, 1986). The power penetration depth is defined as the depth below the surface of the substance where the power density of a perpendicular electromagnetic wave has reduced by 1/e from the surface value (1/e ⫽ 1/2.718 ⫽ 37%). dp ⫽

␭o 2p

2

(5)

1 e⬘  (1⫹ tan2 d) 2 ⫺ 1)   

The dielectric properties of food materials may be determined in frequency intervals from direct current to optical frequencies by various measuring techniques. Lumped circuit methods may be used to determine complex permittivity over the frequency range from zero to approximately 200 MHz. At frequencies from 10 MHz, the capacitance and dissipation factor of samples are generally measured by a capacitance bridge. At frequencies from 104 to 108 Hz, resonant circuits with fixed inductors and variable

Adopting RF heating 453

capacitors may be used, with resonance indicated by voltmeter deflection. Resonance methods also measure the material’s capacitance. However, the loss component is obtained from the width of the resonance curve, determined by either susceptance or frequency variation of the material. Dielectric constants are obtained by either method from their relationship with the material’s capacitance (Mudgett, 1985). The choice of processing frequency for a particular unit operation may be critical because the dielectric behaviour and heating characteristics of foods vary with frequency and temperature in patterns which are significantly affected by moisture and salt contents. Ionic losses for a particular product are much higher and dipole losses are much lower at 915 MHz than at 2450 MHz and vice versa. At higher ISM (industrial, scientific and medical) microwave frequencies (5800 MHz), dipole losses for most products are much greater and ionic losses become negligible. In contrast, ionic losses are increasingly greater as frequency decreases at sub-microwave frequencies and dipole losses for free-water become negligible. The effects of frequency variation in the radio-frequency region on the dielectric constant are negligible because the dielectric constant of water in this region is close to its static value. The dielectric properties of semisolid food products are primarily determined by their chemical composition in terms of moisture, salts and solid contents and to a much lesser extent, by effects of physical structure. Moisture and dissolved salts are the major determinants of dielectric activity in the liquid phase of such products as modified by volumetric exclusion effects of an inert solids-phase containing colloidal or undissolved lipid, proteins, carbohydrate, ash, or bound water. Many reports have been made on the dielectric properties measurement of a variety of products (Mudgett, 1985; Nelson, 1991; Ryynanen, 1995; Funebo and Ohllsson, 1999; Wang et al., 2003a, c and numerous others) and a recent review has been prepared by Piyasena et al. (2003) for reference.

4 Adopting RF heating Viewed from their domain of application, radio frequencies form an ensemble. The interaction with the product is achieved by phenomena of ionic conduction and polarization in the high or radio frequency (HF or RF) domain and principally by rotational polarization in the microwave (MW) domain. We are dealing with relaxation phenomena stretching over a very large domain of frequency. This explains the fact that many types of chemical bonds are affected by these frequencies in each product and that often the criteria for choice between the two techniques are related to the appropriateness between the technology and the character of the product, the industrial environment and the investment cost. It is at the level of the technology that microwave and high frequencies differ (Bialod, 1985). The essential problem in the applications of radio frequencies is the transfer of energy from the generator to the product especially when placed in an industrial environment. The efficiency of the generator being around 60 per cent, and taking into account the high investment cost per usable kilowatt, the major part of the emitted energy must be absorbed by the product with acceptable uniformity.

454 Radio-Frequency Processing

For new applications, the design and installation of radio-frequency equipment consist of the development of equipment specific to the needs of the product and of the application. Each new equipment represents a challenge and the designed prototype requires extensive testing which is translated into large investments and industrial risks.

4.1 The standardized 50 ⍀ RF technology The 50 technology uses a fixed frequency quartz oscillator with subsequent amplification through a vacuum amplifier. This technology is gaining popularity because, although more expensive than a class C oscillator, it offers superior frequency stability and better compliance with EMC regulations (electro-magnetic compatibility) with the overwhelming increase of radio-frequencies for telecommunication purposes (Marchand and Meunier, 1990; Anon, 1994). The standardized 50 technology is composed of: 1 a generator with an adjustable output power in a standard load with 50 impedance 2 standard coaxial lines with characteristic impedance of 50 to carry the RF power 3 matching boxes using adjustable capacitors or inductors located between the coaxial line and the applicator 4 on-line measurement of the incident and reflected RF powers (Marchand and Meunier, 1990; Bernard, 1997). In a general way, an optimized design is obtained if the air gaps between electrodes and product are minimal; if the parasitic capacitances are minimal; and if the feeding conductors are as short and wide as possible (limiting power losses, Cable, 1954). The air gap between the heated product and the electrodes should be minimized. In the presence of an air gap, there are two homogeneous electric field distributions, in each medium, but the corresponding values are not independent: the electric field in the air is equal to the electric field in the product multiplied by its permittivity (usual values of ␧⬘ for food, at radio-frequencies and room temperature are from 1 to 100). The voltage applied is then the sum of two voltages: one creates the electric field through the product and the other through the air. In most applications with a flat plate configuration, there often exists the presence of an air gap between the product and the top electrode. The field in the air is always higher than in the product, hence it is important to minimize the air gap to limit energy wastage. The air gap is, however, an integral part of the tuning to account for runaway heating. Runaway heating takes place when the warmest part of a product takes more and more of the available power at the expense of the coldest parts. In most applications of dielectric heating to food, runaway heating is unavoidable if contact with both electrodes is maintained. In practice, an air gap is introduced between the top electrode and the upper surface of the material being heated (Sanders, 1966). A part of the voltage appears in this gap, the amount depends on the relative heights of the gap and of the material and on the dielectric properties of the material. In a series arrangement, there is no effect on the relative power absorption by the two constituents as the current through both is identical. In a parallel arrangement,

Adopting RF heating 455

the voltage across the two constituents is no longer identical. The voltage and the power absorption are decreased to the greatest extent in the material of highest conductivity. An air gap of one-tenth the height of the material reduces the voltage across the material to 5–48 per cent of that across the plates. It is this difference which makes a continuous process possible where different materials are present between the electrodes at the same time (Sanders, 1966). The air gap becomes an integral part of the equipment design considerations. Knowledge of the RF impedance of the applicator is essential. It can be measured using a network analyser (Metaxas and Clee, 1993; Neophytou and Metaxas, 1997). This impedance, which is a function of the applicator design, obviously depends on the dielectric properties of the heated product. The temperature and moisture content of the product may change during the heating cycle. Therefore, even without any variation of the electrode configuration, the impedance of the applicator may deviate. This requires to be monitored with a matching box which acts as an impedance balancer. At the nominal operating frequency, the impedance of the applicator must be matched, through the matching box to the nominal output impedance of the generator, 50 . In order to minimize the parasitic power losses, the matching device must use reactive components such as capacitances and inductances. To match the impedances of the load to that of the generator, additional units of electrical capacitance or inductance must be placed either in series or parallel, or in a combination of both, with the load (Cottee and Duncan, 2003). The physical dimensions of such components vary with frequency and, therefore, the selected frequency for a given heating application must be chosen to provide matching impedance components of practical physical size. It is often required to adjust these matching impedances to compensate for changes of load impedance with different materials to be heated or different masses of the same material (Metaxas, 1985, 1987).

4.2 Design of a simple applicator Basic radio-frequency applicators are presented in Figure 17.3 and consist of parallel plates (throughfield applicator) for bulk loads, rodded strayfield applicators for thin planar dielectrics and stagger-through field rodded applicators (Metaxas, 1988). The materials chosen for the construction of an applicator and its enclosure are more or less arbitrary; the principal requirements being that they are good conductors (for optimal energy transfer and for the purpose of electromagnetic shielding) at the selected operating frequency and resistant to processing chemicals and temperatures. Enclosure of the applicator in a metal walled compartment increases the electrical safety and isolates the RF radiation produced around the electrodes from the outside environment. It is generally accepted that a level of radiation equivalent to that emanated from the human body in a normal sedentary state (100 W/m2) is safe for permanent exposure (Assenheim et al., 1979). A simple standard parallel plate applicator design is schematically presented in Figure 17.4. The electrodes are square in shape to ensure adequate temperature distribution in the product mass contained in cylindrical-shaped Teflon or borosilicate glass

456 Radio-Frequency Processing

Electrode

Power source and impedance matching

Power source and impedance matching

Material

E-field

E-field

(a) Throughfield applicator (parallel plate)

Material (b) Stayfield applicator

Electrodes Power source and impedance matching

Material

E-field

(c) Stagger-throughfield applicator

Figure 17.3 Schematic presentation of the choice in electrode configurations for the design of an RF applicator.

400 mm

Perforated aluminium plates: oven boundary

Connectors (good electrical conductivity)

Ground electrode

200 ⫻ 200 ⫻ 6 mm aluminium

400 mm Hot electrode 80 mm

Metallic door hinge

100 mm

Support plate (metallic base)

Teflon

Figure 17.4 Schematic view of a typical parallel plate applicator with connections and protective grounded cover.

Radio-frequency heating applications 457

containers. In this design, the lower electrode is chosen as the high voltage electrode since the assembly is mounted directly over the matching box, thus making the connection as short as possible. The electrodes are connected to the RF voltage via thin silver-plated copper strips. Access to the electrodes is made simple through a hinged cover on the top of the cabinet. Electrode spacing is ensured with Teflon columns (Orsat and Raghavan, 2004). The radiating qualities of the installation are limited by ensuring the containment of the applicator in a metal cabinet enclosure maintained at ground potential. Once the construction of the applicator is complete, the RF impedance of the applicator has to be determined. Low level measurement of applicator impedance permits a study of the behaviour of the applicator. Values of impedance for the empty and loaded applicator allow verification of the quality of the matching between the electric field and the material. The measurement of the impedance requires the use of a network analyser. This instrument gives the real and imaginary parts of the impedance of a dipole in a given frequency band (Metaxas, 1985; Metaxas, 1987). This impedance measurement allows the tuning of the matching box which acts as an impedance regulator. The matching box is a passive quadri-pole, in place to control the impedance matching of the system. The purpose of the matching box is to bring back any impedance between the applicator’s load to match the standard impedance of the generator. At the nominal operating frequency, the impedance of the applicator must be changed through the matching box, to the nominal output impedance of the generator (50 ). As the impedance of the applicator may vary around its nominal value (because different materials are processed or because the moisture content varies during the process, etc.), the reactive components of the matching box (variable capacitors) readjust during the heating process to match the overall impedance of the system (Metaxas and Clee, 1993; Cottee and Duncan, 2003). The RF power generator is a free running oscillator circuit coupled to a triode valve which is fed by a high voltage power source (220 V). The oscillator circuit produces the oscillations which are sustained by the triode valve. The output power from the generator is indicated and adjusted by a potentiometer placed on the front of the generator. There are two galvanometers located on the front of the generator. One displays the incident power supplied by the generator and the other one displays the reflected power which comes back to the generator when the power is not adequately absorbed by the load in the applicator. If the amount of reflected power is too great, the life of the generator will be significantly reduced.

5 Radio-frequency heating applications When it is appropriate (or necessary) for foods to be heated, high temperature short time (HTST) treatments generally deliver products of a superior quality (Rosenberg and Bögl, 1987; Harlfinger, 1992). For this reason, electromagnetic energy, with its rapid heating potential, may offer a competitive edge in agricultural and food applications.

458 Radio-Frequency Processing

5.1 Thermal treatment of food products Research in RF technology has stated the possibility of sterilizing or pasteurizing a food product at time-temperature values much lower than those now required using conventional heating techniques. Conceivably, the benefit of using RF energy comes from a potential selective killing effect on microorganisms other than that attributable to a heating effect or a targeted localized heating of the microorganisms. The literature on this topic is quite diverse and somewhat variable and, at times, contradictory in nature. Selective heating of insects in cereals has been shown to be possible and permits killing of the pests without entailing unfavourable effects on the milling and baking properties (Nelson et al., 1966; Fleurat Lessard, 1989). However, some literature suggests that selective heating of pests may only be possible in the highfrequency range from 1 to 100 MHz and not in the microwave range (Rosenberg and Bögl, 1987; Nelson et al., 1998). For successful selective heating of the pests in the carrier material, the ratio of the dielectric properties is of importance and ␧⬙insect/␧⬘food must be as small as possible and tan ␦insect/tan ␦food as high as possible. It may therefore be concluded that a lower water content of the products is more advantageous for selective heating of the pest (Nelson and Stetson, 1974). In recent years, there has been a general consensus that the effect of RF processing on microorganisms is thermal and the benefit of RF heating lies in the time/temperature combination. Beckwith and Olsen (1931) reported significant reductions in the numbers of Saccharomyces ellipsoideus and other yeast suspensions irradiated with RF for 15 min. Temperatures of the irradiated suspensions were not allowed to exceed 39°C. Fabian and Graham (1933) treated broth suspensions of Escherichia coli with 7.5, 10 and 15 MHz RF energy in a combination condenser-cooler apparatus which maintained the medium at about 19°C by circulation of cold water in the jacket of the condenser. They found that destruction of the bacteria occurred at the three frequencies with lethal effect greatest at 10 MHz. About 88 per cent destruction of E. coli occurred after 8 h of treatment. Fleming (1944) irradiated E. coli with RF energy of various frequencies from 11 to 350 MHz; 10 W power was used and the time of exposure for all treatments was 1 min. Maximum temperature reached during any treatment was 30°C. All frequencies tested had a lethal effect on the bacteria with the greatest effect, about 98 per cent destruction, occurring at approximately 60 MHz. Nyrop (1946) applied RF energy of 10–100 kHz to E. coli in broth suspensions. Nyrop observed that 99.6 per cent kill was achieved with a field strength of 205 V/cm in 5 s and 99.8 per cent kill in 10 s exposure. Brown and Morrison (1954) studied the effect of RF energy at 50 Hz, 190 kHz and 25 MHz on E. coli. The bacteria were irradiated in nutrient broth by means of a capsule electrode assembly. Their initial experiments disclosed many instances of destruction of E. coli. They found, however, that a thermal effect was responsible as temperatures in the capsule reached 55°C. They repeated their earlier work and concluded there was no significant killing effect in most treatments unless the final temperature exceeded about 50°C. If heat can be generated faster in the microbial cell than in the suspension medium, the cell might be destroyed thermally at a comparatively low heating rate of the suspension medium. This possibility is dependent upon the chemical composition of the suspension medium and of the microbial cells. Since most microbial cells bear an

Radio-frequency heating applications 459

electrical charge, usually negative, there exists the possibility of mechanically disrupting the cell by causing it to oscillate rapidly in the high-frequency field. If these oscillations are rapid enough, or of a large enough displacement, or both, the elastic limits of the cell structure might be exceeded, thus causing the cell to rupture and die (Carroll and Lopez, 1969). In recent years, the general opinion is that the effect of RF energy on the inactivation of microorganisms is due to heat (Decareau, 1985; Brunkhorst et al., 2000; Geveke et al., 2002). However, it is often interpreted that the lack on non-thermal effects is due to the low electric field strength tested (Geveke et al., 2002). Although there is no apparent synergistic effect of combined heat and RF energy on microorganism inactivation, a good number of recent applications have shown interesting results. Research was conducted on the electric heating of fat/muscle layers of ham for pasteurizing with capacitive dielectric heating up to 100 MHz (Bengtsson et al., 1970). Power efficiency was higher at 60 MHz than at 35 MHz. The size of the air gap was critical to power efficiency and had to be small. Temperature distribution in the ham was improved by using lean hams of uniform salt content, by increasing the frequency from 35 to 60 MHz and by good thermal insulation of the moulds. Horizontal layers of fat were always overheated, while vertical layers (perpendicular to the electrode) and embedded balls or cylinders of fat were not (Bengtsson et al., 1970). Significantly lower juice losses were obtained with RF-processing than in hot water processing and treatment time was less than half. Sensory evaluation showed a general tendency towards better quality of RF-processed hams, particularly for juiciness. Microbiological examination after prolonged storage showed considerably higher total counts for RF-processed hams, indicating a need for higher final temperatures or supplemental heat treatment. Total counts decreased with increasing salt content and final water temperature. Stationary heating tests with sausage emulsions at varying formulations stuffed in tubes were performed at 27 MHz by Houben et al. (1991) in a heating unit consisting of a cylindrical borosilicate glass tube through which a sausage emulsion was passed. The rapid heating rates resulted in considerably reduced cook values (cook values represent integrated heating times at a reference temperature (80–113°C) with respect to physical and chemical changes of product quality) as compared to conventional heating methods (Houben et al., 1991). Sausage products heated well and had a good appearance without release or loss of moisture and fat. Temperatures in the pasteurization region (80°C), could easily be reached, yielding promising results. Laycock et al. (2003) studied the RF cooking of meat products. RF cooking resulted in reduced cooking time, lower juice losses, acceptable colour and texture and competitive energy efficiency. The French RF and MW equipment manufacturer, Sairem (Neyron, France), has worked with the fish industry to develop RF-thawing techniques in the fish-processing sector. The 50 technology for fish tempering is improving the productivity and lowering the manufacturing costs while promoting the development of more innovative products for the benefit of the fish industry and its consumers (Bernard, 1997). A study was conducted by Orsat et al. (2001) to develop a processing method for the RF treatment of fresh-like carrot sticks to reduce their microbial load and their

460 Radio-Frequency Processing

enzymatic activity while ensuring their quality. Results showed that when compared to chlorinated water dipping and hot water dipping, RF-treated carrot sticks had better quality in terms of colour and taste.

5.2 Seed treatments Radio-frequency heating has been proposed in the past for the treatment of seeds. Problems with poorly or slowly germinating seeds are common for many growers of field, horticultural and ornamental crops. In the case of seed-coat impermeability, hard seeds will eventually germinate and grow, however, it may be too slow or too uneven (Nelson, 1976). The common practice used to increase the permeability of seeds is called scarification. This is an abrasive process that has a damaging effect on seeds which can no longer be stored. To help solve this problem, RF heating can come into play. When a seed is exposed to RF fields of sufficiently high frequency and intensity, its temperature will rise due to dielectric heating, its germination will increase to some maximum as exposure increases then, with continued increasing exposure, germination will decline (Nelson, 1976). Direct comparison of 39 MHz and 2450 MHz exposures on germination of alfalfa seeds of three different varieties was made by Stetson and Nelson (1972). The two frequencies were equally effective for reducing hard-seed percentages and increasing germination when the resulting seed temperatures were comparable. The most important temperature influence appears to be that of the final temperature to which the seed is raised by RF treatment. The maintenance of high quality in RF-treated seed lots for several years after treatment is an important advantage over mechanical scarification (Nelson and Stetson, 1985). Nelson et al. (2002) studied the radio-frequency heating of alfalfa seeds for reducing human pathogens. RF exposures that provided the required reduction in pathogens caused significant damage to seed germination. Reduced RF intensity treatments provided a moderate reduction in pathogens while improving seed germination by reducing hard seed percentages. On the other hand, high-frequency heating has also been suggested as a pre-treatment to kill the germination potential of seeds and weed seeds (Rodionova et al., 1990). De-germination treatments are of interest to bird-seed producers and for nursery producers interested in limiting weed propagation (Barker and Craker, 1991; Pyon et al., 1997). RF treatments would offer an interesting alternative to steam-sterilization or high intensity treatments such as roasting. Lambert et al. (1950) conducted RF treatment (15 MHz) on devitalization of wheat seeds. Their results indicated that a combination of high plate voltage (⬎2500 V) with a minimal treatment time of 4 min, successfully devitalized the seeds. Pyon et al. (1997) proposed that RF radiation offers an environmental friendly alternative in weed management practices.

5.3 Product disinfestation or disinfection Many studies have been conducted for the use of RF heating to control product pests in a variety of agri-food products such as cherries (Ikediala et al., 2002), walnuts

Radio-frequency drying applications 461

(Wang et al., 2001), stored grain (Nelson, 1996) and for numerous other food products (Flores et al., 2003). Nelson and Stetson (1974) studied treatments at 39 MHz and 2450 MHz to control rice weevils in wheat. Their results indicated that 39 MHz treatments were more effective with complete insect mortality at a treatment temperature of 40°C, whereas 2450 MHz required a treatment temperature of 80°C to achieve complete mortality. Dwinell et al. (1994) conducted a study to evaluate an RF/vacuum dryer for the eradication of pinewood nematode in green sawn wood. The electromagnetic radiation was provided by a 10 kW-output radio-frequency generator operating at 13.56 MHz under vacuum. Their research pointed out that the temperature needs to reach a certain level in order to achieve eradication. It appears that nematodes were eradicated in boards where the wood temperature exceeded 48°C. This lethal temperature is about the same as that of a conventional steam heat treatment. In an experiment by Pohleven et al. (1998), RF heating at 4.75 MHz was used to eradicate pine wood decay fungi. The eradication was dependent on the fungus species (Coniophora p., Lentinus l. and Gloeophyllum t.), temperature (75–90°C) and duration (4–12 min) of exposure to RF. At low temperatures, the time of exposure had to be adequately longer.

6 Radio-frequency drying applications The R&D work which has been undertaken so far in radio-frequency drying applications is constrained by the small size of the corresponding equipment manufacturing industry and the limited market demand, causing the investment costs to remain high. The present situation may change when more resources are dedicated to the study of the technique and its applications and when the size of the industry increases. However, there are some well-known applications of RF energy to drying, especially in wood, textile (Pai et al., 1989) and post-baking drying (Mermelstein, 1998). The most successful applications often combine two or more drying techniques (heat pump, forced air with RF, infrared, microwave, etc.). With radio-frequency energy, which generates heat within the product, there is an improvement in the drying performance. It has clearly been shown that the ability of RF to target the internal moisture content brings a significant performance increase to a drying process (Marshall and Metaxas, 1999).

6.1 Wood drying In RF wood drying, since the average dielectric constant (␧⬘) for water is about 20 times larger than that of a dry cell wall for the same frequency range (10–30 MHz) and temperature; under radio-frequency water heats at a much more rapid rate than wood. Water is therefore selectively heated internally more than the surrounding cellwall material thus eliminating the slow conduction from the surface to the core of the lumber that occurs in conventional kiln drying processes.

462 Radio-Frequency Processing

When an RF field is combined with low ambient pressures, high temperature and pressure gradients can develop in both the longitudinal and transverse lumber direction. Since both types of gradients develop toward the same direction, outwards from the centre, moisture is driven out rapidly in both liquid and gas phase during the initial stages of drying. The steep gradients increase the rate of bound water diffusion at moisture contents below the fibre saturation point. In laboratory trials conducted by Avramidis and Liu (1994), drying at 13.56 MHz frequency and 2.7 kPa absolute pressure, the total drying time at 0.6 kV was 24 h from an initial moisture content of 38 per cent to a final moisture content of 15 per cent, whereas with 1 kV, the total drying time was reduced to 14 h. It is interesting to note that the same cedar would normally require more than 25 days of drying time in a conventional ‘heat and vent’ kiln for the same initial and final moisture content levels, whereas western hemlock requires approximately 15 days.

6.2 Agricultural product drying Murphy et al. (1992) conducted experiments on RF drying (27 MHz) of alfalfa. To dry alfalfa from 80 to 12 per cent moisture content requires the removal of 3.5 kg water per 1.0 kg of dried alfalfa. Assuming that the water removed is free water, that the alfalfa is initially at 20°C and that the water is vaporized at 100°C, the energy required is 8.8 MJ or 2.4 kWh. Typically, the efficiency of RF ovens is about 50–60 per cent, so 4.9 kWh of input energy would be required to produce 1 kg of dried alfalfa. At a rate of 4 cents per kWh, the energy costs alone would amount to $200 per t of dried alfalfa, which is more than the current market value of about $US150 per t. It is clear that the only economically feasible application of RF power to the drying of alfalfa is as a supplementary rather than a major power source. Based on experimental data, Knipper (1959) established that at a high frequency (10–15 MHz) and field intensity, drying of grain may be completed within 20–25 min, but the seed quality deteriorates. At a lower frequency (1–5 MHz) and field intensity, the seed quality of the grain is preserved but the drying period is increased to 40–60 min. As with conventional hot air drying, there is a limit of heating intensity in high-frequency heating beyond which the seed quality deteriorates. In hot air drying, this limit is reached when the temperature of the drying medium is about 80–90°C. A valuable merit of high frequency drying of grain is the absence of a drying medium with a high temperature and the good uniformity of drying throughout the entire grain mass (Knipper, 1959).

6.3 Food drying RF drying, with regards to food, has mainly been used for post-bake drying of cookies, crackers and pasta (UIE, 1992; Mermelstein, 1998). Cookies and crackers, fresh out of the oven, have a non-uniform moisture distribution which may yield to cracking during handling. RF heating can help even out the moisture distribution after baking, by targeting the remaining moisture pockets.

Nomenclature 463

Recent development trends are investigating hybrid drying systems involving radio frequency to cater for the special needs of heat-sensitive food stuffs (Zhao et al., 2000; Chou and Chua, 2001; Vega-Mercado et al., 2001).

7 Conclusions The advantages and disadvantages of RF are the following. The advantages of RF are: 1 2 3 4 5 6

Overcomes many heat transfer problems as water is preferentially heated A considerable decrease in process time Acts as a moisture levelling process Good overall energy efficiency No surface over-drying or over heating Low maintenance costs.

The disadvantages of RF are: 1 2 3 4

High initial capital cost of equipment Subject to the fluctuations of electrical costs Skilled labour is required for the tuning All generators and applicators must be properly shielded and specially designed to meet the product specific requirements.

The success of an RF heating set up based on the 50 system, lies in its design and in the impedance matching between the power generator and the applicator. The quality of the applicator’s design is very important for its efficiency. Attention must be given to the choice of materials (quality of the electric contacts, resistance to corrosion, dielectric behaviour of insulators, etc.) and to the set up as a whole (rigidity, durability, electrode set-up and practicality, efficiency of the enclosure and grounding, etc.). The development of new applications of RF heating requires targeted equipment design, specific fine tuning of the applicator design, high tech tools and highly skilled technicians. These boundaries are perhaps the reason why only product-oriented applications have successfully been developed to reach the marketplace. Nonetheless, the industrial potential of radio-frequency processing is interesting with its greater penetration depth than microwave with well designed applicator and heating/drying applications. The potential of RF is even better with hybrid systems which take the volumetric heating advantages of dielectric heating and couple them with conventional processing for efficient, rapid and quality results.

Nomenclature co 299 792 458 Speed of propagation of electromagnetic waves in free space (m/s) c specific heat (J/kg ⬵ K)

464 Radio-Frequency Processing

D dp dT/dt E f P T ␧o ␧ ␧abs ␧⬘ ␧⬙ tan ␦ ␳ ␭ ␭o

electric flux density field penetration depth (m) time rate of temperature rise (°C/s) electric field strength (V/m) 1/T frequency (cycles/s, Hz) power (W/m3) period (s) 8.854188 ⫻ 10⫺12 absolute permittivity of vacuum (F/m) relative complex permittivity (F/m) absolute permittivity (F/m) relative real permittivity (dielectric constant) (F/m) relative dielectric loss factor (loss factor) (F/m) ␧⬙/␧⬘ dielectric loss tangent density (kg/m3) wavelength (m) 0.122366 m at 2.45 GHz wavelength in free space

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Nelson SO, Stetson LE, Rhine JJ (1966) Factors influencing effectiveness of radio frequency electric fields for stored grain insect control. Transactions of the ASAE, 9 (6), 809–816. Nelson SO, Bartley PG Jr, Lawrence KC (1998) RF and microwave dielectric properties of stored-grain insects and their implications for potential insect control. Transactions of the ASAE, 41 (3), 685–692. Nelson SO, Lu CY, Beuchat LR, Harrison MA (2002) Radio-frequency heating of alfalfa seeds for reducing human pathogens. Transactions of the ASAE, 45 (6), 1937–1942. Neophytou RI, Metaxas AC (1997) Characterisation of radio frequency heating systems in industry using a network analyser. IEE Proceedings Science, Measurement and Technology, 144 (5), 215–222. Nyrop JE (1946) A specific effect of high frequency electric currents on biological objects. Nature, 157, 51. Orsat V (1999) Radio-frequency thermal treatments for agri-food products. McGill University, PhD thesis. Montreal, QC, Canada. Orsat V, Gariépy Y, Raghavan GSV, Lyew D (2001) Radio-frequency treatment for readyto-eat fresh carrots. Food Research International, 34 (6), 527–536. Orsat V, Raghavan GSV (2004). Review of the design aspects of radio-frequency heating for development of agri-food applications. In Dehydration of Products of Biological Origin (Mujumdar AS, ed.). New Delhi: Oxford & IBH Publishing Co. Pai GA, Mock GN, Grady L, Graham RW, Crabtree KK, Moore EJ (1989) Radio frequency drying in the textile industry. IEEE 1989 Annual Textile Industry Technical Conference 89CH2697-1 P9/1-9/3. Piyasena P, Dussault C, Koutchma T, Ramaswamy HS, Awuah GB (2003) Radio frequency heating of foods: Principles, applications and related properties – A review. Critical Reviews in Food Science and Nutrition, 43(6):587–606. Pohleven F, Resnik J, Kobe A (1998) Eradication of wood decay fungi by means of radio frequency. International Research Group on Wood Preservation IRG/WP/98-10292. Stockholm: IRG Secretariat. Pyon JY, Guh JO, Ku YC (1997). Environment-friendly cultural and mechanical practices for weed management. Korean Journal of Weed Science, 17 (1), 124–134. Risman P (1991) Terminology and notation of microwave power and electromagnetic energy. Journal of Microwave Power and Electromagnetic Energy, 26 (4), 243–250. Rodionova OP, Troshina GA, Fedorova IG, Shvartsman MM (1990) Use of radiofrequency electromagnetic field energy for soil sterilization. Tekhnika V Sel’Skom Khozyaistve, 1, 62–63. Rosenberg U, Bögl W (1987) Microwave pasteurization, sterilization, blanching and pest control in the food industry. Food Technology, 41 (6), 92–99. Rosenthal I (1992) Electromagnetic radiations in food science,Vol 19. Advanced Series in Agricultural Sciences. Berlin, Heidelberg, New York: Springer Verlag. Ryynanen S (1995) The electromagnetic properties of food materials: a review of the basic principles. Journal of Food Engineering, 26 (4), 409–429. Sanders HR (1966) Dielectric thawing of meat and meat products. Journal of Food Technology, 1, 183–192.

468 Radio-Frequency Processing

Stetson LE, Nelson SO (1972) Effectiveness of hot air, 39 MHz dielectric and 2450 MHz microwave heating for hard-seed reduction in alfalfa. Transactions of the ASAE, 15 (3), 530–535. UIE, Union Internationale d’Électrothermie (The International Union for Electroheat), 1992. Dielectric heating for industrial processes. UIE working group, Paris, France. Vega-Mercado H, Gongora-Nieto MM, Barbosa-Canovas GV (2001) Advances in dehydration of foods. Journal of Food Engineering, 49 (3), 271–289. Wang S, Ikediala JN, Tang J et al. (2001) Radio-frequency treatments to control codling moth in in-shell walnuts. Postharvest Biology & Technology, 22 (1), 29–38. Wang S, Tang J, Johnson J et al. (2003a) Dielectric properties of fruits and insect pests as related to radio-frequency and microwave treatments. Biosystems Engineering, 85 (2), 201–212. Wang Y, Wig TD, Tang J, Hallberg LM (2003b). Sterilization of foodstuff using radiofrequency heating. Journal of Food Science, 68 (2), 539–544. Wang Y, Wig TD, Tang J, Hallberg LM (2003c) Dielectric properties of foods relevant to RF and microwave pasteurization and sterilization. Journal of Food Engineering, 57 (3), 257–268. Zhao Y, Flugstad B, Kolbe E, Park JW, Wells JH (2000) Using capacitive (radio frequency) dielectric heating in food processing and preservation – a review. Journal of Food Process Engineering, 23 (1), 25–55. Zhong Q, Sandeep KD, Swartzel KR (2003) Continuous flow radio-frequency heating of water and carboxymethylcellulose solutions. Journal of Food Science, 68 (1), 217–223.

Ohmic Heating Adeline Goullieux1 and Jean-Pierre Pain2 1

Université de Picardie Jules Verne, Laboratoire des Technologies Innovantes, IUT-GB, Amiens, France 2 Université Montpellier II, Département Agro-Ressources et Procédés Biologiques UMR1028, Montpellier, France

This chapter describes the basic principles and physical modelling of ohmic devices designed to heat Newtonian and non-Newtonian fluids and food mixtures. Technical aspects are discussed and various industrial applications are presented. Potential future applications are also introduced. For the successful applications of ohmic heating, careful formulations of the food mixture, in terms of electrical and rheological parameters, should be carried out in order to prevent particle/liquid slip for the same electrical conductivity.

1 Introduction Heating is an important step in food processing. Heat treatment has always been the most common method in the food industry for the conservation, cooking and enzymatic inactivation of raw biomaterials. Conventional processes essentially use heat transfer by conduction, convection and radiation in both steady and non-steady state operations. However, internal resistance is often the limiting factor compared with external resistance by convection, which results in very heterogeneous treatment and a notable loss of product quality. Food heat treatments can be classified into two groups: sterilization and cooking (combined with sterilization). Low viscous liquids (milk, fruit juices, etc.) are usually sterilized using an HTST process (high temperature short time), which minimizes changes in taste. Fluids to be cooked are usually more viscous, with non-Newtonian rheological behaviour and can contain a high percentage of large particles. These liquids are usually processed in a scraped surface heat exchanger, or by canning. When food particles are large in size or have poor heat transfer properties, processing times are lengthy and so product quality is reduced. The main requirement of food heaters is that temperature and residence time distributions have to be narrow to prevent over-cooking, or worse, incomplete sterilization in HTST treatments. Heat treatment for complex food fluids is considerably improved when newer systems such as microwave heating, inductive or ohmic (or direct electrical) heating are used. These volumetric heating methods generate heat inside the food and depend less on thermal conduction and convection and so cause fewer temperature gradients. Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

18

470 Ohmic Heating

Inductive heating is restricted to heating metal plates, tubes, screws and coils and the industrial applications are very limited (extrusion cooking, cookers for caramel, hobs) and information on them extremely limited. The volumetric heating is low, with a poor energy conversion. This chapter therefore focuses on ohmic heating. Ohmic heating technology is considered a major advance in the continuous processing of particulate food products. Ohmic heating is direct resistance heating by the flow of an electrical current through foods, so that heating is by internal heat generation. One advantage of ohmic heating is that large sized heating tubes with lower shear rates can be used. This allows heating of fragile particles. In the nineteenth century, several patents were filed for the use of direct resistance heating for the sterilization of static liquid foods (Jones, 1897) or in-can foods (Roberts, 1900). However, owing to a lack of packaging methods and the required complex and expensive can design (Hull, 1925; Bohart, 1933), these patents did not lead to industrial applications. In the case of pasteurization, aseptic packaging is not necessary and Beattie (1914) used a resistive heating method for infant milk pasteurization. A few years later, still in dairy science, Anderson and Finkelstein (de Alwis and Fryer, 1990) sterilized milk using two parallel walls of graphite across which a set voltage was applied. These experiments allowed the electro-pure process to be used industrially in the 1930s. Process optimization required strict control of the operating parameters (temperature, intensity, voltage, etc.) and inert materials for the electrodes. These requirements could not be met by the technology of the time. In the 1980s, the Electrical Council Research at Capenhurst (UK) registered a patent for a continuous ohmic heating apparatus and gave APV Baker Ltd the exclusive licence for international use. In the UK, the first industrial unit (75 kW) was used in 1989 and the technology was approved to stabilize low acid food in 1991. In 1993, another important step was taken, with the Food and Drug Administration (FDA) approval, to process stable low acid food at ambient temperature. Since then, ohmic heating has been used commercially in Japan, the USA and Europe and much research is still being carried out to improve product quality and process control.

2 Fundamentals of ohmic heating 2.1 Basic principles The basic principle of ohmic heating, or Joule effect, is the dissipation of electrical energy in the form of heat using an electrical conductor. This energy generation is proportional to the square power of the local electric field strength and the electrical conductivity. The power generated, ⌬P, in a homogeneous element conductor of given length ⌬z in the z direction and area ⌬A, perpendicular to the electrical field E, with constant physical properties, is given by
P 


U 2  E 2
A
z  E 2
V


(1)

Fundamentals of ohmic heating 471

where the potential difference in direction z is ⌬U  E
z and the ohmic resistance is defined by
 
z/
A. Equation (1) shows the volumetric nature of the ohmic heating process. The heat power per unit volume inside the material is G  E2. When an electrical potential is applied to a non-homogeneous material of local electrical conductivity , the potential field is obtained by applying Laplace’s law: (U)  0

(2)

An electrical field E is created and generates a local heat power per unit volume G inside the material (E  U and G  E2). In the medium, composed of different phases and electrical conductivities, the E and G expressions are valid only locally and their values depend on physical and electrical homogeneities. Ohm’s law establishes the relationship between current density and electrical field strength. For a homogenous element, Ohm’s law states: J

E 

(3)

The local temperature of an element is determined by its thermal conduction  and volumetric heat capacity G, therefore in a conductive medium: CT  ( T ) G t

(4)

where T is the local temperature at time t,  is density and C is specific heat. If the thermal conduction is neglected and in the case of constant physical properties, the local heat is deduced by:
T   E2
t C

(5)

For a convective medium, Bird et al. (1960) presented a more general form of energy conservation. It includes all the quantities involved in the heat exchanges and is expressed by the following equation:  p  D CT  ( T )  T   v S  T  Dt 

(6)

where p is absolute pressure and v velocity. The source term S represents the exchanges with the external phase and includes the local heat generation through electrical current flow. To solve Equation (4) or (6), the initial and boundary conditions are needed. In ohmic heating, two parameters control the quality of the treatment: heat generation (mainly related to the electrical conductivity) and residence time distribution for flowing products.

472 Ohmic Heating

2.2 Electrical heat generation The effect of cubic particle orientation on the effective electric resistance and the heating rate of a suspension has been studied by Sastry and Palaniappan (1992a). They made a first approach to model such systems in a static form (1992b). Only a weak influence of particle orientation on the electric field at high concentration was observed. In a numerical study of spherical particle heating organized in two different spatial systems, Zhang and Fryer (1993) found a strong distortion of the internal generation, which exhibited a dependency on the conductivity ratio of the two phases and on the solid concentration. Sastry (1992a) established a model for cubic particle heating at high volume fraction (0.8) with constant electrical field and heat transfer coefficients. He also showed that particles with the same conductivity as the liquid in which they were placed (and even if they were less conductive) were heated faster than the liquid itself. The experimental study of Fryer et al. (1993) on the ohmic heating of an electrically insulating particle showed a liquid temperature homogenization role of natural convection, which decreased strongly when the liquid viscosity became high. An ‘enhanced’ conduction model was proposed. It overcame the difficulty involved in the convective term of the Fourier equation by integrating this term to the diffusive term using the Rayleigh number. The resulting electrical field created in the homogeneous medium, without particles (or a case of zero solid concentration), is expressed as: (7)

E0  U

where U is the electric potential. The heat generation per unit volume thus corresponds to: G0  L(E0)2

(8)

where L is the dispersed (or liquid) electrical conductivity. The variation of the electrical conductivity with temperature for both food solids and liquids is often considered in a linear form   in[1 m(T  Tin)]

(9)

where in is the electrical conductivity at the starting temperature Tin and m a proportionality constant (°C1). A numerical study conducted by Zhang and Fryer (1993) characterized the heat generation evolution in two phases as a function of (i) the local particle/liquid conductivity ratio R with R  P/L, (ii) the local solid fraction act, and (iii) the electrical heat generation in the liquid without particles G0(L,act  0). The electrical generation in the liquid and solid phases, GL and GP respectively, is expressed in terms of the functions RGL (L,P,act) and RGP (L,P,act) as follows: GL  RGLG0

and

GP  RGPG0

(10)

A FORTRAN numerical code (Benabderrahmane, 2001) was implemented based on the finite volume method. This method computes centred cubic and cubic systems which are shown in Figure 18.1 and systems of particles in an electrical field normal

Fundamentals of ohmic heating 473

(a) Figure 18.1

(b)

The arrangement of cubic system and centred cubic system: (a) cubic system, (b) centred cubic system.

Table 18.1 Heat generation component functions required in Equation (11) in cubic systems for spherical particles Cubic system

Centred cubic system

Liquid

a

Particles

Liquid 1  1.31act

1  1.91act 1  1.38act 

0.992act

3.002act

Particles

1  0.81act  0.612act

0 5.513act

b

1  1.01act 

c

5.59act  44.422act

167.61 3act  297.174act

199.215act

d

e



2.23  2.38act

1.672act

1  1.16act 

4.013act

0

2.012act

2.25  2.19act 1.172act

4.75act  30.082act

88.353act  120.714act

62.315act

0

1  0.62act  3.522act 5.293act (1 2.14act 0.732act)1

1  0.75act  2.702act

4.133act

(1 0.93act

3.672act)1

a b c1d

a b c1d

0.24  0.40act 1 3.40act  3.462act

0

0.25  0.52act 1 4.36act  8.332act

to two of the six planes of that system. The results correlated numerically with an overall correlation coefficient ␨2 of 0.9999 and yielded the heat generation ratio functions RGP and RGL respectively for the particles and the liquid, as: RG 

a bR cR2 1 dR eR2

(11)

The components a, b, c, d and e are functions of the local concentration act and are given for each phase in Table 18.1 with the corresponding correlation coefficients. The local concentration act ranged between the values 0 act 0.52 for a cubic system with spherical particles and 0 act 0.68 for a cubic centred system with spherical particles. These functions are necessary before undertaking complete two-phase-flow ohmic heater modelling to simplify calculations.

474 Ohmic Heating

3 Electrical conductivity Electrical conductivity is the main parameter in heating rate and in ohmic heating treatment. The conductivity is measured by the quantity of electricity transferred across a unit area, per unit potential gradient and per unit time. It is defined for electrolytes, isotropic solids and liquids. Biological materials are one of the largest classes of poor heat conductors. Typically, the electrical conductivity of a solution is the sum contribution of individual ions, molar equivalent concentrations of individual ions and molar equivalent conductivity (Robinson and Stokes, 1959). These authors (Robinson and Stokes, 1959) present a few values of molar equivalent conductivity obtained by electrical measurements of the conductivity of ionic solutions. The electric conductivity of food products usually increases with water content and temperature (Figure 18.2, Table 18.2). This variation is due to increased ionic mobility at elevated temperatures and is a function of the concentration of individual ions represented by the diffusion coefficient. There is a linear relationship between temperature T and electrical conductivity  (S/m) as indicated in Equation (9). At 25°C, m in Equation (9) is 0.016°C1 for acid solutions, 0.018°C1 for alkaline solutions and 0.019  m  0.022°C1 for salts, underlining the large conductivity variation for different materials. Distilled water, lipids, crystallized products and gases are excellent electrical insulators. Food composition is generally complex, often composed of insulators and ionic components that enable ohmic heating treatment of the product. For complex mixtures, this effective electrical conductivity of mixtures is approached by some authors (Sastry, 1992a) using a percolation approach (Kirkpatrick, 1973), which uses a set of parallel and series resistances giving a mean generation in the mixture or a model of this mixture. The interactions between particles with different 1

Conductivity (S/m)

0.8 0.6 0.4 0.2 0 30

40

Salt content

50 60 Temperature (°C) 0 g/L

4.67 g/L

0.89 g/L

6.55 g/L

70

80

2.78 g/L Figure 18.2

Electrical conductivity of a meal with cocktail tomatoes versus temperature and salt content.

Electrical conductivity 475

conductivities generate shadow effects and so strong temperature heterogeneities occur in the mixture, mainly in unmixed batch systems (Davies et al., 1999). Three electrical conductivities for food products are defined as follows:   0.05 S/m good conductivity: condiments, eggs, yoghurts, milk desserts, fruit juices, wine, gelatine, hydrocolloids, etc. 0.005    0.05 S/m low conductivity requiring high electrical field strength: margarine, marmalade, powders, etc.   0.005 S/m poor conductivity requiring very high electrical field strength and often difficult to process by ohmic heating: frozen foods, foam, fat, syrup, liquor, etc. The exceptions to the above rule are cellular food products (such as vegetables, fruits and meat) and dehydrated products in solution. Although many vegetable and animal tissues contain between 60 and 90 per cent water, some tissues contain less water and ions than others. As the cell membrane is an electrical insulator, current flow is confined principally to the intercellular fluid. Cellular foodstuffs display high extracellular and intracellular ion concentrations, which lends a high electrical conductivity to the solution progressing through the cell wall into the microtubules (Hoff and Castro, 1969). The cell wall is relatively rigid, but highly permeable to solutes and water. Since the electrical resistivity of cell wall is high and its thickness, which can reach several micrometres, varies among plant cells the equivalent mean electrical conductivity is therefore very low for the cell wall compared with the solution P  L. The local heating rate for solution in microtubules and cell wall for the same local electrical field strength can be compared by the following equation: dTP       C  dTL P

      C 

or L

 dTP ⬇ P L dTL

(12)

Table 18.2 Electrical conductivity of vegetables and mixtures Product

Conductivity (S/m)

Temperature (°C)

Brine 0.5 g NaCl/l

0.42

22

Potato Bintje

0.05 0.07 0.09 0.10

40 45 50 55

Potato cubes (40%vol) tap water (60%vol)

0.19

22

Potato cubes blanched in brine (5 g NaCl/l, 95°C, 40%vol) tap water (60%vol)

0.33

21

Potato cubes precooked in brine (28 g NaCl/l, 55°C, 40%vol)

salted water (0.5 g/l, 60%vol)

0.44

22

Cauliflower florets precooked in brine (80 g NaCl/l, 55°C, 70%vol)

salted water (5 g/l, 30%vol)

0.44

30

French beans

0.013

22

French beans (70%vol) tap water (30%vol)

0.032

20

French beans (70%vol) salted water (10 g/l, 30%vol)

0.74

22

Cocktail tomatoes (60%vol) tap water (40%vol)

0.015 0.049

22 65

476 Ohmic Heating

where dTP  dTL. The local temperature rise in microtubules is very high and so ionic mobility is increased, contributing to local plasmolysis (thermal electropermeabilization) and increasing both porosity and mass transfer. The high electrical field can increase the conductivity, particularly with large cells, high frequencies can also improve electrical conductivity in cellular foodstuffs, causing some current flowing through the cell membranes into the cell interior (Pain, 1997). Generally, for dehydrated foodstuffs (e.g. dry vegetables, powder and starch) their conductivity is poor. However, the conductivity increases in solution or molten phase. With the effect of temperature, the hydration and sol/gel transition is accentuated and this leads to non-linearity for electrical conductivity with temperature, notably for high solid concentrations. Several authors (Wang and Sastry, 1997) have used these effects to characterize the sol/gel transition or the reaction progress in extrusion, starch gelatinization, emulsions stabilization, ice cream making, cooking, etc, and sometimes to control unit operation.

4 Generic configurations In ohmic heating, the product (liquid, solid or multiphase mixture) is the conductive medium. Solid electrodes are usually used. These electrodes are in intimate contact with the product. They are fed by an electric power supply. The electrodes are generally separated by a tube or plate space that is electrically insulated. There are three generic configurations (Figure 18.3): 1 without flow, discontinuous, usually called batch configuration, in which the electrodes are coaxial (cylindrical geometry), or plane and parallel 2 transverse configuration (or constant electric field), in which the product flows parallel to the electrodes and perpendicularly to the electric field and the electrodes are generally plane or coaxial and slightly spaced 3 collinear configuration (or constant current density), in which the product fluid flows from one electrode to the other, parallel to the electric field and the electrodes are generally widely spaced. Each configuration has its advantages and disadvantages according to specific technical considerations and applications.

4.1 Batch configuration (Figure 18.3a) For a static medium in a batch system that is thermally and electrically insulated (neglecting the thermal conduction and free convection) and that presents a linear variation of electrical conductivity with temperature, the temperature time course is given by the following equation: T (t)  Tin 

1 [exp(t)  1] m

with

  E2

min C

(13)

Uo

~

~ Flow

Uo

(a) Batch Figure 18.3

Uo

(b) Transverse

~

Flow

Generic configurations 477

(c) Collinear

Generic configurations.

where Tin is the starting temperature. The heating rate is then: T E2 in  exp(t) C t

(14)

The batch or static heater has been widely used in the literature for (i) observations and model validations of solid/liquid samples or complex mixture behaviour in ohmic treatment, (ii) adjustment of formulation on electrical parameters, and (iii) as an HTST simulator. When particle and liquid conductivities differ, particle shape and orientation to the electric field in the liquid produce an overheated or an underheated liquid and shadow effects for more particles (de Alwis et al., 1989; Davies et al., 1999). This batch heater allows for fundamental parameters to be calculated, such as product electrical conductivity, heating time and process homogeneity. It thus provides a tool to test the optimum initial product composition or to monitor processing effects on the quality of end products. This equipment is efficient and it is easier to find the best conditions for a continuous ohmic heating process in order to control accurately the three stages of continuous processing: heating, holding and cooling (Goullieux et al., 1997). For application in laboratories the main advantages are low product quantity, simplicity of application and ability to treat large items. However, the discontinuous operation is limited in industrial applications and its proposed uses are for the thawing of fish and meat (Naveh et al., 1983).

4.2 Transverse ohmic heating (Figure 18.3b) In transverse ohmic heating, the applied electric field and current flux are at right angles to the mass flow and the electrical field strength is constant. If the electric field strength is constant E(z)  Ec, the temperature increase is given by: T (z)  Tin 

1 ( exp( z)  1) m

with

  E2c

min t CP L

(15)

478 Ohmic Heating

Stirling (1987) presents a 50 kW transverse-mode ohmic heater for use with a standard plate-type regenerative cooler. The electrodes are parallel, slightly spaced between plastic insulating spacers and channelled internally to fluid flow. As the surface in contact with the product is large and the voltage between the electrodes is low (96 V), the current is high. Therefore overheating, boiling and electrode erosion have been observed. The applications have thus been restricted to fluids containing no particles such as milk and beverages. Berthou et al. (2001) present a heater for resistive heating of a fluid with different plate configurations.

4.3 Collinear ohmic heating (Figure 18.3c) In a collinear ohmic heating device, the electric field and current flux are parallel to the flow. For an isotropic product flowing through thermally insulated pipes or between plates (adiabatic boundary conditions) of length L and uniform residence time ␶, if the heat axial diffusion (high Peclet number) and free convection are neglected, the solution of Equation (5) can be obtained by variable separation and integration. If the current density is constant J(z)  Jc, the temperature increase is: T (z)  Tin 

1 m

(

)

1 2z  1

with

 

Jc2 m t in C L

(16)

By comparing Equations (15) and (16), it is noticed that the same heat electrical generation will give a higher elevation of temperature when the electric field strength is constant than when the current density is constant. The flowing cross-section area may be constant or variable. A jet heater is normally used for heating fluid. Each jet is an electrical resistance that heats up when electric current flows through it. Generally, the electrodes are widely spaced and assembled in electrode housings (cantilever tube) or are annular on each side of the spacer tube. The food-compatible spacer tube is an excellent electrical isolator (e.g. fluorinated or composite plastics). The surface area in contact with the product is small and the voltage is high (up to 4500 V). This configuration is used for horizontal, vertical or inclined tubes (Reitler and Rudolph, 1986). APV Baker – England developed a commercial process mainly for HTST applications (Skudder and Biss, 1987; Biss et al., 1989). Recently Emmepiemme – Italia proposed annular electrodes enabling integral current flow adapted to large-particle food mixtures. Reznik (1997) patented a spacer tube with a convergent, straight and divergent section adapted solely to liquids without particles (liquid egg products, juices, etc.). The conduit course of relatively small crosssection areas produces a flash treatment in a turbulent flow regime with high heating rate (550°C/s). Therefore, during heating, temperature rises quickly and is uniform. Bibun – Japan proposed ohmic heaters for continuous cooking of salt-solubilized washed fish mince and surimi in which the thin surimi paste moves on a conveyor and the conveyor roll is fed by an electric current. The band between paste and roll is continuously cleaned and moistened.

Modelling 479

4.4 Technical considerations Though technologically simple, ohmic heating has some technical limitations in its electrical, mechanical and hygiene design. As the electrodes are in direct contact with the product, consequently from the food and hygiene viewpoint, corrosion has to be prevented or remain quantitatively and compositionally harmless with respect to the product. Unlike pulsed electric fields, in ohmic heating devices the signal is always an alternating, bipolar (and symmetrical) current. At a low frequency, partial electrolysis of the solution and electrode corrosion have to be prevented in food treatment. These faradaic reactions are generated through electrode/product interfaces and enhanced by a low frequency periodic signal, high current density, inappropriate electrode material, high temperature or aggressive product. To prevent electrolysis at low frequency (50 or 60 Hz) alternating current, Stirling (1987) and Berthou et al. (2001) use titanium electrodes coated with platinum or ruthenium and limit the current density to below 5000 A/m2. Consequently, the electrodes used are expensive; but the power supply is a conventional low-cost transformer. The high frequency limits electrolysis especially for high current densities on the electrodes. Emmepiemme – Italia and Reznik (1996) use frequency up to 100 kHz with conventional stainless steel or graphite electrodes. The signal is sinusoidal or rectangular in shape and the power is supplied on the signal fraction. This power supply is an electronic transformer. Reznik (1997) adds an electrolyte between the electrode and the product to prevent product contamination.

5 Modelling 5.1 Treatment of non-Newtonian liquid This section discusses a vertical tubular ohmic heater: a collinear heater. Figure 18.3c shows a typical apparatus consisting of two basic parts: an electrode housing with electrode and a spacing tube placed between the electrode housings. Usually several housings and tubes are placed in series. The electrode is a ring in the wall or a cantilever tube. The spacing tube is usually made from plastic-lined steel to ensure that the electric current flows only through the liquid product. Most of the heating takes place in this tube. Here, the tube is assumed to be a perfect electrical insulator and a single spacing tube is modelled (Figure 18.4). The electric field near the electrode is nonuniform, but it can be shown that the field at the inlet and the exit of the spacing tube is approximately uniform (Muller et al., 1993). The electric field between two electrodes is described by the Laplace equation: 1  U   U     0  r r r  r  z  z 

(17)

The electrical conductivity  is temperature dependent. Hence the solution of the Laplace equation is dependent on the temperature distribution in the spacing tube.

480 Ohmic Heating

z  L; U  Uo v T 0  r r

and

U 0 r

R rR L

~

Cooling mantle T  Tcl

r0

z r

z0

uv0 T  Nu (T(R)  Tcl)/R r U 0 r v (0,r )  v (0,0) 1 

r R

1 1/n

u  0 and T  Tin z  0; U  0 Figure 18.4 conditions.

Model for a collinear ohmic heater and flowing liquid – thermal and electrical boundary

The boundary conditions for Equation (17) are (i) axial symmetry, (ii) insulating wall and (iii) top and bottom with fixed potential (Figure 18.4). The volumetric heat generation is:  2  2  U U G            r   z    

(18)

The following Fourier equation describing the heat transfer in the liquid has 3 terms: (i) thermal conduction in the radial direction, (ii) the volumetric heating term and (iii) a convection term: 1  T   G   Cv   T  r r r  r 

(19)

The conduction in the axial direction is neglected because the axial conduction is very small compared with the axial convection. The boundary conditions for Equation (19) (Figure 18.4) are (i) the inlet temperature is constant, (ii) axial symmetry and (iii) non-adiabatic wall. In several previous studies (Muller et al., 1993) the tube wall was considered to be a thermal insulator; i.e. the wall was assumed to be adiabatic. However, wall temperatures were shown to increase by up to three times the average temperature rise. Since high wall temperatures lead to fouling, wall cooling is indispensable for fluid volumetric heating. The equation for describing the flow of non-Newtonian liquid is the Ostwald de Waele model which is given below (Bird et al., 1960): n  1   v   p    g (1(T   T ))   v  v  rK   r r   r   z

(20)

Modelling 481

where v is the axial velocity, K (Pa sn) the consistency index and n the flow behaviour index (dimensionless) for non-Newtonian liquids of the Ostwald de Waele model (or power law model). In Equation (20) the natural convection term is included, which is due to the temperature differences between the wall and the centre of the tube. As the radial velocities in the system are smaller in two orders of magnitude than the axial velocities, viscous dissipation due to the radial velocities can therefore be neglected. Instead, the radial velocities can be found from the continuity equation which is given below. The boundary conditions are (i) axial symmetry; (ii) zero velocity at the wall; and (iii) fully developed flow at the inlet v  vc (1  (r/R)1 1/n) (Bird et al., 1960) where vc is the centreline velocity (Figure 18.4). The continuity equation is: v 1  (ru)   0

 z r r  r 

(21)

Therefore the model of the heater is described by Equations (17) to (21) which requires the electrical conductivity , viscosity (K, n) and thermal conductivity  to be known over a range of temperature. The numerical method is used to solve the heater model. Figure 18.5 shows the temperature and axial velocity profiles for various liquids predicted by the above heater model, with relevant parameters given in Table 18.3. A flat temperature distribution is a very important requirement for food heating. The temperature profile typical for ohmic heaters causes the flow profile to flatten because of (i) natural convection and (ii) a large viscosity difference between the wall and the centre. The cooling causes two important improvements of the temperature profile: (i) the maximum temperature decreases when the Nusselt number increases, due to low heat transfer inside the tube; (ii) the maximum temperature moves towards the centre of the apparatus. Consequently, the system with the cooling mantle will be much less affected by fouling. Although the axial velocity profiles differ from liquid to liquid, these variations tend to be small and difficult to characterize. Velocity profiles are mainly determined by the difference in viscosity between the wall and the centre of the heater. Non-Newtonian behaviour only becomes important when the consistency index (n) remains less than 0.6. For low viscosity liquids natural convection improves heater performance. Numerical analysis shows that the axial velocity distribution oscillates due to natural convection: the centreline velocity oscillates around the mean velocity with a decaying amplitude and frequency. This frequency is mainly dependent on (i) the pipe length; (ii) the mean liquid residence time; and (iii) the volumetric heat generation. The exponential decay of the amplitude of the oscillations increases with both tube and liquid residence time. More details and results are presented elsewhere (Muller et al., 1994). By numerical modelling of a Newtonian liquid flow heating, Muller et al. (1993) showed the presence of marked natural convection effects, particularly near the walls, causing a strong flattening of the liquid velocity profile, initially parabolic at the tube inlet. They also concluded that the assumption of a uniform electrical field in the cross-section of the tube causes only small errors. Further theoretical research carried out by Muller et al. (1994) showed the presence of instability, also observed experimentally, due to a mixed convection effect that creates a decreasing periodic fluctuation of the longitudinal centreline velocity, with analogous behaviour of the thermal field.

482 Ohmic Heating

Dimensionless temperature (T Tin)/(Tout Tin)

2.0

1.5

1.0

0.5

0.0 0.0

0.2

0.4

0.6

0.8

1.0

Dimensionless radial position r/R

(a)

Dimensionless velocity v/vmean

2.0

1.5

1.0

0.5

0.0 0.0

0.2 0.4 0.6 0.8 Dimensionless radial position r/R

(b) Water

Figure 18.5

Glycerol 95%

1.0

CMC high viscosity 1.5%

(a) Temperature and (b) flow profiles for various liquids at the outlet of the heater.

Table 18.3 Parameters used in the calculation for results in Figure 18.5 Property

Symbol

Value

Units

Density Specific heat Thermal conductivity Expansion coefficient Water viscosity parameters Glycerol (95%) viscosity parameters Carboxymethylcellulose high viscosity parameters Liquid electrical conductivity at 25°C Temperature coefficient Heat transfer coefficient Tube radius Tube length Fluid velocity Fluid inlet temperature Fluid outlet temperature Mean electrical field

 C   K/n/Ea K/n/Ea K/n/Ea L m hcl R L v Tin Tout — E

1000 4120 0.6 5.6  104 0.001/1/7.6 0.5/1/26 14/1/30 0.5 0.1 1000 0.025 0.75 0.05 20 45 2000—3000

kg/m3 J/kg/K W/m/K /K Pa sn/-/kJ/mol Pa sn/-/kJ/mol Pa sn/-/kJ/mol S/m /K W/m2/K m m m/s °C °C V/m

Modelling 483

5.2 Treatment of solid/liquid mixtures The application of ohmic heating to solid-liquid food mixtures gives rise to a more complex model. The solid particles in the mixture are generally large (approximately 25 mm) and broadly diverse in shape, density, surface roughness and compressibility. Therefore, their presence induces marked hydrodynamic disturbances. Very high solid concentrations are usual, which increases inter-particle and particle-wall interactions. These effects also apply to the electrical field if electrical conductivity of the solid is different from that of the liquid. Direct influences on the thermal fields of the components also arise from the liquid-particle, particle-wall and liquid-wall exchanges and by the coupling imposed by the dependency of physical property on temperature. Many authors (Sastry, 1992a; Quarini, 1995) assume that the mixture’s behaviour at very high solid concentrations is very similar to that of plug flow (Figure 18.6). This assumption is confirmed by the results of Muller et al. (1994), Quarini (1995) and Muller et al. (1993) indicating that the liquid profile flattening should increase by a higher volume fraction of the particles. This effect is moreover confirmed by Palmieri et al. (1992) and Sandeep and Zuritz (1995). Experiments conducted by Lareo (1995) and Liu et al. (1993) on such mixture flows in ducts showed, by residence time analysis, a slip between the solid and the liquid phases. However, none of these studies allows the evaluation or the calculation of this slip velocity in terms of the operating parameters as such calculation involves heat transfers between the dispersed and carrying phases and the particle Reynolds number being different from zero. The impact of cubic particle orientation on the effective electric resistance and on the heating rate of a suspension has been studied by Sastry and Palaniappan (1992a) and the first approach to modelling such systems in a static form was achieved by Sastry and Palaniappan (1992b). Only a weak influence of particle orientation on the electric field at high concentration was observed. Zhang and Fryer (1993), in their

U  U0 zL

T 0 z

D L 0zL 0rR

z vL

vP z0

J  Jc vP for particles vL for liquid

U0 T  Tin

Figure 18.6 Thermal and electrical conditions for modelling a collinear ohmic heater and flowing solid/liquid mixture.

484 Ohmic Heating

numerical study of spherical particle heating organized in two different spatial systems, found a strong distortion of the internal generation, which exhibited a dependency on the conductivity ratio of the two phases and on the solid concentration. Sastry (1992a) devised a model for the heating of cubic particles at high volume fraction (0.8) with constant electrical field and heat transfer coefficients. He also showed that particles with the same conductivity as the liquid (and even less conductive) were heated faster than this liquid. Nevertheless, a short particle residence time increases the probability of incomplete sterilization. Another model has been adopted by Zaror et al. (1993) of a mixture of uniformly sized spherical particles in plug flow with equal phase velocities and constant material physical properties. The heat generation was held constant and independent of the spatial position for both liquids and solids. The simulations were executed taking into consideration the Fourier and Reynolds numbers and no reference was made to the particle-liquid convection coefficient. In their simulation of an analogous suspension sterilization, Zhang and Fryer (1994) found a clear influence of the particles and the electrical conductivity variation on the mixture heating kinetics. Unfortunately, the assumption of a negligible particle temperature gradient in the heating section leaves their sterilization time course unclarified: the hypothesis of a cold point located in the centre of the particle may be invalid and needs verification. Mankad et al. (1995) developed a model, based on the slip velocity observation, describing a wall-heating sterilization system for different mixture concentrations. The problem was considered as a plug flow with heat transfer inside the particles and between the two phases. They concluded that the dispersed and carrying phase residence times needed for the sterilization were very sensitive to the slip velocity and thermal properties. The same approach was developed for a twolayer model by Mankad and Fryer (1997), in which two different slip velocities and two different local concentrations were induced by mass conservation. Two sterilization and cook values were imposed on the heated wall and the holding sections. The impact on the process duration was also studied. The slip velocity parameter affects the thermal behaviour of the mixture during ohmic sterilization through (i) the convective heat transfers involved between the two phases, (ii) the conductive heat transfers inside the particles, (iii) the sensitivity of electrical field to the presence of particles, (iv) the influence of delivered solid concentration influence and (v) the particle/tube diameter ratio. 5.2.1 Electrical heat generation

The evolution of the volumetric weighted mean heat generation in the two phases is expressed as a function of the mean electrical generation in the liquid without particles G0, the local particle/liquid conductivity ratio and the local solid volume fraction. This expression is obtained with the help of the heat generation ratio functions RGL and RGP as: GL  RGLG0

and

GP  RGPG0

(22)

5.2.2 Mass conservation

A mass balance approach to mixture flow has been developed by Mankad et al. (1995). They were motivated by the observation of residence time differences between the

Modelling 485

solid and liquid phases (Liu et al., 1993; Lareo, 1995). These authors (Liu et al., 1993; Lareo, 1995) attributed these residence time differences to a mean velocity difference in term of slip velocity. The assumption of plug flow will be made, according to Palmieri et al. (1992) and Sandeep and Zuritz (1995), who concluded that the higher the concentration, the smaller the particle residence time standard deviation. Applying the mass conservation law to the studied mixture, the relation between  P) and liquid mass flow rates (m  L) can  m), the particle (m the mixture mass flow rate (m    be obtained as mP ⫹ mL  mm. The corresponding relations between the velocity of each phase v*P  vP/vm and v*L  vL/vm, the mass flow rates and the delivered (del) and actual (act) volume fractions are thus given by Mankad et al. (1995) as: 1 * * 2 *   1 v slip (1 v slip ) 4delv slip  2

(23)

1 *  * * 2  (1 v slip ) (1 v slip )  4(1 del )v slip  2

(24)

vP*  vL* 

act 

(

1 * 2v slip

)

 (v *  1) (1 v * )2 4 v *   slip slip del slip  

(25)

where v*slip  v*L  v*P is the dimensionless slip velocity and v*slip  vslip/vm. 5.2.3 Heat transfer

5.2.3.1 Reference temperature In the case of single phase flow of the liquid at constant electrical conductivity in, mean residence time ␶ and electrical potential U applied on the length L, the increase in liquid temperature
Tref  Tout  Tin, with Tin the liquid inlet temperature and Tout the reference outlet temperature, can be determined by:
Tref 

inE20 t (C)L

and

E0  U/L

(26)

5.2.3.2 Energy equation Applying the energy conservation equation to a liquid control volume bounded by the tube wall and the particles in a steady state, for a particle of diameter DP, the following equation can be obtained: vL

TL    2TL   

SL ;  C  z 2 z L

TP        C  t P

 1  T   2 P    r 2 r  r r  SP  

(27)

486 Ohmic Heating

where ,  and C are respectively the thermal conductivity, density and specific heat of the media. The source term S represents the heat exchanges with the external media and includes the local internal heat generation which can be obtained by applying an energy balance to each of the phase control volumes. SL  (1  act)GL + ahP(TP  TL);

SP  GP

(28)

5.2.3.3 Liquid-solid heat transfer coefficient The study of Mankad et al. (1997) shows that the relation of Ranz and Marshall (1952) correlates well with available experimental results for ReP  100, with NuP, Pr and ReP being respectively the particle Nusselt number, the Prandtl number and the particle Reynolds number; which is given below: 1

1

NuP  2 0.6Pr 3 ReP 2

(29)

5.2.4 Effects of parameters

Table 18.4 gives two case studies A and B. All the lengths (particle, tube) are dimensionless with reference to the tube diameter. The following dimensionless temperature difference between the particles and the liquid is considered to measure the quality performance of the ohmic treatment.
TPL 

TP  TL
Tref

(30)

with two considerations:
TPL(DP/2) for the particle surface temperature and
TPL(0) for the particle centre temperature. 5.2.4.1 Slip velocity effect These results on the effect of slip velocity are based on a delivery volume fraction of 30 per cent (v/v), a particle/tube diameter ratio of 0.175 and the same conductivity function for each phase (case A). Figure 18.7(a) shows the enhancement of the dimensionless temperature difference between the particles and the liquid. Good formulations of the mixture for the electrical and rheological parameters prevent particle/liquid slip and allow the same electrical conductivity: (i) the difference with particle/liquid temperatures is minimal and (ii) the heat transfer coefficient is not dominant. For a small particle the temperature is practically uniform (Nu ⬃ 2 and Bi  0.1). The cold point is the liquid, which is easily monitored with a temperature sensor. If the slip velocity is high (gravity effect, low liquid viscosity), the difference in temperature is considerable between particle centre, particle surface and liquid. The particle residence time is higher than that of the liquid. The particle temperature is always higher than the liquid temperature. This temperature heterogeneity leads to quality loss. The difference decreases when the heater length is more than 15 times the pipe diameter.

Modelling 487

Table 18.4 Characteristic values used for the case study of the model Property

Symbol

Value

Units

Density Specific heat Thermal conductivity Viscosity Particle electrical conductivity at 25°C, temperature coefficient Liquid electrical conductivity at 25°C, temperature coefficient Mixture mass flow rate Delivered concentration Mixture inlet temperature Reference temperature difference Mean electrical field

␳ C   P, m

m del Tm
T — ref E

1000 4184 0.645 0.025 Case A: 0.6386, 0.01 Case B: 0.3308, 0.03 0.6386 0.01 0.0255 10—55 15.0 106.6 1047—1234

kg/m3 J/kg/K W/m/K Pa s S/m, K1 S/m, K1 S/m K1 kg/s % °C °C V/m

Dimensionless parameters Particle diameter Tube length Slip velocity

D*p L* v*slip

0.088—0.526 70.175 0—0.50

— —

L, m

Particle-liquid temperature difference

0.08 0.07

v *slip

0.06

0%, 1%, 25%, 50%,

: surface, : surface, : surface, : surface,

: centre. : centre. : centre. : centre.

0.05 0.04 0.03 0.02 0.01 0.00

0

10

(a)

20 30 40 50 Dimensionless tube length (z*)

60

70

Particle-liquid temperature difference

0.08 1%, 10%, 30%, 55%,

φdel 0.06

: surface, : surface, : surface, : surface,

: centre. : centre. : centre. : centre.

0.04

0.02

0.00 0

(b)

10

20 30 40 50 Dimensionless tube length (z*)

60

70

Figure 18.7 Particle-liquid temperature difference ⌬TPL. (a) slip velocity effect, (b) delivery volume fraction effect.

488 Ohmic Heating

5.2.4.2 Volume fraction effect The increase in delivery volume fraction has a similar effect on the temperature time course as shown in Figure 18.7(b). While the core solid is always hotter than the liquid, the temperature difference between the two phases decreases with an increasing solid volume fraction. The temperature difference decreases when the heater length is more than 25 times the pipe diameter but always exceeds 1.5 per cent. 5.2.4.3 Particle diameter effect The particle diameter is the parameter affecting the temperature time course. The temperature gradient inside the particle increases with decreasing particle diameter as shown in Figure 18.7(c). Even for a long heater, the particle/liquid temperature difference is high. 5.2.4.4 Electrical conductivity effect Case B corresponds to large variations between solid and liquid electrical conductivity (L  2P). The particle/liquid temperature difference may be negative. For a tube

Particle-liquid temperature difference

0.24

0.088, 0.175, 0.351, 0.526,

D*P

0.20

: surface, : surface, : surface, : surface,

: centre. : centre. : centre. : centre.

0.16 0.12 0.08 0.04 0.00

0

10

(c)

20 30 40 50 Dimensionless tube length (z*)

60

70

Particle-liquid temperature difference

0.04

 0.03

0.02

0.01

: surface,

: centre.

: surface,

: centre.

0.00

0.01

(d)

Case A, Case B,

0

10

20 30 40 50 Dimensionless tube length (z*)

60

70

Figure 18.7 (continued) (c) diameter ratio effect, (d) electrical conductivity effect – case B. Reference parameters: del  30%, D*p  0.175, v*slip  25% and case A.

Treatment of products 489

length the particle temperature gradient is zero. The liquid is hotter for a short tube heater. If the tube heater is long the conductivity effect is less marked.

6 Treatment of products 6.1 Product suitability – formulation and pre-treatment Ohmic heating is particularly suitable for viscous products and foods containing particulates because the heat is generated inside the food product. The critical property influencing the rate of ohmic heating is the electrical conductivity, which depends on a number of factors including temperature, ionic constituents, material microstructure and field strength (Sastry, 1992b; Wang and Sastry, 1993). As most solid vegetable particles have lower electrical conductivities than liquids (Zoltai and Swearingen, 1996), particular care must be taken in the design of formulations to eliminate temperature changes. One way to obtain solid/liquid heating homogeneity is to use a pilot-scale batch ohmic heater (Goullieux et al., 1997) and to seek the optimum salt concentration within the liquid phase, thereby minimizing the average difference in heating rate between solid and liquid. Figure 18.8 shows the determination of the optimum liquid phase composition and the batch simulation of the processing of a meal with potato cubes. The closer the match between the electrical conductivities of the different phases, the more even the current flow will be; this still may not lead to uniform heating because the thermal capacities of the phases may be different and the temperature dependence of the thermal conductivities may also differ (Fryer and Davies, 2001). Thus food treatment by ohmic heating often needs a pre-treatment in aqueous NaCl solution (Wang and Sastry, 1993; Eliot et al., 1999a) to increase the electrical conductivity of the solid phase. Also, high temperature short time processing of vegetables requires a blanching pre-treatment to degas and inactivate enzymes. It is well 160

(a)

Holding

140 0.12 Optimum concentration

0.08 0.04 0 0 1

2

3 4

5 6 7

Temperature (°C)

Average difference of heating rate between solid and liquid (°C/s)

0.16

8 9 10 11 12

Cooling

100 80 60 40

Liquid phase Potato cubes

20

0.04

0

Salt concentration in liquid phase (g/l) 0.08

Heating

120

0 (b)

500

1000

1500

Time (s)

Figure 18.8 Ohmic heating optimization of a meal with potato cubes: (a) determination of the optimum liquid phase composition, (b) batch simulation of the processing of the cooked meal.

Cutting work (%)

200 150 100 50 0 fresh

55

60

95

Pre-treatment temperature (°C) (a) Potato cubes

Forcemax of compression (%)

490 Ohmic Heating

120 100 80 60 40 20 0 fresh

40

50

95

Pre-treatment temperature (°C) (b) Cauliflower florets

Figure 18.9 Textural quality of potato cubes and cauliflower florets after pre-treatment in salted water and ohmic heating. Each textural value is expressed as a percentage of the same value measured with fresh product. 䊏 after pre-treatment, 䊐 after ohmic heating.

known that fruit and vegetable texture is profoundly altered by thermal processing (Paulus and Saguy, 1980; Rodrigo et al., 1997); firmness decreases as temperature increases. However, a low-temperature long-duration pre-treatment can improve the final texture of treated vegetables (Hoogzand and Doesburg, 1961; Aguilar et al., 1997). The mechanism claimed to explain the firming effect is the demethoxylation of pectic materials of the cell wall catalysed by the pectin methylesterase. Thus a combination of low temperature long-duration pre-treatment and salted water offers an opportunity to improve the electrical conductivity and the final quality of the product after ohmic heating processing, even for fragile products. However, little is published in this particular field of research. Eliot et al. (1999b), Eliot and Goullieux (2000) have evaluated and optimized low temperature long-duration pre-treatment before ohmic heating in a batch unit with potato, a vegetable commonly used in prepared meals, and cauliflower, a brittle product that does not resist conventional thermal treatments. When a pre-treatment in salted water was carried out by immersion of Bintje cultivar potato cubes (30 min at 40 and 50°C, [NaCl]  80 g/kg) and of Brassica oleracea cultivar cauliflower florets (60 min at 60°C – 22.5 g NaCl/kg and 55°C – 28 g NaCl/kg), as shown in Figure 18.9, the intermediate quality, evaluated by mechanical firmness measurements, exhibited:

• in the case of potato cubes, a significant loss compared with fresh product after conventional blanching at 95°C, but a better firmness after pre-treatment at 60°C and 55°C (the improvement reached up to 90 per cent for 55°C versus fresh). • in the case of cauliflower florets, a significant loss was observed compared with fresh product after conventional blanching and a low fall in firmness after pre-treatment at 50°C and 40°C. After pre-treatment and cooling, potato cubes and cauliflower florets were placed in an ohmic heating pilot plant successively reproducing the heating, holding and cooling stages of an ohmic heating process (Figure 18.8b). Heating was obtained by a steady electric field (between 1 and 30 V/cm) from a 50 Hz AC 5 kW power set connected to a rototransformer. The volume of the batch was made up to 1 l with a NaCl

Treatment of products 491

solution (0.5 g/kg for the cubes, 5 g/kg for the florets) to maintain the homogeneity of the electrical conductivity of the vegetable mixture/NaCl solution (0.40–0.45 S/m) and to apply ohmic treatments with similar heating rates. The mixture was then heated to 135°C and held at this temperature for 30 s. After ohmic heating, the final quality was again evaluated by mechanical firmness measurements. Potato cubes pre-treated at 60°C exhibited higher firmness, although there was no significant difference from pre-treatment at 55°C. The textural quality of cubes pre-treated at low temperature was better than those of cubes pre-treated at 95°C. As illustrated in Figure 18.9(a), an increase of 77.6 per cent and 100 per cent was observed for the cutting work, respectively at 60°C and 55°C. The cutting work decreased between the first thermal treatment and the ohmic heating. The most important drop was measured for samples pre-treated at 55°C and 60°C, with a value of the parameter reduced by a factor of 4–5, when they had undergone an increase in firmness during pre-treatment. The value of this parameter was only halved for blanching at 95°C. However, compared with fresh product, cubes pre-treated at low temperature and ohmically heated held 35–40 per cent of their initial firmness versus 20 per cent for conventional blanching. The textural quality of florets pre-treated at low temperatures was better than that of those pre-treated at 95°C (Figure 18.9b). The highest compression force was obtained with the pre-treatment at 40°C, resulting in a firmness gain of 230 per cent, compared with conventionally blanched samples. As in the potato case, the textural parameter decreased between the first thermal treatment and the ohmic heating. The greatest drop was again measured for samples pre-treated at 90°C, with a value of the parameter reduced by a factor of 4, against 2–2.5 in other cases. Compared with the fresh product, florets pre-treated at low temperature and ohmically heated held 30–35 per cent of their initial firmness versus 10 per cent for conventional blanching. These results show the importance of pre-treatment before ohmic heating. When potato cubes and cauliflower florets have undergone a low temperature pre-treatment, their residual firmness after ohmic heating is multiplied by 2 to 3 compared with the firmness of samples blanched for 5 minutes at 95°C. This type of pre-treatment is therefore better than conventional blanching for obtaining high quality products by ohmic heating. To examine the feasibility of sterilizing cauliflower continuously, experiments were conducted in a 10 kW APV continuous ohmic heating pilot plant (Eliot-Godéreaux et al., 2001b). Cauliflower florets were variously pre-treated as described above, mixed with starch solutions (carrier fluids prepared to obtain various electrical conductivities) and processed with different flow rates and holding times. After aseptic packaging, the floret texture was evaluated by compression testing and compared with fresh product. Several aseptic bags were incubated at 25°C, 37°C and 55°C for one week to conduct a microbiological examination, as affected by pH, changes in external package, product appearance and microbial flora morphology. Low temperature pretreatment, high flow rate and adequate adjustment of the electrical conductivity of the two phases seem to be optimal conditions. Attractive appearance and valuable firmness properties and stabilities (at 25°C and 37°C) were obtained for cauliflower.

492 Ohmic Heating

The above reported results highlight the importance of food pre-treatment before ohmic heating. A well-conducted pre-treatment minimizes texture loss and adjustment of electrical conductivity to obtain homogeneous heating rates between each phase of the mixture.

6.2 Thermal treatments 6.2.1 Stabilization

As stated above, ohmic heating is a thermal process that has quite naturally found a place among unit operations involving heat transfer. Its ability to generate the heat directly inside products allows the temperature of solid food to be raised rapidly, unlike the conventional thermal processes involving a conductive and so slower transfer. In addition, after an appropriate pre-treatment, it is possible to control the speed of heating of the various essential phases of a food mixture to ensure the homogeneity of the ohmic treatment. In the food industry, ohmic heating was first applied to the HTST stabilization of food containing pieces. As an aseptic process, it first has to ensure the microbiological quality of products. It was thus important to conduct research to validate and optimize its action on microorganisms. 6.2.1.1 Quantification of lethal effect Quantifying the effect of thermal treatment on the microbiological or sensorial attributes of food products is a key to process optimization. The kinetics of thermal destruction of target microorganisms or components (e.g. vitamins and enzymes) are usually used to quantify the lethal effect of conventional aseptic processes. Kim et al. (1996a) investigated lethality within food particles undergoing ohmic heating using an alginate bead/meatball system containing spores of Bacillus stearothermophilus. However, the destruction of microorganisms at high temperatures occurs so quickly that the residual count is often below the detection level inherent in the method. Consequently, only the minimum sterilizing value received by the product was obtained. Tucker (2000) and Tucker et al. (2002) used an -amylase time-temperature integrator (TTI) in silicone particles to estimate the pasteurization value achieved at the centre of a moving fruit piece in an industrial ohmic heater. Owing to low electrical conductivity of the silicone insulating the amylases from the electrical heating effects, the authors (Tucker, 2000; Tucker et al., 2002) obtained conservative pasteurization values. The method could not be used for process optimization but it should be useful for confirmation of a minimum pasteurization value. The decimal reduction time of these amylases at sterilization temperatures was insufficient for it to be used as a marker for sterilization processes. Kim et al. (1995) have suggested using the formation of a target molecule as a time-temperature integrator to quantify the effect of thermal processing. The formation of M-1 (2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one) in food products as the product of non-enzymatic browning between reducing sugar and amine was demonstrated during thermal processing. This chemical marker formation was used to evaluate the distribution of lethality among different particles together

Treatment of products 493

with the contribution of various holding sections in two ohmic heating installations. Correlations between the chemical marker yield and bacterial destruction have been obtained at both 118°C (Kim et al., 1996b) and 121°C (Kim et al., 1996a). EliotGodéreaux et al. (2003) have used the basic idea developed by Kim et al. (1996a, b), but with a simple glucose/serine system placed in small glass flasks. The kinetic modelling of the formation of the marker showed that it could be used as a TTI to quantify the thermal treatment imposed upon food products in HTST processing. However, the sterilization values obtained were conservative because the glass did not conduct electricity and so the TTI escaped the electrical heating and was heated only by conduction and convection. The foregoing shows that research is still necessary to devise a TTI that undergoes real ohmic heating and thus gives a more accurate estimation of ohmic heating intensity. 6.2.1.2 Mechanisms of microbial lethality The aim of pasteurization and sterilization is to use heat to kill microorganisms. Several studies (Palaniappan and Sastry, 1990; Parrott, 1992) have been carried out to investigate the microbial lethality. A summary of the kinetic data available (Sastry and Barach, 2000) shows that the ohmic D, z and activation energy value kinetics are similar to conventional ones. There are exceptions for rate constants, which may be enhanced by the electric field. More specifically, Palaniappan and Sastry (1992) subjected yeast cells to both conventional and ohmic heating under identical temperature histories and found no difference in kill rate. Hence they concluded that if there is any microbial mortality due to electricity, it should be small compared with that produced by heat. However, an ohmic pre-treatment on Escherichia coli cells before conventional heating resulted in an increase in bacterial inactivation under certain conditions. Hence the electrical action had to be elucidated. By studying growth kinetics of Lactobacillus acidophilus under ohmic heating, Cho et al. (1996) showed that the bacteriocin production was delayed and its activity was lower than that of conventional heating methods. Spores are the resistant form of microorganisms and they can usually be destroyed by severe thermal treatment. Ohmic heating of suspensions of Bacillus subtilis spores resulted in a higher lethality and a greater Tyndallization effect than conventional heating (Cho et al., 1999). The authors (Cho et al., 1999) postulated that the electrical treatment injured spores and thereby made them more vulnerable to the associated ohmic heating. Imai et al. (1995) investigated the effect of AC frequency on plant tissue and found that the electroporation of the cell membrane was caused by the induced membrane potential. The samples heated more rapidly at a constant voltage of 40 V/cm with a low frequency (50 Hz) and a proton nuclear magnetic resonance image analysis (1H-NMR) showed that the cell membrane was damaged, increasing its permeability. Thus under the given conditions, ohmic heating of white radish led to electroporation. This effect was also reported by Yoon et al. (2002) on yeast cells of Saccharomyces cerevisae. The leakage of cellular materials increased significantly with electric field strength (10–20 V/cm). It was concluded that ohmic heating induced irreversible electroporation of cell membranes. Based on this electric breakdown of cell membranes, Uemura and Isobe (2002, 2003) developed an apparatus for inactivating Escherichia

494 Ohmic Heating

coli and Bacillus subtilis spores, respectively, in saline water and orange juice with a high alternating current electric field (20 kHz, 7–17 kV/cm). 6.2.2 Cooking

The ability of ohmic heating to rapidly generate heat inside products can be used to accelerate cooking. The fast food sector usually cooks patties frozen to prevent bacterial contamination during thawing. The long time needed to reach 70°C at the centre is due to the high latent heat of ice melting. Consequently, this cooking method entails high power consumption, alters organoleptic properties and does not guarantee the required kill level for all pathogenic microorganisms. Özkan et al. (2004) reported a new method of hamburger patty cooking combining ohmic and plate heating. At 50 V AC, the required cooking was reduced by around 30 per cent, while the taste and texture were not affected. The authors (Özkan et al., 2004) concluded that this combined ohmic and plate heating was very successful in reducing cooking time and producing safer products. 6.2.3 Thawing

Thawing is a critical operation for the microbial quality of a foodstuff. It must be carried out rapidly at low temperature. The conventional process is to place the frozen product in circulating water (temperature below 20°C), thereby reducing moisture loss. However, there are several disadvantages: possible microbial growth on the product surface, reduction of nutrient quality due to leaching of soluble proteins, high consumption of fresh water and consequently large quantities of loaded wastewater. Dielectric or microwave thawing avoids these problems, but the product surface may be cooked before its centre is thawed. Liquid water absorbs microwaves more rapidly than ice and so the peripheral front of thawing prevents microwave penetration in the non-thawed central region. An intermittent exposure, allowing temperature stabilization by thermal conduction, is usually applied. Ohmic heating is another alternative to thaw frozen food blocks, but its adoption is hindered by hot spots. Electrical conductivity increases with temperature and is approximately two orders of magnitude lower for frozen food than for thawed food. Thus the thawed portion of a block can cook while the rest is still frozen. Roberts et al. (1998) developed an ohmic thawing unit with surface temperature sensing and computer-automated capabilities that can thaw shrimp blocks without runaway heating problems. The ability of liquid-contact thawing of frozen meat by ohmic heating was reported by Naveh et al. (1983) and Wang et al. (2002). The latter (Wang et al., 2002) used brine as a carrying fluid and found that the thawing process was faster when the brine concentration increased and the largest surface of the sample was perpendicular to the electrical field. No significant changes in sample colour, pH or brine electrical conductivity were observed after ohmic thawing. Therefore ohmic thawing is a rapid method worth developing in the future. 6.2.4 Blanching

Blanching is an important operation in food processing. According to what the next step is, it has different functions: enzymatic or microbial destruction, improved

Treatment of products 495

rehydration, removal of gas inside tissues, correction of bad taste or cloudiness, removal of reducing sugars, etc. Blanching is conventionally effected in a water bath and results in a loss of soluble solids, which considerably increases the BOD (biological oxygen demand) of the plant wastewater. Mizrahi et al. (1975) were the first to study the feasibility of uniform heating by electroconductive blanching. Application of an AC field of 0.2–20 V/mm at a frequency of 50–60 Hz in a water bath containing sliced potatoes reduced deep-frying time by 10 to 50 per cent, improved colour throughout the chips, reduced blistering and gave a crispier texture after deep-frying (Vigerstrom, 1976). However, the leaching of water-soluble substances was increased. In hot water or ohmic treatment, the extent of solute loss is practically linearly proportional to the surface, to the volume ratio of the product and to the square root of the processing time (Mizrahi, 1996). A reduction of one order of magnitude in solute losses could be achieved and the process time shortened if large vegetables, without dicing, were immersed in a saline solution and subjected to ohmic heating. Cousin et al. (2001) showed that the application of a high direct electric field on whole potato tubers resulted in clean cutting due to tissue softening. Also, an ohmic pre-treatment without a liquid medium may result in a decreased oil uptake during frying of potato slices (Salengke and Sastry, 2001).

6.2.5 Evaporation

Water has to be removed to concentrate a wide range of products. Evaporation is the most popular method used but it may entail quality loss. Owing to poor heat transfer of an increasingly viscous product, process time is long and organoleptic and nutritional properties are adversely affected. Ohmic heating is an alternative method that has been studied for vacuum evaporation of orange juice by Wang and Chu (2003), in which they showed that vacuum evaporation by ohmic heating could evaporate more moisture than a conventional process in the same time. Moreover, the final product was brighter and kept more aroma. Application of ohmic heating for pasteurization, HTST sterilization, cooking, thawing, blanching and evaporation of food products mainly uses its thermal contribution. However, the non-thermal contribution of ohmic heating also has wide applications as discussed in the following sections.

6.3 Pre-treatment on mass-transfer operations The improvement of mass transfer is important in separation processes such as extraction, expression or diffusion to improve yield and product quality. Product size reduction and/or pre-heating are the pre-treatments usually used to enhance mass transfer. Several studies (Mizrahi, 1996; Wang and Sastry, 2002) of ohmic heating have shown an increase in leaching of soluble solids due to reversible or irreversible electroporation of cell membranes. It was therefore useful to examine the effects of an ohmic pre-treatment on mass transfer operations.

496 Ohmic Heating

6.3.1 Diffusion and extraction

In the early years of ohmic heating application, experiments were carried out to compare the taste and texture of electrically- and conventionally-processed food. Particular attention was paid to nutrient or flavour diffusion during heating. Schreier et al. (1993) showed that the mass transport increased linearly with the applied electric field strength and was proportional to the surface area of the sample. Lima and Sastry (1999) showed that lowering the frequency of alternating current significantly improved apple juice extraction yields. Wang and Sastry (2002) also observed a strong improvement in apple juice yield by ohmic pre-treatment. Moreover, ohmic heating involved a lower pressing energy than microwave heating. The authors (Wang and Sastry, 2002) postulated that under low frequency treatment, cell membranes may undergo electroporation and thermal denaturing. These two mechanisms may also be operational during the extraction of mint from fresh mint leaves (Sensoy and Sastry, 2001; Sensoy, 2002). In the case of lipid extraction from rice bran, Lakkakula et al. (2004) observed that the improvement of extraction depended on frequency and electric field strength but not on temperature. Hence ohmic pre-treatment is a valid method for heat-labile components. Food processes involving mass transfer can be enhanced by choosing conditions in which the conductivity of a sample under ohmic treatment is maximized (Lima et al., 2001) and the threshold potential for permeabilization is minimized by a low frequency (Kulshrestha and Sastry, 2003).

6.3.2 Dehydration

The influence of ohmic heating on mass transfer can also be useful to enhance water removal. The hot-air drying rate can be significantly increased during most of the drying process by low frequency and high electric field strength (Lima and Sastry, 1999). In addition, Wang and Sastry (2000) have observed a shift of sorption isotherms for ohmic pre-treated samples, indicating a permeabilization of the structure and a redistribution of water within the vegetables. The drying processes are time and energy intensive and so ohmic heating has been studied as a possible accelerating pretreatment. Zhong and Lima (2003) obtained a 24 per cent decrease in vacuum drying time with minimal ohmic heating (50 V/cm, 45°C) but at final moisture level no difference from that of raw sweet potatoes. In this case, the mild electroporation known to occur during ohmic heating was not evident.

6.4 On-line treatment validation Among many studies on ohmic heating, most of them have been conducted on batch units or on model foods (de Alwis et al., 1989; Halden et al., 1990; Palaniappan and Sastry, 1991; Schreier et al., 1993; Marcotte et al., 1998; Eliot and Goullieux, 2000). To be useful, this process had to be validated in real industrial conditions, i.e. continuous industrial scale unit, aseptic packaging and meals with complex formulation. These conditions were used in the following work.

Treatment of products 497

Ohmic processing was evaluated in 1992 by a consortium of 25 partners. A wide variety of shelf-stable low- and high-acid products (broccoli in cheese sauce, shrimp gumbo, strawberries in glaze, chicken and pasta) were developed on a 5 kW unit. Texture, colour, flavour and nutrient retention were comparable to or exceeded that of traditional processing (Zoltai and Swearingen, 1996). The US Army Natick Research Development & Engineering Center validated on-line the technology for self-stable military rations using a 25 kW ohmic system. The results show that the overall product quality was enhanced; the sensory score was 7.3 on a 1–9 hedonic scale where 7 was ‘good’ and 8 ‘very good’ (Kim et al., 1996a). Also, using a commercial-size APV 45 kW unit, Yang et al. (1997) examined the microbiological safety and sensory quality of six ohmically heated stew type foods (sauce, meat, soup, vegetables and dishes) initially and after 3 years’ storage at 27°C. The commercial sterility was verified before and after the 3-year storage. Following the storage, the sensory evaluation indicated a significant darkening of sauce colour, but no significant change in sauce consistency, flavour intensity or texture. A significant decrease in overall quality was noted for three products due to colour darkening or an off-spice component, but the others showed very good quality retention. Zuber et al. (2000) showed that the stabilization of a soup containing potato particles was conclusive on a 10 kW APV installation. They obtained a final product of pleasant texture and high viscosity. Using this same unit, Eliot-Godéreaux et al. (2001b) worked on stabilizing a meal containing cauliflowers florets. These two studies (Zuber et al., 2000; Eliot-Godéreaux et al., 2001b) highlight the pre-treatments required to adapt the product to this technology.

6.5 Other aspects The modelling of ohmic treatment of solid/liquid mixture shows the effect of slip velocity, solid fraction and particle and tube size. The flow behaviour of food mixtures becomes a critical factor as (i) the residence time distribution of particles and liquid, (ii) the consequences for the outlet temperature of liquid and solid and (iii) the product quality. The prediction of particle passage times is required to ensure sterility, cooking and optimized product quality. It is important to be able to predict minimum and maximum passage times of particles. More detailed reviews and discussion of recent work and implications for thermal treatment can be found elsewhere (Lareo et al., 1997; Fairhurst and Pain, 1999). A positron emission particle tracking (PEPT) technique, Hall effect sensors and visual tracers were used to determine the particle trajectories and passage times in non-Newtonian viscous solutions on experimental loop and ohmic heating pilot plants (Fairhurst et al., 2001; Eliot-Godéreaux et al., 2001a). The industrial development of a food process is only possible if the product’s organoleptic and sanitary quality is ensured. Ohmic heating, like any HTST aseptic process, requires the calculation of sterilization conditions at the cold spots within the food product during heating. Several studies (Kim et al., 1996a, b; Tucker, 2000; Tucker et al., 2002) carried out to quantify microorganism destruction, used TTI and

498 Ohmic Heating

demonstrated ohmic heating efficiency. However, these approaches were global and did not permit an accurate understanding and control of the process. Thus it is necessary to design methods to determine spatial and temporal temperature distribution within mixtures. Ye et al. (2003) used magnetic resonance imaging (MRI) temperature thermometry, a non-destructive, non-invasive and high spatial resolution technique, to examine local temperature distribution during ohmic heating. Experiments were carried out in a static ohmic heater on whey gel and NaCl solutions and on potato cylinders in carboxymethylcellulose (CMC) solutions (Ye et al., 2004). A near plug flow of foods with high solid content and a modified heating section were the reasons justifying the use of the static apparatus. The data acquisition time for one image was 0.64 s and the spatial and temporal resolution of the temperature maps were respectively 0.94–1.25 mm and 0.64 s, with a proton resonance frequency (PRF) shift method into a FLASH (fast low angle shot) sequence. The MRI temperature maps obtained provided accurate temporal and spatial distribution for ohmic model development and validation. This method therefore has a high potential for the validation of product organoleptic and sanitary quality and for process and product design.

7 Conclusions Ohmic heating is of growing interest for the treatment of viscous products and products containing large particles within a liquid phase. Internal heat generation, volumetric heating, higher temperature in particles than in liquid and reduced fouling are the main advantages of ohmic heating, which make it possible to apply an HTST process to solid/liquid food mixtures. Thus it is possible to obtain safe ready meals with a high retention of nutrients and vitamins. Depending on the product to be treated, the choice of the configuration is the first to consider in using ohmic heating. Current ohmic heating modelling is very rewarding because the flow behaviour of the products and the invaluable assistance of the control of treatment homogeneity can be better identified. The models can show the influence of the inhomogeneous electrical conductivities on the heating history of the products and their strong impact on the final temperature. On liquid products, modelling also shows that volumetric heaters must be equipped with wall cooling, owing to the low velocities and therefore long heating times near the wall, in order to reduce fouling sufficiently. Furthermore, the modelling indicates that free convection effect increases at the bottom of heater but decreases at the top. In addition, studies carried out to investigate lethality mechanisms during ohmic heating highlighted the electroporation effect, which leads to mass transfer. Consequently, ohmic heating is not only a very useful thermal process in food stabilization, but also a pre-treatment to prepare vegetable tissues before a mass transfer operation (e.g. diffusion, extraction or dehydration). Further work is needed to take into account the mixture formulation (electrical conductivity, rheological properties and particle size) and the changes in the flow parameters and electrical behaviour. Research has so far demonstrated the need for further

Nomenclature 499

understanding of the flows of solid/liquid mixtures of large particles with high concentrations. Adequate techniques of velocity measurement in these opaque media are necessary. A good fit between experimentation and modelling should enhance our knowledge of this technology at full scale and help us to develop an optimization strategy for the treatments by associating the reactions with the state changes encountered.

Nomenclature a,b,c,d,e A C D E g G h J K L m . m n p P r R S t T U u V v– v z

constant (Table 18.1) area (m2) specific heat capacity (J/kg/K) diameter (m) electric field strength (V/m) gravitational acceleration (m/s2) heat generation (W/m3) heat transfer coefficient (W/m2/K) current density (A/m2) consistency index (Pa sn) tube length (m) proportionality constant (/K) mass flow (kg/s) flow behaviour index () absolute pressure (Pa) power generated (W/m3) radial coordinate (m) radius of column (m) thermal source (W/m3) time (s) temperature (K) electrical potential (V) radial velocity (m/s) volume (m3) velocity vector axial velocity (m/s) axial coordinate (m)

Greek symbols  ,  ⌬    

coefficient (/s) coefficient (/m) expansion coefficient (/k) small element, difference thermal conductivity (W/m/K) viscosity (Pa s) resistance () volume fraction (%)

500 Ohmic Heating

␳  ␶

density (kg/m3) electrical conductivity (S/m) residence time (s)

Dimensionless numbers Bi Biot Nu Nusselt Pr Prandtl Re Reynolds Subscripts act c cl del G in L m o out P PL ref R slip

local centre, constant cooling delivery internal generation input liquid mixture zero solid concentration output particle particle-liquid reference ratio slip velocity

Superscripts *

dimensionless variable

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Reznik D (1996) Ohmic heating of fluid foods: various parameters affect the performance of ohmic heating devices used to heat fluid food products. Food Technology, 5, 251–260. Reznik D (1997) Electroheating methods. US Patent No 5863580. Roberts IL (1900) Receptacle for sterilised perishable substance. US Patent No 645 569. Roberts JS, Baladan MO, Zimmerman R, Luzuriaga D (1998) Design and testing of a prototype ohmic thawing unit. Computers and Electronics in Agriculture, 19, 211–222. Robinson RA, Stokes RH (1959) Electrolyte solutions. London: Butterworths. Rodrigo C, Rodrigo M, Fiszman S, Sanchez T (1997) Thermal degradation of green asparagus texture. Journal of Food Proteins, 60, 315–320. Salengke S, Sastry SK (2001) Effect of ohmic pre treatment on oil uptake of potato slices during frying and subsequent cooling. In 2001 IFT Annual Meeting Book of Abstracts, Paper 15D-37. Chicago: Institute of Food Technologists. Sandeep KP, Zuritz CA (1995) Residence times of multiple particles in non-Newtonian holding tube flow: effect of process parameters and development of dimensionless correlations. Journal of Food Engineering, 25, 31–44. Sastry SK (1992a) A model for heating of liquid-particle mixtures in a continuous flow Ohmic heater. Journal of Food Process Engineering, 15 (4), 263–278. Sastry SK (1992b) Advances in ohmic heating for sterilisation of liquid particle mixtures. In Advances in Food Engineering (Singh RP, Wirakartakusumah MA, eds). Boca Raton: CRC Press, pp. 139–147. Sastry SK, Barach JT (2000) Ohmic and inductive heating. Journal of Food Science Supplement, 65 (4), 42–46. Sastry SK, Palaniappan S (1992a) Influence of particle orientation on the effective electrical resistance and ohmic heating rate of a liquid-particle mixture. Journal of Process Engineering, 15 (3), 213–227. Sastry SK, Palaniappan S (1992b) Mathematical modeling and experimental studies on ohmic heating of liquid-particle mixtures in a static heater. Journal of Food Process Engineering, 15 (4), 241–261. Schreier PJR, Reid DG, Fryer PJ (1993) Enhanced diffusion during the electrical heating of foods. International Journal of Food Science and Technology, 28, 249–260. Sensoy I (2002) Extraction from leafy materials using moderate electric fields: effects of field strength and frequency. In 2002 IFT Annual Meeting Book of Abstracts, Paper 91C-9. Chicago: Institute of Food Technologists. Sensoy I, Sastry SK (2001) Extraction using moderate electric fields. In 2001 IFT Annual Meeting Book of Abstracts, Paper 15D-38. Chicago: Institute of Food Technologists. Skudder PJ, Biss CH (1987) Aseptic processing of food products using ohmic heating. The Chemical Engineer, 433, 26–28. Stirling R (1987) Ohmic heating – a new process for the food industry. Power Engineering Journal, 6, 365–371. Tucker G (2000) Estimation of pasteurisation values using an enzymic time-temperature integrator. Food Australia, 52 (4), 131–136. Tucker G, Lambourne T, Adams JB, Lach A (2002) Application of a biochemical timetemperature integrator to estimate pasteurisation values in continuous food processes. Innovative Food Science & Emerging Technologies, 3, 165–174. Uemura K, Isobe S (2002) Developing a new apparatus for inactivating Escherichia coli in saline water with high electric field AC. Journal of Food Engineering, 53, 203–207.

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Uemura K, Isobe S (2003) Developing a new apparatus for inactivating Bacillus subtilis spore in orange juice with a high electric field AC under pressurized conditions. Journal of Food Engineering, 56, 325–329. Vigerstrom KB (1976) Passing an electric current of 50–60 cps through potato pieces during blanching. US Patent No 3 997 678. Wang WC, Sastry SK (1993) Salt diffusion into vegetable tissues as a pretreatment for ohmic heating: electrical conductivity profiles and vacuum infusion studies. Journal of Food Engineering, 20, 299–309. Wang WC, Sastry SK (1997) Starch gelatinization in ohmic heating. Journal of Food Engineering, 34, 225–242. Wang WC, Sastry SK (2000) Effects of thermal and electrothermal pretreatment on hot air drying rate of vegetable tissue. Journal of Food Process Engineering, 23, 299–319. Wang WC, Sastry SK (2002) Effects of moderate electrothermal treatments on juice yield from cellular tissue. Innovative Food Science & Emerging Technologies, 3, 371–377. Wang WC, Chen JI, Hua HH (2002) Study of liquid-contact by ohmic heating. In 2002 IFT Annual Meeting Book of Abstracts, Paper 91F-4. Chicago: Institute of Food Technologists. Wang WC, Chu CY (2003) Study of vacuum evaporation by using ohmic heating. In 2003 IFT Annual Meeting Book of Abstracts, Paper 92B-59. Chicago: Institute of Food Technologists. Yang TCS, Cohen JS, Kluter RA et al. (1997) Microbiological and sensory evaluation of six ohmically heated stew type foods. Journal of Food Quality, 20, 303–313. Ye X, Ruan R, Chen P et al. (2003) Accurate and fast temperature mapping during ohmic heating using proton resonance frequency shift MRI thermometry. Journal of Food Engineering, 59, 143–150. Ye X, Ruan R, Chen P, Doona C (2004) Simulation and verification of ohmic heating in static heater using MRI temperature mapping. Lebensmittel-Wissenchaft und Technology, 37, 49–58. Yoon SW, Lee CYJ, Kim KM, Lee CH (2002) Leakage of cellular materials from Saccharomyces cerevisae by ohmic heating. Journal of Microbiology and Biotechnology, 12 (2), 183–188. Zaror CA, Pyle DL, Molnar G (1993) Mathematical modelling of an ohmic heater steriliser. Journal of Food Engineering, 19 (1), 33–53. Zhang L, Fryer PJ (1993) Models for the electrical heating of solid-liquid food mixtures. Chemical Engineering Science, 48 (4), 633–642. Zhang L, Fryer PJ (1994) Food sterilization by electrical heating: sensitivity to process parameters. AIChE Journal, 40 (5), 888–898. Zhong T, Lima M (2003) The effect of ohmic heating on vacuum drying rate of sweet potato tissue. Bioresource Technology, 87, 215–220. Zoltai P, Swearingen P (1996) Product development considerations for ohmic processing. Food Technology, 50, 263–266. Zuber F, Schietequatte F, Da Silva F (2000) Application du chauffage ohmique à la stabilisation de potages avec morceaux. Informations Techniques n°162, Centre Technique de la Conservation des Produits Agricoles (eds), ISSN 0249 7085, France.

Combined Microwave Vacuum-drying Christine H Scaman and Timothy D Durance University of British Columbia, Food, Nutrition, and Health, Vancouver, Canada

Microwave vacuum-drying is a rapid and efficient dehydration method that can yield unique characteristics and improved quality compared to conventionally dried products. The electromagnetic microwave energy penetrates into the interior of the food, where it is converted to thermal energy, providing a rapid heating mechanism. Vacuum reduces the boiling point of water keeping the product temperature low, as well as creating a pressure gradient that enhances the drying rate. Load size, power level and vacuum pressure influence drying rate. Drying can typically occur in minutes, compared to hours or days for air- and freeze-drying. In situ vaporization of water provides an expansive force to maintain an open cellular structure in the dried product, which translates into excellent rehydration rates. For products consumed in the dry state, a unique crisp texture can be achieved. Because the sample temperatures are kept relatively low during the dehydration process and the dehydration occurs quickly at low oxygen pressure, food components sensitive to oxidation and thermal degradation can be retained to a higher degree than by air-drying. Operating costs and energy costs of microwave vacuum are expected to be somewhat more expensive than those of natural gas fuelled convection hot air dryers. The microwave vacuum driers use electricity rather than the less expensive natural gas but the microwave vacuum is also more energy efficient than convection dryers, especially towards the end of the drying cycle. Optimum product quality and cost efficiency has been demonstrated to occur for several products with a combination of air- and microwave vacuum-drying. Microwave vacuum processing, with its unique characteristics, has also been utilized for tempering and thawing, inactivation or preservation of enzymes and microorganisms, pharmaceutical processing and histochemical processing of samples.

1 Introduction The potential of microwave energy combined with a vacuum environment for rapid low temperature dehydration and high quality products has long been recognized. Microwave vacuum-drying is a dehydration process that uses microwave radiation for heat generation in the absolute pressure (chamber pressure) range from above the triple point of water to less than atmospheric pressure (0.61–101.33 kPa). The earliest related applications involved using a combination of radio-frequency energy and vacuum. Potatoes and cabbage were reported to be rapidly dehydrated using this technique Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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(Rushton et al., 1945) and penicillin was dried using radio-frequency energy (28 MHz) in combination with a vacuum to prevent the thermal degradation of the antibiotic (Brown et al., 1947). While the radiation used in these applications was below microwave frequency, these examples embody the concept of converting electromagnetic energy to heat inside a material rather than relying on conduction and convection for heat transfer and of using vacuum to maintain a low product temperature, resulting in improved product quality. Drying rates by any technology are determined by the relative rates and interactions between energy transfer into the food and mass transfer of the water out of the food and into the surrounding environment. Microwaves provide the fastest means available of transferring energy into the interior of biological solids. Energy transfer is rate limiting for freeze dehydration, in which energy must diffuse into the food by conduction in order to provide the latent heat of sublimation of water. As conduction in most freeze-dryers is from the shelves of the dryer through the food, in the later stages of the process heat must travel through an insulating layer of dry food. In hot-air convection dryers, water evaporates from the surface of the food and energy transfer to the surface is usually efficient. However, mass transfer of liquid water from the centre of the food piece to its surface is slow and rate limiting. In microwave drying, most water is evaporated in situ within the food and diffuses to the surface as a vapour. Once the boiling point of the solution in the food is reached, positive steam pressure quickly develops within the food which forces vapour to the surface. The available microwave power and the dielectric properties of the food, which define the efficiency of conversion of microwaves to heat, primarily determine drying rates of microwave processes. Thus microwave dehydration can proceed very rapidly. Unfortunately, drying by absorption of microwave energy at ambient pressure occurs at too high a temperature for most food products because the food rapidly heats to the boiling point of water. By combining microwave with vacuum one can retain the speed of the microwave heating but avoid the high temperatures. The impact of pressure on the boiling point of water, and by inference, on vacuum microwave-drying temperature is illustrated in Figure 19.1. While Figure 19.1 represents the boiling point of

Boiling point of pure water (ºC)

100

75

50

25

0 0

Figure 19.1

25

50 Pressure (kPa)

75

Relationship between pressure and the boiling point of water.

100

Microwaves 509

pure water, aqueous solutions in foods will have somewhat higher boiling points. Drouzas and Schubert (1996) reported that the temperature of banana slices during microwave vacuum-drying was always greater than the boiling point of water at the chamber pressure, an effect attributed to the presence of dissolved solids in the water that causes an increase in the boiling point.

2 Microwaves Microwaves are electromagnetic radiation with wavelengths from 1 mm to 1 m in length, with frequencies from about 300 MHz to 300 GHz. As with all electromagnetic radiation, microwaves have electric and magnetic components that propagate through space at right angles to each other. They can be transmitted, reflected, or absorbed by materials that they encounter. The energy of the radiation, E, is directly related to the frequency,
, the number of oscillations per second, by Planck’s constant, h  6.63  1034 J/s: E  h


(1)

The frequency,
, is inversely related to the wavelength, , by:


c l

(2)

where c is the velocity of the radiation. The principal frequencies of microwaves used for industrial and domestic heating in North America are 915 and 2450 MHz. Microwaves of household ovens and many industrial applications are produced efficiently by continuous wave magnetrons. A magnetron is a vacuum diode, in which the cathode is surrounded by a coaxial anode (Love, 1995). The anode has an even number of vanes that extend in toward the cathode (Figure 19.2). The open areas between each of the vanes are resonant cavities that determine the output frequency of the magnetron. Alternating segments are opposite in polarity and are connected with conducting straps. A direct current with a high potential difference runs through the cathode causing the filament to heat up and emit electrons. The electrons are deflected by a magnetic field, formed by permanent magnets outside of the vacuum tube, into a circular path between the anode and cathode. As the electrons travel around the cathode, they form a cloud with rotating ‘spokes’ that touch every alternate vane (Figure 19.3). This induces an alternating current in the resonance cavities at microwave frequencies. An antennae located in a resonant cavity transfers the radiofrequency energy into a waveguide, which then transmits the microwaves into the oven cavity. Microwaves do not have intrinsic heat, but rather, the interaction of the rapidly oscillating electronic field with the food material allows the microwave energy to be converted to thermal energy. Both charged/ionic and polar/dipolar material are affected by the electronic field of the microwaves. Ions are linearly accelerated in the direction of the electronic field while polar components rotate to align their dipole with the

510 Combined Microwave Vacuum-drying

Resonance cavity

Anode

Vanes

Cathode

Figure 19.2

Strapping

Simplified diagram of a continuous wave magnetron.

Antenna ⫺



⫺



 Resonance cavity

⫺

⫺ Electron cloud 

 ⫺



⫺

Figure 19.3 Cross-section of a magnetron. Rotating spokes of the electron cloud induce an alternating current in the resonance cavities at microwave frequencies.

electronic field. Since the microwave field oscillates millions to billions of times per second in an arbitrary direction in the oven cavity, the particles are accelerated or rotated in a random pattern, generating heat. The heat is distributed in the food by conduction. Water, because of its permanent dipole and abundance, is often the main component of a food that is affected by the microwave field. The physical state of water has a significant effect on its response to microwaves. Free water exhibits a hydrogenbonded network predominantly to other water molecules and is able to relax at microwave frequencies. The relaxation time is the time for a molecule to reach a disordered state after the microwave field that it was aligned with is removed (Mudgett, 1985a). However, the dipole rotation of water bound to proteins or carbohydrates, or present as ice, is hindered (Hasted, 1961), reducing the frequency at which it relaxes to

Dielectric properties of food 511

below the microwave range. Therefore water in these states will have a negligible contribution to microwave heating.

3 Dielectric properties of food The permittivity, or the dielectric properties of foods describe the ability of the material to store or dissipate energy from the electromagnetic radiation (Mudgett, 1985a). The dielectric properties determine the reflection of radiation at the material surface and the transmission and absorption of radiation by the material. The complex permittivity or complex dielectric constant, , is defined as (von Hippel, 1954):     j

(3)

where  is the permittivity or dielectric constant and the ()0.5 is equivalent to the microwave index of refraction,  is the dielectric loss factor, which is a measure of the microwave absorptivity of the material, and j is an imaginary portion used for calculation of the sinusoidal behaviour of electromagnetic waves. The dielectric properties of a material will vary with temperature and moisture content. The temperature dependence of the dielectric constants and loss factors of some foods (Bengtsson and Risman, 1971) and the dielectric properties at 2450 MHz of some select food materials including water are available (Buffler, 1993; Yaghmaee and Durance, 2003). Generally, as the product temperature is increased from the frozen state to 0°C, the dielectric constant and loss factor increase rapidly. Then, above freezing, the dielectric constant and loss factor decrease with increasing temperature. However, as the salt concentration increases, ionic effects can compensate for this decrease and, for a sample with a high salt concentration such as ham, the dielectric constant increases from 0°C to 60°C. The dielectric constants of foods tend to decrease as the moisture content decreases. Under conditions of microwave heating, the loss factor for several fruits and vegetables was shown to reach a slight but significant maximum value at intermediate moisture level (2.0–0.8 kg H2O/kg dry matter, dry basis) while the dielectric constant decreased with decreasing moisture content in a relatively linear manner (Funebo, 2000). This maximum of  at an intermediate moisture content has also been reported by Favreau et al. (1997) and Mudgett et al. (1980), who suggested that it was due to the concentration of salts. Funebo (2000) hypothesized that the shift in the relaxation frequency towards the microwave radiation frequency was responsible for the observed maximum in  , as the maximum amount of microwave energy is converted to heat at the relaxation frequency. Other studies of the dielectric properties at different moisture contents have noted a direct relationship between moisture content and  (Sun et al., 1995; Tulasidas et al., 1995; Lian et al., 1997). As with other forms of radiation, microwaves can be transmitted, reflected, or absorbed by material that they interact with. As a microwave is directed onto the surface of a material at a right angle, the portion of the energy reflected from the surface is dependent mainly on , the dielectric constant. As  increases, so does the proportion

512 Combined Microwave Vacuum-drying

of reflected energy. The energy that is not reflected from the surface is transmitted into the material where it continues its propagation through the material, losing energy as it is absorbed (Mudgett, 1985b). Materials with a high loss factor absorb a high proportion of microwave power per unit of material. These materials may not heat or dry evenly using microwave energy, as the microwaves may not be able to penetrate to the interior. Penetration depth is defined as the depth at which power available has decreased to 1/e, or 36.8 per cent of its original power (Buffler, 1993). Penetration depth will vary inversely with microwave frequency; at the same moisture, penetration is approximately 2.7 times higher at 915 MHz compared to 2450 MHz (Mudgett, 1985b). Therefore, at lower frequencies, thicker materials can be heated with greater uniformity. For microwaves that strike the surface of a material at an angle, a portion can be reflected at the same angle. The remaining energy will be transmitted into the material at an angle inversely related to the ()0.5. If some energy penetrates through the material to the opposite side, a portion of that energy will be both reflected internally and transmitted out of the material. This internal reflection can be partly responsible for the uneven heating characteristic of microwave processing, as these internal waves can create hot spots in regions where microwave reflections are additive.

4 Thermal properties of food While the dielectric constants determine the absorption of microwave power, the thermal properties of thermal conductivity and heat capacity and physical properties of density and viscosity determine how a food will heat once microwave energy has been absorbed. The heat capacity and density are inversely related to the rate of temperature rise in a food exposed to microwave energy. Viscosity of the material will determine how easily heat will flow by convection through the product. The thermal diffusivity, , is defined as



k Cp

(4)

where k is the thermal conductivity (W/m K), Cp is the heat capacity (J/kg K) and  is density (kg/m3). The thermal diffusivity of most foods is similar, however, ranging from 0.9  107 to 11.7  107 m2/s, while the dielectric constants and loss factors can vary by a factor of 34 to 20 000, respectively (Buffler, 1993). Therefore, different food materials can exhibit relatively large differences in the absorption of microwave energy and conversion of that energy to heat, compared to the differences in how the material will heat once the microwave energy has been absorbed.

5 Characteristics of microwave vacuum-drying There are three major aspects of a dehydration process that can be used to compare different techniques: the rate of dehydration, the quality and characteristics of the

Characteristics of microwave vacuum-drying 513

final product, and the economic and energy costs of the process. The limitations of conventional dehydration technologies, such as air- or freeze-drying, in these areas can be addressed by microwave vacuum-drying.

5.1 Drying rate The combination of vacuum and microwave allows solid food pieces to be dried more rapidly than with any other method, while maintaining the product temperature relatively low during most of the process. A comparison of the drying curves of replicate batches of 3 mm thick carrot slices treated by three drying technologies illustrates the rapid dehydration rate that can be achieved using microwave vacuum-drying (Figure 19.4). Freeze-drying required approximately 3 days to reach the end point of 9.9 per cent moisture (dry basis), air-drying required 8 hours, while microwave vacuumdrying required 33 minutes. Drying time becomes a controllable variable in microwave vacuum-drying, determined predominantly by the ratio of microwave power to the amount of water to be evaporated. Therefore, to increase the rate of drying, either the load size can be reduced or the microwave power can be increased. For example, drying avocado, mushroom and strawberry pieces at high power (850 W) versus low power (425 W) resulted in a faster drying rate and also higher rehydration rates for the dried product (Pappas et al., 1999). Limitations are imposed only by the microwave power density (W/kg) within the chamber and the limits of microwave penetration into the load of wet food material. Excessively high power density may cause microwave arcing or plasma discharge in the vacuum chamber, particularly if the load of microwave absorbing material in the chamber is small. In practical terms, power densities greater than 10 000 W/kg frequently leads to arcing in moderate vacuum systems where absolute pressure is 4–10 kPa. The rapid rate of microwave vacuum dehydration can be attributed to effects on each of the three distinct drying rate regions that characterize the process. First, there

Moisture (% dry basis)

1200

Moisture (% dry basis)

1000 800

1200 800 400 0 0

0.1

0.2

0.3

Time (h)

600 400

AD FD MVD

200 0

0

20

40

60

Time (h) Figure 19.4 Dehydration curves of air-dried (AD), microwave vacuum dried (MVD) and freeze-dried (FD) carrot slices. Inset: Expanded dehydration curve of microwave vacuum-dried carrot slices.

514 Combined Microwave Vacuum-drying

is a short increasing rate region, where the temperature of the product reaches the boiling point of the water when it is first exposed to microwave energy. This temperature will depend on the pressure of the system. Compared to hot air-drying, this increasing rate region occurs more quickly with microwave vacuum-drying due to the efficient energy transfer of microwaves. Second is the constant rate region, where free water is driven off at a constant rate. The temperature of the product is relatively stable during this period, as the latent heat of vaporization of water maintains the temperature at the boiling point. This constant rate period may extend to lower moisture levels than occurs with air-drying, reducing the drying time. In Figure 19.4, the drying rate of the air-dried carrots decreased as the process proceeded while the slope of the microwave vacuum curve changed only very slightly. In another study, the drying rate of chillies under microwave vacuum conditions was reported to be constant until about 0.75 per cent moisture, dry basis (Kaensup et al., 2002). Because of this extended constant rate region, Kim and Bhowmik (1995) suggested that microwave vacuum-drying was simpler to use to obtain isothermal drying curves of materials than conventional drying methods. Product temperature could be easily maintained within ⫾1°C, after a short initial heating phase. The isothermal drying data could then be used to estimate diffusivity values, used for modelling dehydration or other mass transfer processes. Third is the falling rate period, where the rate of moisture loss slows as more tightly bound water is removed. This falling rate period can be the most time-consuming region of conventional air-drying processes. It has been estimated that two-thirds of the time in conventional drying may be dedicated to removing the last one-third of moisture (Schiffman, 1987). Microwave vacuum-drying can provide a significant advantage in this falling rate period for two reasons: (1) this region may be reduced, as more moisture is lost in the constant rate period; and (2) the in situ evaporation of water that occurs in microwave vacuum-drying is much more rapid than diffusion of liquid water that occurs during air-drying. In addition to microwave power and load size, chamber pressure can affect the rate of drying. For dehydration to occur, it is necessary to supply energy to raise the product temperature to the boiling point of water and to provide the latent heat of water vaporization. As pressure is decreased, the latent heat of vaporization increases slightly. This contributes to a slight decrease in the rate of drying at the same microwave power. However, the lower the pressure in the drying chamber, the greater the driving force for water vapour diffusion from the product (Wei et al., 1985). Therefore, moisture may be removed more rapidly at a lower operating pressure. The relative magnitude of these two opposing effects may depend on the configuration of the equipment used for the dehydration, as well as the characteristics of the sample. Cui et al. (2004a) showed a slight positive relationship between chamber pressure and dehydration rates of thin carrot slices using equipment where the product was rotated but not thoroughly mixed. Under these conditions, the driving force of the pressure differential between the inside of the chips and the chamber may be less important than the influence of drying temperature which was increased with lower chamber pressure. However, there are many reports that document an inverse relationship between chamber pressure and drying rate (Wadsworth et al., 1990; Kiranoudis et al., 1997; Drouzas et al., 1999;

Characteristics of microwave vacuum-drying 515

Lin et al., 1999; Pappas et al., 1999), suggesting that the pressure differential between the water vapour in the tissue and the drying chamber is the dominant effect. Kiranoudis et al. (1997) modelled the effects of microwave power (425–850 kW) and chamber pressure (2–7 kPa) on the drying kinetics of three different fruits (apple, kiwi, pear), in equipment where the product was static. Their results showed that microwave power had a direct relationship with the drying rate, while chamber pressure had a relatively minor and slightly negative effect. During dehydration of parboiled rice, the effect of pressure on drying rate was more pronounced at lower power levels (Wadsworth et al., 1990). This was interpreted as an indication that the adsorption/desorption rate of water on the surface of the sample was more important in determining the overall drying rate, rather than the diffusion rate of water to the surface. Drouzas et al. (1999) found that the drying rate constant increased significantly with decreasing pressure between 5 and 3 kPa and increasing microwave power from 640 to 710 watts. However, it was noted that the equipment and operating costs associated with the higher vacuum may not be justified for all products.

5.2 Quality attributes of microwave vacuum-dried products 5.2.1 Rehydration potential

Many dried foods are rehydrated before consumption and therefore rehydration behaviour is a critical functional property of these products. Although the rehydration ideal differs from product to product, in many instances, a rapid rate and complete hydration are desirable. Freeze-drying is generally acknowledged to allow the greatest rehydration potential of any food drying technique, both in terms of the rate and the amount of water uptake (Barbosa-Canovas and Vega-Mercado, 1996). Because water is removed by sublimation while tissues are at a low temperature, the original cellular structure is largely retained. However, careful design of the microwave vacuum process can often lead to products that rehydrate very nearly as fast as freezedried (Figure 19.5). Barriers to water uptake in air-dried products include collapsed cellular structure within the tissue and case hardening, the surface layer of precipitated tissue solutes left behind when water evaporates from the surface. Microwave vacuum-dried products generally exhibit rehydration properties intermediate to those of freeze- and air-dried products (Drouzas and Schubert, 1996; Lin et al., 1998; Pappas et al., 1999). This is ascribed to two, or possibly three, mechanisms: puffing, reduced case hardening and low drying temperatures. During microwave vacuum-processing, water vapour pressure inside the tissue is substantially higher than chamber pressure, resulting in a force that opposes the tendency of the tissue to collapse as water is removed. This force can be controlled to some extent during the microwave vacuum process as it is a function of evaporation rate and the chamber pressure. Evaporation rate is, in turn, a function of microwave power density (W/kg). Chamber pressure is controlled by the vacuum system. Timing of the processing steps can also influence puffing. If the dry tissue is allowed to cool and become rigid before the vacuum is released, the expanded structure may be largely retained and a puffed dry tissue is produced. Periodic fluctuations in chamber

516 Combined Microwave Vacuum-drying

10

Rehydration ratio

8

6

4 AD FD MVD

2

0 0

30

60

90

120

150

180

190

Rehydration time (minutes) Figure 19.5 Rehydration curves of air (AD), microwave vacuum (MVD) and freeze-dried (FD) carrot slices at 25°C.

pressure and/or microwave power during the process can also improve product expansion (Durance and Liu, 1996). When chamber pressure is increased, the temperature of liquid water is also increased in the drying tissue because the boiling temperature is influenced. When chamber pressure is abruptly reduced, the liquid water becomes super-heated, is quickly evaporated and produces vapour pressure inside the tissue. If a microwave vacuum product of higher density is required, the microwave vacuum chamber may be pressurized while the product is still hot, in which case the internal structure may collapse. Puffing can be demonstrated by product density changes or by microscopic structural studies (Yousif et al., 2000; Sham et al., 2001). As illustrated in Figure 19.6, scanning electron micrographs of microwave vacuum dried plant tissues are similar to those of freeze-dried tissues, with the original open structure of the tissue clearly visible. By contrast, air-dried samples are dense and collapsed. Microwave vacuum dehydration eliminates or greatly reduces case hardening, which is recognized as a barrier to rehydration. Because water is believed to be largely evaporated in situ within the tissue and to diffuse out of the food as vapour, solute migration to the surface is minimal. Finally, rehydration of some tissues, such as potato, is known to be hindered by drying temperatures above approximately 60°C (Talburt and Smith, 1967). The low temperatures of microwave vacuum-drying, often less than 45°C, may contribute to better rehydration of those products. 5.2.2 Texture modification

Microwave vacuum-drying can be used to create a desirable crisp texture in foods that are consumed in the dehydrated form, such as snack foods or crackers. This contrasts with the spongy or tough textures that characterize freeze- and air-dried materials.

Characteristics of microwave vacuum-drying 517

a

b

c

d

e

f

Figure 19.6 Scanning electron micrographs of (a) air-dried apple; (b) microwave vacuum-dried apple; (c) freeze-dried apple; (d) air-dried potato; (e) microwave vacuum-dried potato; and (f ) freeze-dried potato.

One of the first reports of a microwave vacuum-process creating a crisp texture involved potato and apple pieces, air-dried to approximately 45 per cent and 25 per cent moisture (wet basis), respectively. These were then heated to 99°C using microwaves and the pressure in the oven was rapidly reduced to 6.7 kPa to create a product that was described as being porous and crisp (Huxsoll and Morgan, 1968). More recently, microwave vacuum processing was used to produce a crisp potato chip product (Durance and Liu, 1996). A crisp texture is the result of a tissue that consists of cells or cavities that are filled with air, surrounded by a brittle structural phase (Vickers and Bourne, 1976). Factors that maximize the in situ vaporization of water result in increased crispiness; for example, lower chamber pressure during dehydration yielded greater puffing and crisper apple chip texture (Sham et al., 2001). As well, the physicochemical characteristics of the sample also affect textural parameters. Lefort et al. (2003) determined that potatoes with low specific gravity and low starch content produced chips with a desirable texture that required less breaking force. 5.2.3 Retention of chemical components

Flavour, colour, nutrient, or other biologically active chemicals that are sensitive to thermal or oxidative degradation typically exhibit better retention after microwave vacuum-drying compared to air-drying (Table 19.1). Several studies with herbs have demonstrated enhanced retention of flavour volatiles. Microwave vacuum-dried sweet basil (Ocimum basilicum L.) retained 1.5 times more methylchavicol and 2.5 times more linalool, compared to samples air-dried at 48°C (Yousif et al., 1999). The colour and rehydration rate of the microwave vacuum-dried samples were superior to the air-dried product. Similarly, thymol, a key character impact compound in oregano

518 Combined Microwave Vacuum-drying

Table 19.1 Retention of labile chemical components Reference

Bohm et al. (2002) Cui et al. (2003) Cui et al. (2004b)

Durance et al. (2000) Kim et al. (2000a) Kim et al. (2000b) Kwok et al. (2004) Lin et al. (1998)

Source material

Parsley Garlic Carrot slices blanched Carrot slices unblanched Chinese chive St John’s wort Echinacea purpurea flowers E. purpurea flowers Saskatoon berries, Thiessen variety Carrot slices

Mui et al. (2002) Vaghri (2000)

Banana chips Blueberries, Hardy blue variety

Yousif et al. (1999)

Sweet basil

Yousif et al. (2000)

Oregano

Chemical component

Essential oils Pyruvatec Total carotenes Total carotenes Total chlorophyll Hypericin Chicoric acid Caftaric acid Alkamides Anthocyanins Phenolics Ascorbic acid

 carotenes Flavour volatiles Anthocyanins Phenolics Ascorbic acid Linalool Methylchavicol Thymol

Retention Air-dried

Microwave vacuum-dried

30% 54% 86% 71% 38% 35a 254a 61a 285a 50a 640a 38% 81% 4.0d 198a 2150a NDb 62d 65d 23d

93% 89% 95% 96% 97% 45a 1120a 176a 307a 149a 890a 79% 97% 6.4d,e 498a 3350a 7.5a 157d 96d 30d

a

mg/100 g dry weight; b ND  not detectable; c enzymatically generated pyruvate as a measure of pungency; d relative peak area; e 90% air dried  10% microwave vacuum dried.

(Lippia berlandieri Schauer) was significantly higher in microwave vacuum dried samples than in air-dried products and similar to fresh and freeze-dried samples (Yousif et al., 2000). However, four other volatiles ( -myrcene, -terpinene, -terpinene and -cymene) showed no difference in retention between air-drying and microwave vacuumdrying. In another study, parsley dried by air or pulsed microwave vacuum-drying was evaluated for colour, essential oil content and aroma by a sensory panel (Bohm et al., 2002). The microwave vacuum dried product was rated as having better colour and aroma than the air-dried and the essential oil content was approximately 94 per cent of the fresh content, while air-dried retained only 30 per cent. The low drying temperature may enhance flavour volatiles retention, although the vacuum will increase the vapour pressure of these components as well as that of the water. The preferential retention of volatiles may be attributed to a selective diffusion mechanism (Thijssen, 1971), where the diffusion coefficient of the volatiles is decreased to a greater extent than that of water during concentration. Therefore, although volatiles may evaporate within the food, they may not diffuse quickly enough to escape the tissue before drying is complete. Two volatiles in onion, 2-methyl2-pentenal and 1-propenyl Pr disulphide, were found to be lost quickly during the first 20 minutes of microwave vacuum-drying of onions at 600 W and 13.3 kPa chamber pressure (Chen and Chiu, 1999). Each compound then approached a different equilibrium retention in the last stages of drying.

Characteristics of microwave vacuum-drying 519

Retention of volatiles in banana chips was evaluated using a combination of airdrying and microwave vacuum-drying (Mui et al., 2002). Chips were first air-dried to remove 60, 70, 80 or 90 per cent moisture (wet basis) and then subjected to microwave vacuum-drying to 3 per cent moisture (dry basis). Samples that underwent more airdrying and less microwave vacuum-drying, had higher levels of volatile compounds. This was attributed to the increased formation of an impermeable solute layer on the surface of the chips that may have reduced the volatile loss. However, banana chips that were exclusively air-dried had volatile levels significantly lower than the 90 per cent air-dried/microwave vacuum-drying samples, attributed to volatile loss during the relatively long drying time. A study on Saskatoon berries showed that berries dried using microwave vacuum processing exhibited higher total anthocyanin and phenolic levels, which were associated with a greater antioxidant activity, compared to air-dried berries (Kwok et al., 2004). Similarly, microwave vacuum-dried blueberries retained higher amounts of total phenolics and ascorbic acid, compared to air-dried berries (Vaghri, 2000; Durance et al., 2001). Ascorbic acid can be easily oxidized during drying of foods due to enzymatic and chemical processes. Therefore, in addition to being an essential vitamin in human nutrition, it can be used as an indicator for the severity of a thermal process. Lin et al. (1998) observed that ascorbic acid content of carrot pieces dehydrated using microwave vacuum processing, was significantly higher than for air-dried pieces. Echinacea purpurea flowers were microwave vacuum dried at 6.7 kPa with nitrogen gas flushed into the drying chamber (Kim et al., 2000a). The retention of caftaric and chicoric acid, caffeic acid derivatives with potential pharmacological activity, were found to be higher in the microwave vacuum dried samples, compared to samples airdried at 70°C. Although comparable levels of the acids were found when samples were air-dried at 40°C, this process required 55 h and resulted in enzymatic browning of the flowers. Similarly, microwave vacuum-drying of roots of Echinacea purpurea resulted in better retention of alkamides, compared to air-drying, although freeze-drying gave the best retention (Kim et al., 2000b). However, in leaves, surprisingly, air-drying at 50°C was superior to both freeze-drying and microwave vacuum-drying. During dehydration of St John’s wort (Hypericum perforatum L), retention of hypericin was significantly improved by microwave vacuum-drying, compared to air-drying (Durance et al., 2000). Biologically active components present in foods may benefit from the low temperatures, fast processing rates and low oxygen pressure of microwave vacuum-drying. As links between enhanced health effects and consumption of specific food components are confirmed with scientific studies, the market potential for functional foods will continue to grow (Gray et al., 2003). Therefore, the enhanced retention of functional components in food materials as a result of microwave vacuum-drying will be a significant advantage to food manufacturers.

5.3 Dehydration costs The cost of a dehydration process is a function of capital costs, labour costs, energy costs and energy efficiency. Although few data are available on large-scale microwave

520 Combined Microwave Vacuum-drying

vacuum-drying operations, operating costs and energy costs of microwave vacuum are expected to be slightly more expensive than natural gas fuelled convection hot air-dryers. Our experience suggests operating and maintenance cost of a 100 kW microwave vacuum system to be about $12 Canadian per hour in Western Canada. Such a system would remove about 130 kg of water per hour. The microwave vacuum requires electricity rather than the less expensive natural gas but the microwave vacuum is also more energy efficient than convection dryers for food, especially towards the end of the drying cycle. The most efficient strategy appears to be hybrid hot-air/ microwave vacuum-drying in which free and lightly bound water is removed by airdrying while the final portion of more tightly bound water is removed by microwave vacuum (Owusu-Ansah, 1991). Durance and Wang (2002) compared energy consumption per kilogram of water evaporated between a pilot scale natural gas convection air and a 20 kW vacuum microwave dehydrator. Tomatoes, with a moisture content of 93 per cent wet basis were dried using each dryer separately and in various combinations. The energy consumed per kilogram water evaporated was lower for the microwave vacuum process, compared to air-drying. However, when costs of natural gas and electricity were considered, the least expensive method of dehydration was a combination method, where 70 per cent of the initial moisture was removed using air-drying and the remaining moisture was removed via microwave vacuum-drying. Ultimately, the most costeffective process will be determined by the efficiencies of the specific air- and microwave vacuum-dryer designs, as well as the natural gas and electricity costs at the drying site. Reducing the microwave power density and decreasing the chamber pressure have been used to improve the thermal and drying efficiencies of microwave vacuumdrying. Using intermittent or pulsed microwave power to dry cranberries increased the energy utilization factor, defined as total energy absorbed/total energy input, and the drying efficiency, defined as the amount of moisture evaporated per unit of energy input, compared to continuous drying (Yongsawatdigul and Gunasekaran, 1996; Gunasekaran, 1999). It should be noted, however, that these studies were carried out in equipment where the product was static. Development of product ‘hot spots’ due to the non-uniform distribution of microwave energy is well documented in this type of equipment. A smaller effect of microwave power on drying efficiency might be expected in equipment where effective mixing of the product was in place to ensure more even drying of the product. As well, the time required for dehydration increases at lower power and the impact of lower power density on product quality must be considered. Factors such as rehydration rate, the extent of rehydration and puffing are generally improved when products are microwave vacuum dried at higher power densities. Chamber pressure has a negative relationship with the efficiency of microwave vacuum-drying. Mousa and Farid (2002) demonstrated that the thermal and drying efficiency was significantly higher at 30 kPa than at atmospheric pressure, due to the enhanced mass transfer at the lower pressure. At lower pressure, the pressure differential between the interior of the food where water is vaporized and the drying chamber increases, providing a greater driving force for moisture migration from the food. This effect was found to be more pronounced as moisture levels decreased.

Combination of microwave vacuum with other processes 521

There is little information available on the capital costs of microwave vacuum-dryers. There are only a few manufacturers of industrial or pilot plant scale equipment and such equipment is expensive compared to air dryers. Prices vary from approximately $5–12 US per watt of microwave heating capacity, compared to $0.50–1 US per watt of air-drying capacity. Operating costs of freeze-dryers are clearly greater than either air- or microwave vacuum-dryers. In freeze-drying, there is the added energy cost associated with prefreezing of the food and as well as with sublimating water from the frozen state and re-condensing the water vapour upstream from the vacuum pump. Also, industrial freeze-drying is almost always a batch system. Air and microwave vacuum systems can be either batch or continuous, a distinction that substantially reduces labour costs.

6 Combination of microwave vacuum with other processes Microwave vacuum processing has been used in combination with other processes to improve both the cost and energy efficiency of the dehydration and the quality of the final product. The throughput and product quality of air dryers in existing processing operations can be improved by combining air and microwave vacuum-drying. For example, if the carrot slices shown in Figure 19.4 were removed from the air dryer after 4 hours, they could be finished dried to 9 per cent moisture in a matter of minutes using a relatively small microwave vacuum-dryer. The entire drying process would be completed in approximately 4 hours of air-drying time and five minutes of additional microwave vacuum processing, rather than 8 hours. This would effectively double the production capacity of the air dryer. In addition, product quality would be improved. It is well known that product temperature reaches a maximum during the final stages of airdrying, when evaporative cooling is at a minimum and therefore quality loss is greatest at this stage (Karel, 1975). This high temperature portion of the drying process can be eliminated by using microwave vacuum to finish drying. As well, product temperature can be kept low during microwave vacuum-drying by reducing the microwave power during the final stage of the drying process. Air-drying has also been used to finish dry product initially processed by microwave vacuum. Cui et al. (2003) used microwave vacuum-drying to dry garlic slices to 10 per cent moisture (wet basis) and then finish-dried the product using hot air-drying at 45°C. While air-drying was reported to take 6 hours at 60–65°C and freeze-drying took 24 hours, 120 g of microwave vacuum-air-dried product reached the target moisture after approximately 35 minutes of microwave vacuum followed by 25 minutes of airdrying. The pungency, colour, texture and rehydration rate of garlic dried with the combination treatment was reported to be improved over product that was exclusively air-dried. Finish air-drying was used to avoid the temperature rise and product hot spots associated with the last stages of microwave vacuum-drying when using equipment that did not allow the product to be mixed during the drying. Similarly, improvements

522 Combined Microwave Vacuum-drying

to product quality were reported by Krokida and Maroulis (1999) when using a combination of microwave vacuum and finish air-drying, compared to air-drying only. While the moisture content achieved by the microwave vacuum process was not reported in this work, some general trends were observed. The products treated with the combination process had a lower bulk density and higher porosity, lower values for maximum stress and strain during compression and higher elasticity. Hunterlab colour parameters suggested that browning was minimized by the combination process. Osmotic dehydration has also been used in combination with microwave vacuumdrying. Erle and Schubert (2001) used a combination of osmotic treatment with sucrose or sucrose/calcium solutions, followed by microwave vacuum to dehydrate apples and strawberries. The authors noted that the methods were complementary to each other in terms of efficiency of the process as less microwave energy was required and, as well, the quality of the final product was improved. However, the extended time for the osmotic treatment (3–22 hours) nullified the advantage of the rapid dehydration rate typical of microwave vacuum processing.

7 Equipment 7.1 Commercial microwave vacuum-driers There were two commercial scale microwave vacuum-driers developed in the 1970s. The Gigavac, manufactured by Industries Micro-Onde Industrielles, Epone, France, used six to eight magnetrons of 6 kW each, operating at 2450 MHz, at a chamber pressure between 2.7 and 27 kPa. One application was the dehydration of 63°Brix orange juice concentrate to 2 per cent moisture in 40 minutes at a rate of 49 kg/h (Attigate, 1978). The process was less expensive than freeze-drying and excellent retention of ascorbic and dehydroascorbic acid and flavour volatiles was reported. A second drier, developed by the McDonnell Douglas Corporation, was the MIVAC, used mainly for drying of agricultural crops, such as grains (McKinney et al., 1977), rice (Wear, 1982) and peanuts (Pominski and Vinnett, 1989). It was also used for dehydration of grapes to create a puffed product (McKinney et al., 1983). The prototype pilot scale drier was built by the Aeroglide Corp (Raleigh, NC) and typically operated at 3.4–6.6 kPa with two magnetrons of 6 kW each (Gardner and Butler, 1982). The process allowed rapid drying of the grain with a substantially higher germination rate compared to hot air-drying. A summary of the drying times, rates, product initial and final temperatures and moistures for a number of different samples is available (Gardner and Butler, 1982). An analysis of the efficiency of the equipment indicated that less than 5200 kJ of energy was required for every 1 kg of water removed from the samples (average initial moisture level approximately 23 per cent), compared to approximately 7300 kJ/kg for a conventional drier (Keener and Glenn, 1978). In addition to the speed and higher quality product, the microwave vacuum-drying of grains had the added advantage of safety, as the danger of dust explosions was eliminated. Despite these promising early developments, commercial application of microwave vacuum-drying in the food industry has been limited. This may be attributed to

Modelling of microwave vacuum-drying 523

equipment costs, as well as the conservative nature of the food processing industry. However, due to reduced magnetron costs, the speed and energy efficiency of the process and the distinct characteristics of the dried products, there has been a renewed interest in this process for research and commercial applications. Manufacturers of commercial, pilot plant, or lab scale microwave vacuum driers include Puschner Mikrowellen Energietechnik (Bremen, Germany) that produces several models, including batch models and a continuous microwave vacuum belt drier; Enwave Corporation (Vancouver, Canada) that manufactures 1.8, 15 and 20 kW microwave vacuum driers; and Sairem (France) that manufactures laboratory microwave vacuum ovens (Labotron), with up to 0.6 kW of power; INAP (Marzling, Germany) that produces Mivap™, a semi-continuous microwave vacuum processor; and Nanjing Sanle Microwave Technology Development Co, Ltd, (Nanjing, China) that manufactures microwave vacuum-driers and microwave freeze-driers.

7.2 Research microwave vacuum-driers There are several lab scale microwave vacuum-driers described in published reports. The simplest systems are adapted household microwaves into which a vacuum chamber, composed of a microwave transparent material such as glass, has been inserted (Pappas et al., 1999). Non-uniform product heating has been a problem with these simple designs. However, with careful reduction of microwave power, especially during the final stages of drying, high quality product has been reported to be obtained using this approach (Mousa and Farid, 2002). Other designs have tackled this problem in a more direct manner, by incorporating a mechanism for mixing the product during the drying process. A microwave vacuum-dryer, with a perforated high density polyethylene drum in which product could be tumbled, was used for drying several products (Yousif et al., 1999; Kim et al., 2000a). A laboratory model microwave vacuum-dryer used to dehydrate parboiled rice, was designed to use either a rotating drum continuously to tumble the sample, or a revolving turntable, suspended within the drying chamber (Wadsworth et al., 1990). It was noted that the product obtained using the rotating drum produced more uniform drying. Kaensup et al. (2002) used a perforated polypropylene drum with vanes to rotate chillies. In this equipment, the most rapid dehydration was found occur at 20 rpm.

8 Modelling of microwave vacuum-drying Models describing heat and moisture transfer during microwave vacuum-drying can provide valuable information to aid process optimization and equipment design. While there have been several approaches to modelling of microwave heating and microwave drying (as cited in Cui et al., 2004a) relatively little work has been carried out for microwave vacuum dehydration. Lian et al. (1997) described the coupled heat and moisture transfer during microwave vacuum-drying of a porous material. Using the finite element method, equations were

524 Combined Microwave Vacuum-drying

developed to simulate the microwave vacuum-drying of a concentrated water-soluble food paste. It was assumed that the electric field around the product was uniform and decayed exponentially as it penetrated into the material and that dehydration occurred from both capillary liquid and vapour transfer. The equations were in good agreement with experimental data derived from the dehydration of a paste with 65 per cent solid concentration. Rodier and Rizzo (2003) modelled the microwave vacuum-drying of a packed bed of porous beads. They derived an empirical relationship to describe the drying rate as a function of moisture content and vapor pressure inside the beads. The drying was found to occur in a constant rate period at the beginning of the process, followed by a falling rate period in which the rate limiting process was first water loss from capillaries and then water desorption from the bead surface. Cui et al. (2004a) modelled the kinetics of microwave vacuum dried carrot slices from 162.8 to 336.5 W and 30–71 mbar. The model was developed assuming conservation of sensible heat, latent heat and source heat of the microwave power. Dehydration rates were found to be essentially linear to a moisture content of approximately 2 kg/kg dry basis and a correction factor was used to fit data below this moisture level.

9 Microwave freeze-drying An extension of microwave vacuum-drying is the application of microwave energy to facilitate freeze-drying. A considerable amount of research effort has been devoted to modelling and optimization of microwave freeze-drying and overcoming the limitations of the process (Ma and Peltre, 1975a, b; Ang et al., 1977a, b; Chang and Ma, 1985; Arsem and Ma, 1990; Wang and Shi, 1998). Heat transfer to the sample is a rate-limiting step in conventional freeze-drying and becomes progressively less efficient as a dry, insulating layer of material expands around the frozen core of the sample. Microwaves are able to generate heat volumetrically in the sample, significantly reducing drying times up to 75 per cent compared to conventional freeze-drying (Lombrana et al., 2001 and references therein). Therefore, mass transfer resistance is rate limiting during microwave freeze-drying. In addition to faster dehydration rates, enhanced rates of rehydration (Cohen et al., 1992) and greater volatile retention (Chen et al., 1993) have been reported for microwave vacuum freeze-drying compared to conventional freeze-drying. An economic study by Sunderland (1982) suggested that, although capital costs for a microwave freezedrier were higher than for a conventional freeze-drier, the higher production throughput associated with the reduction in dehydration times resulted in a lower unit cost of producing dried food using the microwave heating. The main factors that affect the rate of dehydration are the electric field strength, the chamber pressure and the sample size (Ang et al., 1977b). An increase in field strength decreases drying time, although overheating, melting and a loss of product quality occurs when the power is too high. A smaller sample size (0.64 cm2 versus 1.9 cm2) allows a higher field strength to be used without the sample overheating or

Other applications of microwave vacuum processing 525

melting (Ang et al., 1977b). Drying rate can be increased slightly as chamber pressure increases, however, similar problems of overheating and melting can occur. Therefore, pressure should be kept as low as possible. There are several limitations to the process that have been identified (Gould and Kenyon, 1971; Karel, 1974). Plasma discharges are more likely to occur at the chamber vacuum level used for freeze-drying (0.003–0.2 kPa). These discharges consume large amounts of energy and cause thermal damage to the product surface (Sunderland, 1982). As noted above, a limited amount of microwave energy can be applied to samples. If more energy is added to the material than can be removed by the sublimationmass transfer process, the pressure will rise above the triple point and the ice will melt. This in turn leads to run-away heating, since the dielectric properties of water are much higher than for ice. A third limitation in microwave freeze-drying is the uneven heating of the sample, associated with the uneven distribution of microwave energy inside the drying cavity and variations in sample shape and thickness. Various modifications have been used to overcome these three limitations, including microwave on-off cycling with simultaneous pressure modification (Lombrana et al., 2001); addition of a material with high dielectric properties to the sample prior to freezing to enhance energy transfer into the sample (Wang and Chen, 2003); use of combined radiant and microwave heating during freeze-drying (Chang and Ma, 1985); and use of a two-stage process whereby materials were air-dried to the end of the constant rate period and finish dried by microwave freeze-drying (Wang and Shi, 1999).

10 Other applications of microwave vacuum processing The advantages of rapid energy transfer while maintaining a low temperature that characterize microwave vacuum processing have been exploited for purposes other than dehydration (Table 19.2).

Table 19.2 Other applications of microwave vacuum processing Application

Reference

Histochemical processing of tissue specimens for microscopy Baking of bread dough using a microwave oven with a partial vacuum Drying of pharmaceutical drugs and excipients

Kok and Boon (1995) Acknin (2002) Chatrath and Stanisforth (1990); McLoughlin et al. (2003) Mishenko et al. (2000) Tanner and Leong (1997)

Destruction of microflora and insect pests in grain and feed Drying of marine sediment for determination of moisture content, metals and total carbon Composite dewatering of concrete Detoxification of contaminated soil Preparation of animal hides in the tanning processes

Dongxu and Wu (1994) George et al. (1992) Komanowsky (2000)

526 Combined Microwave Vacuum-drying

10.1 Tempering and thawing Tempering is a process whereby a frozen material is brought to a temperature just below the freezing level of free water, while thawing is the process of increasing the temperature to above the freezing point. Although microwaves at atmospheric pressure have been used for thawing and tempering for many years (e.g. Philippon, 1975), the advantage of working at a reduced pressure is that the product temperature can be kept low and thermal runaway is limited. Ice has a very high thermal diffusivity and a much lower loss factor and lower dielectric constant than water. There will always be some unfrozen water in the sample, which may have high concentrations of solutes. The microwaves, therefore, are preferentially absorbed by these high-solute pockets of water, resulting in rapid temperature rises in small areas of the material (Mudgett, 1985b). Under vacuum conditions, this temperature rise can be mediated to a large degree. James (1984) described a system for thawing frozen meat, where the pressure in a chamber could be reduced to 1 kPa and microwaves were produced by two 2.5 kW magnetrons at 915 MHz. Blocks of meat (61  40  15 cm) stored at 20°C were thawed in less than 2 h and the maximum surface temperature reached ranged from 14.9 to 26.7°C. However, the author noted that, at publication time, the high energy requirements and capital cost could limit the industrial application of the technique. More recently, a patent was issued that described equipment designed to expose frozen foods to alternating cycles of vacuum application and vacuum release, with microwave treatment initiated part way through the vacuum release phase of each cycle (Ito et al., 2003).

10.2 Enzymes and microorganisms Microwave vacuum processing has been used to enhance biological activity, or conversely, for thermal destruction of dried microbial cultures and enzymes, depending on the conditions used. At low chamber pressure and low microwave power, the retention of biological activity may be enhanced by the lower temperature and the shorter dehydration times of microwave vacuum-drying compared to other drying processes. For example, the effect of temperature (30–50°C) and water activity (aw) (0.04–0.996) on the D-values of bacterial cultures in yogurt dried using microwave vacuum-drying was determined (Kim et al., 1997). The 2450 MHz vacuum microwave drier was maintained at 1 kPa and 250 W of power was used. Survival of the bacterial cultures was much higher for microwave vacuum-dried yogurt than spray-dried, or even freezedried yogurt. Similarly, higher levels of -amylase activity were reported to be retained in microwave vacuum-dried rice koji, compared to freeze-, vacuum- and air-drying (Kim et al., 1999). Alternatively, enhanced destruction of enzyme activity or microbes has been attributed to microwave vacuum processing under some conditions. The inactivation of several enzymes ( -amylase, -amylase, glucoamylase and peroxidase) at 60–80°C by microwave vacuum was compared to conventional heating (Moon et al., 1997). It was found that the heating time for complete inactivation was reduced for the microwave vacuum conditions. Rajko et al. (1997) used microwave vacuum processing to inactivate

Conclusions 527

anti-nutritional factors of soybeans, including urease and trypsin inhibitor. Optimal conditions were processing for 6.5–7.7 minutes at 91.8–94 kPa chamber pressure and 275 W per 100 g sample. The advantages of using this process were the rapid inactivation and the simple equipment required compared to other heating processes that have been developed to treat soybeans. There is controversy over whether the destructive effect of microwaves on microorganisms and enzymes is strictly due to thermal effects (Fujikawa et al., 1992) or attributed to additional ‘microwave’ effects (Porcelli et al., 1997; Tajchakavit et al., 1998; Banik et al., 2003). While this is an intriguing area for further research, it is clear that microwave vacuum processing can be effective for controlling enzyme and microbial activity when higher temperatures and chamber pressure are used.

11 Commercial potential Microwave vacuum-drying cannot be expected to provide substantial cost savings over air-drying, although it probably can be much less expensive than conventional freeze-drying. Nonetheless, the greatest potential of microwave vacuum processing is not in cost savings but in the capacity to create new products, or products with unique characteristics, that cannot be replicated effectively with other technologies. If food manufacturers are able to utilize microwave vacuum-systems to provide consumers with desirable food characteristics, such as excellent flavour and texture, better bioactive or nutrient retention and more convenience than competing technologies, then microwave vacuum-technology will grow rapidly and become widespread. If, however, the consumer benefits are not perceived as significant, then microwave vacuum will remain an interesting technology for research purposes but with limited application to commercial food processing.

12 Conclusions Microwave vacuum-dehydration has been shown to be very rapid and to allow drying of foods with less heat and oxygen damage than air-drying. The resulting tissue structure and rehydration potential may be comparable to that of freeze-dried foods. The operating costs of microwave vacuum-drying are expected to be slightly more than those of forced convection air-drying technology. Vacuum microwave-technology is not a new concept but, until recently, it has not been successful in commercial food dehydration operations. The combination of modern computer control systems, more economical microwave equipment and advances in microwave vacuum-food process science, have now made commercial success possible. Whether its use becomes widespread or remains confined to speciality niche products depends upon the ingenuity and success of food scientists and product development specialists in defining the unique benefits of microwave vacuum and developing consumer

528 Combined Microwave Vacuum-drying

products to exploit these benefits. Current public demand for quick-to-prepare meals and functional foods may offer many opportunities.

Nomenclature c Cp E h j k

   


velocity of radiation (m/s) heat capacity (J/kg K) radiation energy (J) Planck’s constant, 6.63  1034 J/s an imaginary value used for calculation of the sinusoidal behavior of electromagnetic waves thermal conductivity (W/m K) thermal diffusivity (m2/s) complex permittivity or complex dielectric constant permittivity or dielectric constant dielectric loss factor radiation wavelength (m) radiation frequency (s) density (kg/m3)

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New Hybrid Drying Technologies Kian Jon Chua and Siaw Kiang Chou Department of Mechanical Engineering , National University of Singapore, Singapore

It is well known that the dehydration process influences the quality of the bioproducts. The current drive towards improving drying technologies is motivated by the economic incentive to produce better quality products at a faster rate and lower operating costs. In recent years, the engineering focus has been to improve the design and operation of dryers to achieve the dried food product with desired characteristics. Even though much has been done in the development of improving individual drying technology, much remains to be achieved in the study of new hybrid systems whereby drying technologies can be combined to evolve new age drying systems. This chapter summarizes some recent developments in hybrid drying technologies of interest to the bioproduct industry. New emerging hybrid drying technologies are listed and discussed. The potential roles of these hybrid technologies in product quality enhancement are also identified.

1 Introduction In many agricultural countries, large quantities of bioproducts are dried to enhance shelflife, reduce packaging costs, lower shipping weights, enhance appearance, encapsulate original flavour and maintain nutritional value. According to Okos et al. (1992), the goals of drying process research in the bioproduct industry are summarized by three issues:

• Economic considerations: to reduce operating costs and improve capacity per unit amount of drying equipment; to develop simple drying equipment that is reliable and requires minimal labour; to minimize off-specification product; and to develop a stable process that is capable of continuous operation. • Environmental concerns: to minimize energy consumption during the drying operation and to reduce environmental impact by reducing product loss in waste streams, i.e. to incorporate the possibility of waste heat recovery systems. • Product quality aspects: to have precise control of the product moisture content at the end of the drying process; to minimize chemical degradation reactions; to reduce change in product structure and texture; to obtain the desired product colour; to control the product density; and to develop a versatile drying process that can produce products of different physical structures for various end-users. Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

20

536 New Hybrid Drying Technologies

Though the primary objective of food drying is preservation, depending on the drying technology employed, the raw material may end up as a completely different material with significant variation in product quality. Therefore, great care must be taken in choosing a suitable dryer considering the closely knitted relationship between the bioproduct and dryer. A mismatch between the product and drying technology would result in dire consequences, often resulting in great financial losses. As new discovery of hybrid bioproducts is constantly being made, the spectrum of dried bioproduct gets wider. When a single drying system cannot handle the stringent quality requirements of these new bioproducts, the need for new hybrid drying systems becomes essential. Therefore, the principal motivation in developing hybrid drying technologies is to meet consumer expectations of quality and yet produce a product with the desired moisture content. Figure 20.1 shows a general classification scheme of hybrid drying technologies. Under the umbrella of hybrid drying are drying techniques that employ multiple modes of heat transfer as well as those that use two or more stages of drying to achieve the desired dryness, product quality, drying time and manufacturing throughout. A more common definition of hybrid drying would consist of a successful integration or intelligent combination of two or more conventional drying know-hows. This alone can constitute the emergence of a wide-range of new hybrid drying technologies.

Hybrid drying technologies

Combined drying technology

Infrared-heat pump drying Infrared-convective drying Microwaveconvective drying Microwave-vacuum drying

Multiple-stage drying

Same drier type at each stage

Drying and cooling

– Two-stage fluid beds – Two-stage vibrofluid beds

Drying and coating

Different drier type at each stage – Flash/fluid bed – Spray/fluid bed – Fluid bed/packed bed Different drying technologies per stage – Superheated steam drying followed by convective air drying – IR/microwave drying followed by convective air drying

Figure 20.1

Multiple-process drying

A general classification scheme of hybrid drying technologies.

Drying and granulation Drying and filtration

Hybrid drying systems 537

This chapter serves to provide an overview of some newly developed hybrid drying technologies applicable for bioproducts that are particularly sensitive to thermal treatment. Drying technologies incorporating convective, radiation and electromagnetic heat transfer modes will be presented along with novel technologies such as pressureswing, vacuum-superheated steam and rotating spouted jet drying. The impact of each hybrid drying technology on various quality parameters will also be briefly discussed in the light of growing interest and demand for high quality dried products.

2 Product quality degradation during dehydration To understand how the employment of hybrid drying technologies can improve product quality, it will be useful first to understand the degradation process of foodstuffs. The quality of many food products degrades during dehydration above room temperature. The added heat and exposure time of the product at elevated temperature affects the rate of nutrient quality degradation. The types of food degradation during drying are listed in Table 20.1. Dried foods are known to be high in fibre and carbohydrates and low in fat, making them healthy food choices. Food scientists have found that by reducing the moisture content of foodstuffs to between 10 and 20 per cent, bacteria, yeasts, moulds and enzymes are all prevented from spoiling it. The flavour and aroma are well preserved and concentrated. The loss of nutrient can be viewed as the decomposition of a particular chemical compound. This decomposition of a single monomolecular reaction may be described using zero or first-order kinetics equations (Chua et al., 2000a). As the temperature of the product increases, the reaction rate constant is increased. The dependence of the reaction constant on temperature implies that a low temperature drying process results in less nutrient degradation. A longer constant rate drying period increases the nutrient retention because, owing to evaporative cooling, the product is at a lower temperature.

3 Hybrid drying systems The diversity of food products has introduced many types of dryers to the food industry. Often the selection of the appropriate dryer is based on the drying characteristics Table 20.1 Change in food quality parameters during dehydration Chemical

Physical

Nutritional

Browning reaction Lipid oxidation Colour loss Gelatinization

Rehydration Solubility Texture and shrinkage Aroma loss

Vitamin loss Protein loss Microbial survival

538 New Hybrid Drying Technologies

of the food product. For heat-sensitive bioproducts, the methods of supplying heat to the product and transporting the moisture from the product become the critical considerations for selecting the right drier to achieve the desired product moisture content. In the following sections, the potential of employing new hybrid driers for drying of bioproducts is presented.

3.1 Heat pump drying There has been a growing interest in recent years to apply heat pump drying (HPD) technology to foods and biomaterials where low-temperature drying and well-controlled drying conditions are required to enhance the quality of food products. High-valued products, which are extremely heat-sensitive, are often freeze-dried. This is an extremely expensive drying process (Baker, 1997). Therefore, there has been great interest in looking at the heat pump drying system as a replacement system for freeze-dried products. Table 20.2 presents a summary of some hybrid heat pump drying systems for selected bioproducts.

Table 20.2 Selected works on hybrid heat pump drying of selected food products Researchers

Hybrid system

Application(s)

Conclusions

Chua et al. (2000b) Chou et al. (2001) (Singapore)

Heat pump/IR system

Agricultural and marine products (mushrooms, fruits, sea-cucumber and oysters)

The quality of the agricultural and marine products can be improved with scheduled drying conditions

O’Neill et al. (1998) (New Zealand)

Modified atmosphere heat pump system

Agricultural food drying (apples)

Modified atmosphere heat pump drying (MAHPD) produces products with a high level of open pore structure, contributing to the unique physical properties, such as floatability and rehydration capabilities. MAHPD drying system affords the possibility of drying with colour qualities comparable to sulphured products

Best et al. (1994) (Mexico)

Solar-assisted heat pump drier

Rice

Advantage of low temperature and better control in the drier results in good quality rice

Rossi et al. (1992) (Brazil)

Heat pump assisted heating drier

Vegetable (onion)

Drying of sliced onions confirmed energy saving of the order of 30% and better product quality due to shorter processing time

Alves-Filho and Strømmen (1996) (Norway)

Heat pump assisted fluidized bed drier (HPFBD)

Marine products (fish)

The high quality of the dried products was highlighted as the major advantage of HPFBD and introducing a temperature controllable programme to HPFBD makes it possible to regulate the product properties such as porosity, rehydration rates, strength, texture and colour

Hybrid drying systems 539

Some of the advantages of the heat pump drier are as follows:

• Higher energy efficiency with improved heat recovery results in lower energy consumed for each unit of water removed.

• Better product quality with well-controlled temperature schedules to meet specific production requirements. • A wide range of drying conditions typically
20–100°C (with auxiliary heating) and relative humidity 15–80 per cent (with humidification system) can be generated. • Excellent control of drying environment for high-value products and reduced electrical energy consumption for low-value products. Along with the advantages are limitations given as follows:

• Requires regular maintenance of components (compressor, refrigerant filters, etc.) and charging of refrigerant. • Increased capital costs. For many of the research studies conducted in Table 20.2, the common conclusion was that the heat pump drier offers products of better quality with reduced energy consumption. This is particularly true of food products that require a closed-control drying environment such as temperature, humidity and/or even a special drying medium (O’Neill et al., 1998). Heat-sensitive food products, requiring low-temperature drying, can take advantage of HPD technology since the drying temperature of the HPD system can be adjusted from
20 to 100°C. With proper control, it is also possible for HPD to produce freeze-drying conditions at atmospheric pressure (Prasertsan and Saen-saby, 1998). So far as food drying is concerned, HPD offers an alternative to improve product quality through proper regulation of the drying conditions. Chua et al. (2000b) have demonstrated that HPD can produce pre-selected cyclic temperature schedules to improve the quality of various agricultural products dried in their two-stage HPD. They have shown that with appropriate choice of temperature-time variation, it is possible to reduce the overall colour change and ascorbic acid degradation by up to 87 and 20 per cent, respectively. Besides yielding better food quality, Rossi et al. (1992) have reported that onion slices dried by HPD used less energy in comparison to a conventional hot air system. Food products with a high water content can be dried efficiently using HPD. As the drying air absorbs more of the latent energy due to vaporization, this energy can be transferred at the evaporators for higher heat recovery. Lower energy input is then required at the compressor to enable sensible heating of the air when it passes through the condenser. Ginger dried in a heat pump drier was found to retain over 26 per cent of gingerol, the principal volatile flavour component responsible for its pungency, compared to only about 20 per cent in rotary dried commercial samples (Mason et al., 1994). The higher volatile retention in heat pump dried samples is probably due to the reduced degradation of gingerol when lower drying temperatures are employed. The loss of volatiles varies with concentration, with the greatest loss occurring during the early stages of drying when the initial concentration of the volatile components is low (Saravacos et al., 1988). Since heat pump drying is conducted in a closed chamber, any compound that volatilizes will remain within the drying chamber and the

540 New Hybrid Drying Technologies

partial pressure for that compound will gradually build up within the chamber, retarding further volatilization from the product (Perera and Rahman, 1990). A recent hybrid system combines radio frequency (RF) and heat pump drying (Marshall and Metaxas, 1998). Such a system has potential application in the food industry. The limitation of relative low heat transfer rates in convective air drying, particularly towards the falling rate period, can be overcome by introducing volumetric heat generation such as the RF technology. The RF field generates heat volumetrically within the material wetted with polar molecules, such as water, by the combined mechanisms of dipole rotation and ionic conduction. The internal heat generation speeds up the drying process because of unidirectional temperature and moisture gradient and internal pressure build-up. Figure 20.2 shows a schematic of a radiofrequency-assisted heat pump drier (RF-assisted HPD). Such a hybrid drier is suitable for food materials that are difficult to dry with convective heating, especially food products that have a film of wax on the surface such as chillies, cherries and tomatoes. With regards to product quality, it appears that RF-assisted HPD reduces colour degradation, surface cracking and differential shrinkage of the product. In terms of energy consumption, RF-assisted drying has been observed to improve the specific moisture extraction rate (SMER) and coefficient of performance (COP) of the heat pump system. Furthermore, the potential for increasing the product throughput is good. For example, in the bakery industry, the throughput for crackers and cookies can be improved by as much as 30 and 40 per cent, respectively (Clark, 1997).

Evaporator

Compressor Expansion valve

Condenser Axial fan

Drying product

Metallic perforated plate

Heat pump drying chamber Figure 20.2

RF generator

Schematic diagram of RF-assisted heat pump drier (Marshall and Metaxas, 1998).

Hybrid drying systems 541

Alternatively, to dry heat-sensitive materials, a combined radiant-convective drying method may be applied. An infrared-augmented HPD drying system could be used for fast removal of surface moisture during the initial stages of drying, followed by intermittent drying over the rest of the drying process. This mode of operation ensures a faster initial drying rate. Therefore, an infrared (IR) assisted HPD would offer the advantage of compactness, simplicity, ease of control and low equipment costs (Mujumdar, 2000). Also, there are the possibilities of significant energy savings and enhanced product quality due to the reduced residence time in the drying chamber. For such an IR-assisted HPD system, it is essential to implement a good control strategy for IR operation in order to achieve the desired results in terms of drying kinetics and product quality, as well as to ensure safe operation. A typical example of a good feedback control is one that enables the IR power source to be cut off if excessively high temperatures are measured in the chamber, which may lead to overheating of the product. Chou et al. (2001) designed a feedback system by coupling a PID controller to the IR lamps and by inserting a type ‘T’ thermocouple needle to the bioproduct, feedback signals were sent to the controller. In comparison to the IR operating in intermittent mode, they found that by pre-programming different food sample temperatures, greater reduction in drying time and improvement in product colour could be achieved.

3.2 Fluidized bed drying Fluidized bed drying (FBD) has found many applications for drying granular solids in the food, pharmaceutical and agriculture industries. For drying of powders in the 50–2000 m range, FBD competes successfully with other more traditional drier types, e.g. rotary, tunnel, conveyor, continuous tray, etc. The advantages of FBD include: 1 high heat and mass transfer phenomena between the particles and the gas 2 closed control product temperature making FBD ideal for processing temperaturesensitive solids 3 highest thermal efficiency of any gas-suspension drying system. The disadvantages of FBD include: 1 it is able to dry only a limited range of materials 2 the size of the product particles is relatively large 3 difficulty involved in processing needle or platelet-shaped particles. Recent hybrid fluidized bed driers incorporating a heat pump drying mechanism have been developed at the Norwegian Institute of Technology (Alves-Filho and Strømmen, 1996) as shown in Figure 20.3. The drying chamber receives wet material and discharges dried product through the product inlet and outlet ducts. The desired operating temperature is obtained by adjusting the condenser capacity, while the required air humidity is maintained by regulating the compressor capacity via frequency control of the motor speed. According to Alves-Filho and Strømmen (1996), this set-up can produce drying temperatures from
20 to 60°C and air humidity spanning 20 to 90 per cent. With these features, heat-sensitive food materials can be dried

542 New Hybrid Drying Technologies

Fluidized bed Wet material

Dry material

Condenser Centrifugal fan

Evaporator

3-way valve

Expansion valve

External condenser Liquid receiver

Compressor

Figure 20.3 Fluidized bed driers incorporating heat pump drying technology (Alves-Filho and Strømmen, 1996).

under convective air or freeze drying conditions. It is also possible to sequence these two operations (convective and freeze drying). This will be advantageous for drying of food and bioproducts since freeze drying causes minimal shrinkage but produces low drying rates while convective air drying can be applied to enhance drying rates. Therefore, a combination of drying processes, e.g. freeze drying at
5°C followed by convective drying of 20–30°C, enables the control of quality parameters such as porosity, rehydration rates, strength, texture, colour, taste, etc. Experiments performed at the department of mechanical engineering, Norwegian University of Science and Technology, on various heat-sensitive materials such as pharmaceutical products, fruits and vegetables have shown that this new hybrid fluidized bed drying offers a better product quality but at higher cost. Even as experimental work is still being conducted with this hybrid drier, a two-stage fluidized bed heat pump dryer has already interested some food industries in Norway (Strømmen and Jonassen, 1996). The two-stage system simply comprises two fluidized beds connected in series. Two heat pumps supply conditioned air independently to each drying chamber. The drying chambers are connected in series so that one receives wet product and discharges the semi-dried product to the next, which produces the final dried product. Such a hybrid two-stage drier is versatile as it allows independent freezing and convective drying to be carried out. The advantages of multiple-stage fluidized bed drying over single fluidized bed heat pump drying include improved product quality and enhanced energy efficiency (Alves-Filho and Strømmen, 1996) but at higher capital cost.

Hybrid drying systems 543

Recently, Taechapairoj et al. (2003) employed a fluidized bed system incorporating superheated steam as the drying medium for paddy drying. They observed that when using superheated steam drying the head rice yield is more sustainable and has higher values than those obtained from hot air drying. However, there was some colour depreciation in terms of the rice whiteness. The main cause of the rapid change in the colour is partly due to the steam condensation on the paddy surface.

3.3 Radio-frequency drying Radio-frequency drying (RFD) is a simple precise process and is common place in the food industry with appropriate and proven processes available for a wide range of applications, such as pre-heating, pre-cooking, sterilization, tempering, post-baking and moisture control. A limitation of heat transfer in conventional drying with hot air alone, particularly in the falling rate period, can be overcome by combining RF heating with conventional convective drying (Thomas, 1996). RF generates heat volumetrically within the wet material by the combined mechanisms of dipole rotation and conduction effects which speed up the drying process (Marshall and Metaxas, 1998). A typical RF-convective drier comprises a convective drying system retro-fitted with an RF generating system capable of imparting radio-frequency energy to the drying material at various stages of the drying process. Biomaterials that are difficult to dry with convection heating alone are good candidates for RF-assisted drying. Food materials with poor heat transfer characteristics have traditionally been problem materials when it comes to heating and drying. Radio frequency heats all parts of the product mass simultaneously and evaporates the water in situ at relatively low temperatures, usually not exceeding 82°C (Thomas, 1996). Since water moves through the product in the form of a gas rather than by capillary action, migration of solids is avoided. Warping, surface discoloration and cracking associated with conventional drying methods are also avoided. The potential for application of RF drying in the food industries can be appreciated for the following reasons:

• RFD prevents over-drying because radio waves concentrate in the wettest and densest areas of the biomaterial. It improves the colour of products, especially those that are highly susceptible to surface colour change, since RF drying starts from the centre and moves to the product surface, minimizing any surface effect. • Cracking, caused by the stresses of uneven shrinkage in drying, can be eliminated by RF-assisted drying. This is achieved in the drier by even heating throughout the product maintaining moisture uniformity from the centre to the surface during the drying process. • Simultaneous external and internal drying significantly reduces the drying time to reach the desired moisture content. The potential for improving the throughput of product is good. • Closer tolerance of the dielectric heating frequency significantly improves the level of control for internal drying and thus has potential in industry that produces food products that require precision moisture removal (Clark, 1997).

544 New Hybrid Drying Technologies

Another potential hybrid system worth mentioning is the combination of RF and vacuum drying. Even though current application of RF-vacuum drying is concentrated mainly in the wood industry (Rasev, 1999; Saito and Sulaiman, 1999), there is great potential for applying it to the food industry. RFD under vacuum condition allows for moisture to be removed at temperatures as low as 30°C. With the ability to improve product quality and nutrient retention, low temperature bulk drying with RF-vacuum technology is ideal for functional food manufacturing processes. Some of the advantages of such a hybrid system would include: 1 lower drying temperature resulting from a reduced boiling point due to a lowering of chamber pressure 2 better improvement of quality parameters such as product colour and shrinkage compared to RF-convection or RFD alone 3 chemical oxidation due to contact with drying air can be eliminated.

3.4 Microwave drying The physical mechanisms involved in heating and drying with microwaves are distinctly different from those of conventional means. Microwaves (MW) can penetrate into dielectric materials and generate internal heat (Jia et al., 1993). The internal heat generated establishes a vapour pressure within the product and gently ‘pumps’ the moisture to the surface (Turner and Jolly, 1991). This moisture pumping effect results in moisture being forced to the surface and preventing case hardening from occurring. Drying rates and product quality are subsequently enhanced. Because of this unique advantage, microwave drying has been used in a number of industries, e.g. timber, paper, textile, food and ceramic industries (Schiffmann, 1987). However, the progress of microwave drying at the industrial level has been relatively slow because of its high initial capital investment and low energy efficiency when compared with conventional drying technologies. To improve on the economic aspects of microwave drying, it is necessary to incorporate energy conservation features. Funebo and Ohlsson (1998) and Prabhanjan et al. (1995) have demonstrated that employing microwave-assisted air dehydration, the drying time for apple and mushroom can be significantly shortened and the products have better quality. The incorporation of MW technology with conventional driers can, perhaps, produce a more commercially viable drying technology. The advantages of microwave drying can be summarized as:

• • • •

Enhancement of heat and mass transfer processes Development of internal moisture gradients which enhance drying rates Increased drying rates without increased surface temperatures Improved product quality.

Currently, industrial microwave driers can be commercially viable for applications in food industries that require short drying time and higher product throughput at the expense of higher energy input. Also, food industries dealing with products that are susceptible to case hardening may consider microwave drying to be a good alternative in

Hybrid drying systems 545

quality enhancement. Recently, several technical staffs at the Dried Foods Technology Laboratory at Washington State University developed a state-of-the-art microwave vacuum drier (Clary, 1999). Figure 20.4 shows a simplified schematic of the drier. The microwave/vacuum process occurs inside large stainless steel vessels under vacuum conditions. Inside, the vessel contains a conveyor, a microwave unit and a radiant heat source. There are three zones in the vessel. As the food product is transported via the conveyor, it enters each zone with different microwave power. In the first zone, the product is subjected to a high level of microwave energy of either 12 kW at 2450 Hz or 30 kW at 915 Hz under vacuum conditions of about 1.3–4.0 kPa. In this zone, the product undergoes rapid dehydration because of the high level of microwave energy. In the second zone, the product is subjected to a moderate level of microwave energy of 6 kW at 2450 Hz. The final zone may or may not be accompanied by even lower microwave energy to ensure equalization of the moisture content. In this zone, the product is cooled and finally transported by the conveyor system for packaging. The incorporation of vacuum to the microwave system minimizes product oxidation and lowers the boiling point of the water in the food making it possible for drying to occur rapidly at temperatures below 55°C. Lower temperature drying allows food to minimize the degradation of quality parameters such as colour, flavour and nutritional value. The subtle raising of the microwave energy in different zones is another distinct feature of this hybrid drier. When the product possesses high moisture during the early stages of drying, it is able to undergo high thermal impact without significant quality degradation. As the moisture is removed, it is more susceptible to thermal-related quality change. Therefore, the microwave energy scheduling ensures rapid drying while minimizing quality degradation. It is also noteworthy that the drier heats food uniformly and thus preserves its

Product infeed Microwave power supplies

ZONE 1 – 12 kW at 2450 MHz or 30 kW at 915 MHz ZONE 2 – 6 kW at 2450 MHz or 30 kW at 915 MHz ZONE 3 – Product rest and equalization

Condenser and vacuum pump Product out-feed Figure 20.4

Combined microwave-vacuum drying system with different zones.

546 New Hybrid Drying Technologies

original shape. Moreover, it was found that microwave vacuum dehydration technology produces food quality superior to that of freeze-dried products and only at a fraction of the cost (Clary, 1999). It has huge potential in the fruit drying industry. Nindo et al. (2003) have recently evaluated several drying technologies namely, tray drying, spouted bed drying, combined microwave and spouted bed drying (MWSB) and freeze drying. Figure 20.5 shows a schematic of the MWSB experimental set-up. From their experiments, they observed that combined microwave and spouted bed drying of asparagus slices at a power level of 4 W/g was at least 5 times faster than tray drying when air temperatures between 50 and 70°C were used. At a drying air temperature of 60°C, the MWSB, operating with a power level of 4 W/g, was observed to be 6.0 and 2.8 times faster than tray and spouted bed drying, respectively. MWSB-dried asparagus had the highest rehydration. Microwave spouted bed drying at 60°C resulted in the highest retention of total antioxidant activity in asparagus.

3.5 Novel drying technologies The following sections contain some novel drying technologies being recently developed that are suitable for bioproducts. Most research and development works conducted for these technologies are still at their infancy stage. Nevertheless, they are worth mentioning in order to present more available options for consideration in choosing the most practical and efficient drying technology for the wide range of bioproducts.

Circulator Wave-guide Microwave magnetron Tub tuners

Directional coupler Microwave power controller Multi-mode microwave cavity

Spouted bed

Sample

Temperature controller Hot air

Ambient air Heater Valve Blower

Figure 20.5

A schematic layout of combined microwave-spouted bed drying system (Nindo et al., 2003).

Hybrid drying systems 547

3.5.1 Combined microwave and superheated steam drying

Microwave (MW) and superheated steam (SHS) are both well-established drying technologies. The advantages of MW drying have been previously mentioned. The advantages of superheated steam drying are: 1 it is a non-polluting and safe drying method requiring low energy consumption 2 it can improve drying efficiency, sometimes as much as 50 per cent greater than a conventional drying system 3 steam is known to be a better agent compared to dry air in destroying all stages of insects, moulds and microorgansims found in foodstuffs. In general, both are known to be more expensive than traditional driers and hence they are only considered in some niche industries where the bioproducts are considered to be of high-value. Shibata et al. (2000) have studied the combined MW-SHS drying technology using sintered glass as a model material. From their experiments, they found that, in comparison to MW-nitrogen drying, the drying rates under MW-SHS drying were higher than those at less than the critical moisture content. The result is a reduction in drying time to achieve the desired moisture content. According to Kudra and Mujumdar (2001), there is good potential for employing such a hybrid system to produce products with low apparent density due to puffing, which may or may not be a desirable attribute. For food products such as cereals and selective snack-food that require both drying and puffing processes, MW-SHS would then present itself as an attractive option. 3.5.2 Pressure regulating drying

A very useful way to enhance the quality of heat-sensitive food products and yet achieve the desired product dryness is through the use of a pressure-regulatory system. The operating pressure range is usually from vacuum to close to one atmosphere. A total vacuum system may be costly to build because of the need for stronger materials and better leakage-prevention. Therefore, the system that is proposed here is recommended to operate above vacuum conditions. The period of operating at lower pressure may be continuous at a fixed level, intermittent or a prescribed cyclic pattern. The suitability of employing the appropriate type of pressure-swing pattern depends chiefly on the drying kinetics of the product and its thermal properties. Maache-Rezzoug et al. (2002) have recommended a pressure-swing drying mechanism for food products requiring the production of homogeneous thin sheets. Their experiments in drying a collagen gel in order to obtain a homogeneous film were recently carried out using a new process: dehydration by successive decompression. This process involves a series of cycles during which the collagen gel is placed in desiccated air at a given pressure then subjected to an instantaneous (200 ms) pressure drop to a vacuum (7–90 kPa). This procedure is repeated until the desired moisture content is obtained. A comparative study between this new pressure-swing drying process and conventional methods indicated that the respective saving in drying time could be as high as 480 and 700 minutes in comparison to vacuum and hot air drying systems. Chua and Chou (2003) studied a successive pressure drops method on the drying kinetics as well as the colour degradation of two bioproducts, namely potato and carrot. The parameters under investigation include the cycle duration, pressure

548 New Hybrid Drying Technologies

levels and chamber temperature on the drying kinetics, product colour and porosity changes. Their experimental results showed that the pressure level has a positive impact on the drying kinetics of heat-sensitive bioproducts. Also, for a given drying period, a shorter drying cycle time was observed to result in better drying kinetics for the agro-samples. Finally, drying conditions employing lower chamber pressure were shown to have a significant impact in reducing the colour change of bioproducts. Based on the studies presented here, the general conclusion is that integrating a pressure-swing system to any convective drier would significantly improve product quality and, at the same time, reduce the drying time which would result in a smaller drying chamber to obtain similar product throughput. 3.5.3 Rotating jet spouted bed

Jumah et al. (1996) implemented the principle of intermittent drying in a novel spouted bed system. They studied the drying kinetics of corns using a rotating jet spouted bed (RJSB). A schematic of their set-up is shown in Figure 20.6. Briefly, the rotating jet spouted bed is formed when the air jet moves circumferentially in the annular region between the chamber wall and the central spout. One distinct advantage of the rotating jet configuration is the prescription of an intermittent spout due to the continuous movement of the air jet. Intermittent drying due to periodic heat supply is then possible. By varying the rotational speed of the spouting jet of heated air, the intermittency frequency and hence the intermittent drying schedule can be varied. Jumah et al. (1996) performed experiments to test the hypothesis that corn, as a slow drying material, could be dried to produce high quality grain with lower energy consumption via prescribing an intermittent air schedule. The intermittent scheduling was achieved by using various drying periods alternated by long tempering periods.

Spouting particles

Centrifugal fan

PID controller Gate valve

Electric heaters Figure 20.6

Fabric filter

Rotating air distributor

Motorized drive

Experimental rotating jet spouted bed with central and peripheral air jets (Jumah, 1995).

References 549

During the active periods, the corn particles were subjected to very intense mixing and circulation due to the hydrodynamics of the rotating spouts. The resulting effect was a period of high intensity heat and mass transfer. During the no-flow periods, the temperature and moisture gradients were effectively relaxed and favourable moisture re-distribution inside the particle occurred. Minimal mechanical damage to the kernels due to reduced attrition caused by inter-particle collisions during spouting was also observed. It was further demonstrated that moisture levelling occurred during the tempering periods with moisture migration to the corn kernel surface. In terms of energy saving, intermittent drying conducted with the RJSB resulted in substantial energy saving of up to 37 per cent when compared to a continuous spouting bed dryer (Jumah et al., 1996).

4 Conclusions One of the main contributions of the twentieth century has been to lay the fundamental platform for new technology to emerge. This review chapter has summarized some of the recent developments of hybrid-drying technologies. As technology advances to new frontiers, the method to dehydrate food is constantly evolving to produce new hybrid drying systems. Some of the hybrid drying techniques, if combined in an intelligent fashion, would promote efficient drying in terms of enhanced product quality and reduction in energy consumption. However, R&D effort is still required to study system scale-up, optimization and control of these hybrid systems. This chapter may not have covered all novel drying technologies available, but it is hoped that those hybrid technologies presented would give dried food manufacturers a better understanding of the technologies available to improve their drying processes. Since product quality and energy consumption are usually the primary concerns during food dehydration, there is still scope for discovering new drying technologies. It is hoped that in the coming years more hybrid systems can be developed to handle even the most complex bioproduct drying problems.

References Alves-Filho O, Strømmen I (1996) Performance and improvements in heat pump dryers. In Drying ’96 (Strumillo C, Pakowski Z, eds). Krakow: Elsevier, pp. 405–415. Baker GJ (1997) Industrial Drying of Foods. London: Chapman and Hall. Best R, Soto W, Pilatowsky I, Gutierrez LJ (1994) Evaluation of a rice drying system using a solar assisted heat pump. Renewable Energy, 5 (1–4), 465–468. Chou SK, Chua KJ, Mujumdar AS, Tan M, Tan SL (2001) Study on the osmotic pre-treatment and infrared radiation on drying kinetics and colour changes during drying of agricultural products. ASEAN Journal on Science and Technology for Development, 18 (1), 11–23.

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Chou SK, Chua KJ (2001) New hybrid drying technologies for heat sensitive foodstuffs. Trends in Food Science and Technology, 12 (10), 359–369. Chua KJ, Chou SK (2003) Evaluating a pressure regulatory system for drying of heat-sensitive bioproducts. Journal of Food Engineering, 63 (3), 151–158. Chua KJ, Chou SK, Ho JC, Mujumdar AS, Hawlader MNA (2000a) Cyclic air temperature drying of guava pieces: Effects on moisture and ascorbic acid contents. Transactions of IChemE, Part C, 78, 72–78. Chua KJ, Mujumdar AS, Chou SK, Hawlader MNA, Ho JC (2000b) Convective drying of banana, guava and potato pieces: Effect of cyclical variations of air temperature on convective drying kinetics and colour change. Drying Technology, 18 (5), 907–936. Clark TD (1997) The current status of radio frequency post-baking drying. In Proceedings of the 72nd Annual Technical Conference of the Biscuit and Cracker Manufacturers’ Association, USA: Forth Worth, Texas, pp. 104–107. Clary C (1999) Microwave vacuum drying. Washington State University Today, 15 (9), 3–4. Funebo T, Ohlsson T (1998) Microwave-assisted air dehydration of apple and mushroom. Journal of Food Engineering, 38, 353–367. Jia X, Clements S, Jolly P (1993) Study of heat pump assisted microwave drying. Drying Technology, 11 (7), 1583–1616. Jumah RY, Mujumdar AS, Raghavan GSV (1996) A mathematical model for constant and intermittent batch drying of grains in a novel rotating jet spouted bed. In Mathematical Modelling and Numerical Techniques in Drying Technology (Turner I, Mujumdar AS, eds). New York: Marcel Dekker, pp. 339–380. Kudra T, Mujumdar AS (2001) Advanced Drying Technologies. New York: Marcel Dekker Inc. Maache-Rezzoug Z, Rezzoug SA, Allaf K (2002) Development of a new drying process – Dehydration by successive pressure drops: Application to the drying of collagen gel. Drying Technology, 20 (1), 109–129. Marshall MG, Metaxas AC (1998) Modelling the radio frequency electric field strength developed during the radio frequency assisted heat pump drying of particulates. International Microwave Power Institute, 33 (3), 167–177. Mason R, Britnell YG, Birchall S, Fitz-Payne S, Hesse BJ (1994) Development and application of heat pump dryers to the Australian food industry. Food Australia, 46, 319–320. Mujumdar AS (2000) Dryers for particulate solids, slurries and sheet-form materials. In Mujumdar’s Practical Guide to Industrial Drying: Principles, Equipment and New Developments (Devahastin S, ed.). Thailand: Thananuch Business Ltd Publication, pp. 37–61. Nindo CI, Sun T, Wang SW, Tang J, Powers JR (2003) Evaluation of drying technologies for retention of physical quality and antioxidants in asparagus (Asparagus officinalis L). Lebensmittel Wissenschaft und Technologie, 36, 507–516. O’Neill MB, Rahman MS, Perera CO, Smith B, Melton LD (1998) Colour and density of apple cubes dried in air and modified atmosphere. International Journal of Food Properties, 1 (3), 197–205.

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Okos MR, Narsimhan G, Singh RK, Weitnauer AC (1992) Food dehydration. In Handbook of Food Engineering (Heldman DR, Lund DB, eds). New York: Marcel Dekker, pp. 437–562. Perera CO, Rahman MS (1990) Heat pump drying. Trends in Food Science and Technology, 8 (3), 75–78. Prabhanjan DG, Ramaswamy HS, Saravacos GSV (1995) Microwave-assisted convective air drying of thin layer carrots. Journal of Food Engineering, 25, 283–293. Prasertsan S, Saen-saby P (1998) Heat pump drying of agricultural materials. Drying Technology, 16 (1&2), 235–250. Rasev AI (1999) Particular features of dielectric and vacuum wood drying. In Proceedings of the 6th International Wood Drying Conference (Vermaas HF, ed.). Stellenbosch, South Africa: Stellenbosch Publisher, pp. 37–41. Rossi SJ, Neues C, Kicokbusch TG (1992) Thermodynamics and energetic evaluation of a heat pump applied to drying of vegetables. In Drying ’92 (Mujumdar AS, ed.). Amsterdam: Elsevier Science, pp. 1475–1483. Saito S, Sulaiman IB (1999) Radio frequency vacuum (RF/V) drying of small diameter Keruing. In Proceedings of the 6th International Wood Drying Conference (Vermaas HF, ed.). Stellenbosch, South Africa: Stellenbosch Publisher, pp. 234–238. Saravacos GD, Marousis SN, Raouzeos GS (1988) Effect of ethyloleate on the rate of airdrying of foods. Journal of Food Engineering, 7, 263–267. Schiffmann RF (1987) Microwave and dielectric drying. In Handbook of Industrial Drying (Mujumdar AS, ed.). New York: Marcel Dekker Inc, pp. 345–372. Shibata H, Iwao Y, Ide M (2000) Combined superheated steam and microwave drying of sintered glass beads. In Proceedings of 12th International Drying Symposium (Coumans WJ, Kerkhop PJAM, eds). Noordwijkerhout, The Netherlands: Elsevier Publication, Paper no. 363. Strømmen I, Jonassen O (1996) Performance tests of a new 2-stage counter-current heat pump fluidised bed dryer. In Drying ’96 (Strumillo C, Pakowski Z, eds). Krakow: Elsevier, pp. 563–568. Taechapairoj C, Dhuchakallaya I, Soponronnarit S, Wetchacama S, Prachayawarakorn S (2003) Superheated steam fluidised bed paddy drying. Journal of Food Engineering, 58, 67–73. Thomas WJ (1996) Radio frequency drying provides process savings: New systems optimize radio frequency drying for the ceramic and glass fibre industries. Ceramic Industry Magazine, April, 30–34. Turner IW, Jolly P (1991) Combined microwave and convective drying of a porous material. Drying Technology, 9 (5), 1209–1270.

Monitoring Thermal Processes by NMR Technology Nanna Viereck, Marianne Dyrby and Søren B Engelsen The Royal Veterinary and Agricultural University, Quality and Technology, Frederiksberg, Denmark

Nuclear magnetic resonance (NMR) possesses some unique properties for studying food while it undergoes processes such as thermal processes. This chapter begins with an introduction to the basic principles and techniques of NMR and magnetic resonance imaging (MRI). The current status of and future prospects for the use of NMR and MRI in applications of interest to food scientists and process engineers is also summarized. Both low field NMR and MRI can be used to investigate changes in food during processing, as the technique can non-invasively detect changes in the distribution and mobility of water. This application has increased considerably during the last decade mainly due to a rapid development of the NMR techniques. Furthermore, high field NMR has a large, but until now poorly utilized potential for exploring chemical, rheological and textural transformation processes in liquids and semi-solids.

1 Introduction Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) are based on the magnetic properties of the atomic nucleus and many elements have isotopes with such properties, above all the omni-abundant proton. NMR and MRI have some distinct advantages over other instrumental methods: they are noninvasive and non-destructive; most systems are transparent for the excitation; they measure volumes instead of surfaces; and the methods allow the extraction of both physical and chemical information. By exploitation of magnetic resonance it is possible to obtain unique knowledge about, for example, flow, diffusion and water distribution without perturbing the system. Similarly, processes such as heating, freezing, salting, hydration and dehydration can be monitored absolutely non-invasively. The theory of NMR spectroscopy was developed in the mid-1930s by the Dutch physicist Gorter, but it was not until 1945 that the American physicists Bloch and Purcell discovered NMR in the form that is known today (Bloch et al., 1946; Purcell et al., 1946). The possibilities of using NMR in chemistry were not appreciated until Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

21

554 Monitoring Thermal Processes by NMR Technology

1950, when Proctor and Yu discovered the so-called chemical shift which enabled elucidation of molecular structures of organic compounds (Proctor and Yu, 1950). Although originating in physics, it is in chemistry that NMR has attracted the greatest interest. The main field of use is within structural elucidation of complex molecules and NMR is an indispensable tool, especially for protein analysis in solution. The use of NMR as a general analytical tool has been developed particularly during the last 15 years. However, exploitation of NMR in food processing requires the quantitative aspect of NMR to be fully utilized. Quantitative NMR data analysis presents a new frontier and the application of NMR to complex food matrices has thus far been hindered by the lack of data analytical tools capable of utilizing the complex data that most foods generate. The development of MRI can be seen as an extension of NMR spectroscopy, where the main advantage is that MRI furthermore provides a spatial resolution, however, often at the cost of spectral resolution. In 1973, Lauterbur reported the first proton density map using NMR and in the same year Mansfield and Grannell independently demonstrated the Fourier relationship between the spin density and the NMR signal acquired in the presence of a magnetic field gradient (Lauterbur, 1973; Mansfield and Grannell, 1973). In medicine, MRI is widely established as a powerful tool for many diagnoses; however, in recent years MRI has made progress in food science as well.

2 Basic theory of NMR and MRI 2.1 Nuclear spins and energy levels About two-thirds of all isotopes possess a spin angular momentum, the magnitude of which is dependent on the size of the angular momentum quantum number (I), which is commonly referred to simply as spin. The spin of a nucleus depends on the mass of the isotope and nuclei with even mass and even charge numbers possess no spin angular momentum, i.e. I ⫽ 0. Such nuclei cannot be detected by NMR, since it is the nuclear spin property that enables NMR. When a nucleus with a non-zero spin is placed in a magnetic field, the nucleus will occupy one of a number of energy levels where the number of levels available depends on the value of I. The proton (1H) is the most abundant NMR nucleus and has spin I ⫽ 1⁄2. For such nuclei there are two different energy levels that the spins can occupy when placed in a magnetic field: I ⫽ ⫺1⁄2 and I ⫽ 1⁄2. This corresponds to an orientation parallel (I ⫽ 1⁄2) or anti-parallel (I ⫽ ⫺1⁄2) to the applied magnetic field. Figure 21.1 shows the two possible energy levels for protons and the dependence of the applied external magnetic field Bo. In the rest of this section of the NMR theory protons will be used as a model nucleus. The difference in the energy levels shown in Figure 21.1 is equal to: ⌬E ⫽ h ⭈ n

(1)

Basic theory of NMR and MRI 555

E I ⫽ ⫺½ llel

ara

i-p Ant

∆E ⫽ h ν

0

Par

alle

l I⫽½

Magnetic field strength

Bo

Figure 21.1 Representation of the two possible energy levels for nuclei with spin ⫽ 12⁄ and the influence of the strength of the applied magnetic field (Bo) on the energy difference ⌬E.

where h is Planks constant and  is the frequency of the excitation pulse at which transition between the two energy levels is induced. The frequency  is referred to as the resonance frequency or the Larmor frequency and will depend on the type of nucleus and the magnetic field strength as given by the following equation: ␯⫽

␥ ⭈ Bo 2⭈␲

(2)

Here  is the gyromagnetic ratio, which is a constant for a given nucleus; for protons ␥ ⫽ 2.6752 ⭈ 108/T ⭈ s. When at equilibrium in a magnetic field, the protons will be distributed between the two energy states according to the Boltzmann distribution: N⫺ 1

 ⌬E   ⫽ exp ⫺  k ⭈ T 

2

N1

2

(3)

In Equation (3) N ⁄ and N⫺ ⁄ represent the populations of the protons in the lower and upper energy levels, respectively, k is the Boltzmann constant and T the temperature. According to this distribution, there will be a small excess of protons in the lower energy state, since this is the energetically more favourable state. It is this small difference in population levels that is measured in NMR and a simple calculation shows that at room temperature and a magnetic field strength of 14.1 T (equal to ␯ ⫽ 600 MHz for protons), the excess in the lower energy state will be approximately 50 protons out of one million, i.e. N ⁄ /N⫺ ⁄ ⫽ 1000000/999950. It is therefore obvious why NMR is commonly described as insensitive compared to other spectroscopic methods, since a large number of protons are required to generate an appreciable signal. In addition, raising the temperature further decreases the excess in the lower energy state according to the Boltzmann distribution (Equation (3)), introducing a disadvantage when monitoring thermal heating processes. 1

1

2

2

1

1

2

2

556 Monitoring Thermal Processes by NMR Technology

Bo

M

Figure 21.2 Precession of individual spins around the external magnetic field (Bo) and the resulting net magnetization vector (M).

2.1.1 Precession and net magnetization vector

When the sample is unaffected by an external magnetic field, the orientation of the spins will be randomly distributed in all directions. However, when the sample is placed in a magnetic field, Bo, the spins will align as described in Equation (3). Under the influence of the external magnetic field the spins will start to precess about the direction of the magnetic field, as shown in Figure 21.2 for four spins in the spin-up state and two in the spin-down state. The net magnetization is the sum of all single spins and, since the spins are randomly distributed about Bo, the net magnetization vector, M, is positioned exactly on the axis of the external magnetic field. The bold arrow in Figure 21.2 represents this net magnetization vector, which is characterized by a small positive component on the axis of the external magnetic field corresponding to the small excess of protons in the spin-up state and no components on the other axes of the coordinate system. In order to facilitate the description of the spin manipulation that gives rise to the NMR signal it is normal to ascribe axes to the NMR instrument as shown in Figure 21.3. Thus, the z-axis is normally ascribed to the direction of the magnetic field (Bo) and the net magnetization vector at equilibrium, while in modern NMR instruments, both the x- and the y-axis are equipped with coils for applying radio frequency (RF) pulses and for detection. 2.1.2 RF pulses

When the sample is at equilibrium in the magnetic field there is no observable signal, since the net magnetization vector, M, has no component in the xy-plane where the signal is to be detected. By applying RF pulses at exactly the Larmor frequency, the net magnetization can be rotated from the z-axis towards the xy-plane and thus obtain an observable signal. An RF pulse of a duration precisely long enough to flip the net magnetization vector into the xy-plane will generate the largest possible signal

Basic theory of NMR and MRI 557

Bo

z

M 90°

y Detector coil x

RF-coil

Figure 21.3 Diagram of the coordinates normally ascribed to the NMR instrument including the RF coil. The drawing also depicts a perturbation of the equilibrium system by an RF pulse denoted a 90° pulse.

(the horizontal bold arrow in Figure 21.3). This pulse is referred to as a 90° or reading pulse, while a pulse with a duration long enough to flip the net magnetization vector along the negative z-axis is referred to as a 180° pulse. Once in the xy-plane the magnetization vector will continue to precess about Bo, thus inducing an oscillating signal in the detectors along the x- and y-axes.

2.2 Relaxation After a perturbation by a 90° RF pulse the spin system will begin to lose coherence in the xy-plane, a process known as spin-spin or transverse relaxation. The loss of coherence is exponential and is described by a time constant called T2. This process is due to energy exchange between spins as well as inhomogeneities in the magnetic field, which will particularly influence molecules with mobile protons and high diffusion rates, such as water. Simultaneously with the loss of coherence in the xy-plane the protons will seek to regain equilibrium orientation along the z-axis due to the influence of the magnetic field. The time it takes for the protons to regain equilibrium distribution between the two energy states depends on the probability of energy exchanges occurring between the spins and their environment (the lattice). This is characterized by a relaxation mechanism referred to as longitudinal or spin-lattice relaxation described by a time constant T1. T1 relaxation is enhanced by interactions with less mobile species and thus provides an indication of the degree of water mobility in the vicinity of macromolecules and surfaces. A long T1 is suggestive of greater mobility.

2.3 Chemical shift Protons in different positions of a molecule do not experience exactly the same magnetic field due to the effect known as shielding. When brought into a magnetic field, the motions of the electrons orbiting around an atom are perturbed in such a way that

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a magnetic field is induced in the sample that opposes the external field. Hence, the sample becomes magnetized and modifies the field. In a molecule, electrons are hindered in their rotation around a particular atom by the presence of other atoms and are therefore not capable of exerting their maximum shielding effect. Thus, differences in shielding reflect local differences in geometry and electron density in a molecule. Therefore, protons in different chemical environments experience different effective magnetic fields and thus resonate at slightly different frequencies, a phenomenon known as chemical shift. The relationship between the degree of shielding and the resulting resonance frequency is: ␯⫽

␥ ⭈ Bo * (1⫺ ␴) 2⭈␲

(4)

The term  is known as the shielding constant and is a small dimensionless number. The existence of chemical shift is what enables the NMR spectroscopist to distinguish between nuclei in different chemical environments and makes NMR spectroscopy a powerful tool for the determination of the configuration of molecules and as a general chemical analytical tool. The net magnetization vector thus consists of a number of spins that, once in the xy-plane, will precess with slightly different frequencies close to the Larmor frequency. The signal detected in NMR is the current induced in the coils by the precessing magnetization. Since we are not interested in the Larmor frequency, but in the difference in shielding and thus chemical shift, in the receiver the Larmor frequency (some MHz) is ‘subtracted’ from the detected frequency for each spin, resulting in what is called the audio signal (chemical shift frequencies, some kHz). In practice, a frequency called the carrier frequency positioned in the middle of the range of frequencies of interest is subtracted instead of the Larmor frequency, yielding both positive and negative frequencies of the different spins in the sample. The frequencies in the Fourier transformed spectrum are dependent on the size of the applied field and thus an alternative axis is defined relative to a reference compound, resulting in the chemical shift axis (in units parts per million, ppm) on which a given spin always has the same value independent of the size of the applied magnetic field. The most widely used reference substance is tetramethyl silane, Si(CH3)4 (abbreviated TMS) or in aqueous solutions the water-soluble sodium salt of trimethylsilyl proprionic acid (abbreviated TSP). TMS/TSP is a suitable reference compound, because it exhibits almost maximum shielding, so that most sample NMR peaks have a smaller shielding constant () and thus a positive value on the axis compared with the reference compound (per definition 0 ppm). The chemical shift of a sample spin can be calculated as: ␦⫽

␥sample ⫺ ␥reference ␥reference

⭈ 106 ppm

(5)

In cases where the addition of TMS/TSP to the sample for some reason is not an option, other reference compounds can be used, providing that the chemical shift value is well known.

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2.4 Detection and Fourier transformation The presence of spins moving both clockwise and counter-clockwise in the xy-plane is the reason for using a detection system with two simultaneous channels which are mutually 90° out of phase. This system is called quadrature detection and enables the discrimination between a spin with a frequency ⫺␯ and one with a frequency +␯, something which is not possible when detection is only done on one axis. During a modern NMR experiment, the signal is measured in the time domain, i.e. as a function of time. This signal, a superposition of sinusoids damped due to relaxation, is Fourier transformed to obtain the spectrum in the frequency domain. The signals from the two detectors are handled as the real and imaginary parts of a complex signal, enabling the use of complex Fourier transformation. Prior to and after Fourier transformation several cosmetic operations are performed that influence the signal-tonoise ratio of the spectrum, the resolution between peaks and the correction of artefacts such as deviations from a flat horizontal baseline. These include zero filling, application of window functions and baseline corrections, topics that will not be discussed further here.

2.5 Pulse sequences The possibility to design pulse experiments for the selection of specific signals is the basis for the enormous diversity of NMR applications. A few selected pulse experiments will be presented which cover the common experiments used in the applications described in Section 3. As a basis, the simplest unselective pulse experiment, the free induction decay (FID), will be described, followed by experiments evaluated to measure relaxation times as well as an example of a pulse experiment designed to measure self-diffusion of molecules in solution. Finally, a short review will be given of solvent suppression techniques which can be implemented in both the simple and the more complex pulse experiments described in the literature. 2.5.1 Free induction decay – FID

This pulse experiment is used when the purpose of the experiment is to view all nuclei of a given type in the sample. It is the simplest pulse experiment possible and employs the application of a short unselective 90° RF pulse (also termed a hard pulse) and the measurement of the decreasing signal in the xy-plane due to relaxation following the pulse. The duration of a 90° pulse is typically in the order of a few microseconds to excite the spin system unselectively over a given bandwidth, whereas the data acquisition time may be as long as seconds. All resonances are excited simultaneously and the FIDs from all components superimpose in the time domain, but may be separated in high field NMR according to chemical shift by the Fourier transform. 2.5.2 Measurement of relaxation times

Measurement of T1 and T2 relaxation times can be accomplished through two designed pulse sequences. The inversion recovery pulse sequence is typically used to

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measure T1. It consists of a 180° RF pulse, followed by a 90° RF pulse, with a variable delay, , in between. After application of a perfect 180° pulse, the magnetization will lie exactly along the z-axis, maintaining a magnitude M. If now the delay between the pulses is zero, a 90° pulse will rotate the magnetization to the xy-plane, detected as an inverted magnetization of magnitude M. The magnitude of the detected magnetization following the 90° pulse varies with the delay, , as:   ␶  M(␶) ⫽ M0 1 ⫺ 2 exp ⫺   T1   

(6)

where M( ) is the magnetization in the longitudinal orientation at time and M0 is the initial equilibrium magnetization. Therefore, T1 can be determined by measuring multiple 180° – – 90° sequences with varying . Since T2 describes the decay of the magnetization in the xy-plane, measurement of T2 should be straightforward as identical to the time constant of the FID following a single 90° pulse. However, unlike T1 the measured value of T2 is severely affected by instrumental imperfections, diffusion, etc. The decay rate of the FID, termed 1/T 2* (experimental spin-spin relaxation), is always greater than 1/T2, since magnetic field inhomogeneity is an important contributor. Hence, nuclei in different parts of the sample experience slightly different values of the magnetic field and thus precess at slightly different frequencies. The result is a loss in phase coherence similar to that induced by real T2 processes. Nevertheless, it is possible to measure the real T2 by using the Carr-Purcell-Meiboom-Gill pulse sequence (Carr and Purcell, 1954; Meiboom and Gill, 1958). This sequence creates a spin echo in the xy-plane, where effects due to magnetic field inhomogeneity and diffusion are refocused. The decay of the magnetization in the xy-plane is simply plotted as a function of the length of the spin echo sequence determined by the repetition of the number, n: 90° ⫺ ␶ ⫺ (180° ⫺ 2␶)n ⫺ 180° ⫺ ␶ ⫺ measure. 2.5.3 Diffusion-editing

The purpose of diffusion-edited NMR experiments (also known as diffusion ordered spectroscopy, DOSY) can be many, including to determine self-diffusion coefficients of molecules in solution, to monitor intermolecular binding, to separate signals from molecules with similar or overlapping NMR signals but different size, to edit the NMR spectrum to represent molecules of a specific size only or to suppress signals from fast moving species such as water. Diffusion-editing is usually acquired as a 2D spectrum consisting of a series of 1D spectra through which the intensity of signals decreases exponentially due to diffusion. The relationship between signal intensity, diffusion coefficient and gradient strength is: I ⫽ q ⭈ M0 ⭈ exp[⫺D ⭈ ␥2 ⭈ ␦2 ⭈ g 2 ⭈ ⌬⬘⫺ R]

(7)

where I is the measured signal intensity, M0 is the equilibrium magnetization, D is the diffusion coefficient (m2/s), ␥ is the gyromagnetic ratio (/s/T), ␦ is the time duration of the applied gradient pulse (s) and g is the applied gradient strength (T/m). The term

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 is equal to ⌬ ⫺ ␦ ⭈ k, where ⌬ is the diffusion time (the time between the two gradient pulses) and k a correction factor which depends on the shape of the gradient pulses. The term R is a constant that accounts for relaxation and the constant q is a correction factor accounting for the loss of signal when using pulse experiments based on the stimulated echo sequence. 2.5.4 Water suppression

Many biological samples contain large amounts of water, which gives a very strong signal in NMR of 110 molar in protons. The purpose of water suppression is to obtain agreement between, on one hand, the dynamic range of the interesting NMR signals and, on the other hand, the dynamic range of the analogue-to-digital converter and of the receiver. In some cases, the purpose may also be to avoid the disappearance of small signals on the huge water peak. Various means of suppressing the water signal exist. Only two methods will be discussed here: Presaturation and WATERGATE. One of the simplest, most robust and widely used methods is presaturation, where the water signal is dephased through a long weak pulse (also termed a soft pulse), a pulse which affects only the water peak. The presaturation pulse is applied at the beginning of a pulse sequence and can thus be implemented into practically any pulse experiment. The method is simple and reasonably effective, but has the disadvantage of also partly suppressing other signals close to the water peak. The effectiveness is impaired in long pulse sequences where the water may regain some phase coherence and thus detectable signal. A more complex – but also very effective – water suppression technique is the WATERGATE (water suppression by gradient tailored excitation) sequence (Piotto et al., 1992), which is usually implemented as the last part of a pulse sequence. It employs two gradient pulses of equal strength and polarity with a sequence consisting of three pairs of symmetric hard pulses in between. The hard pulse sequence has no effect on the water resonance, but the effect of a 180° pulse for all other resonances. Thus, the water signal is further dephased by the second gradient pulse, while all other resonances are refocused.

2.6 Magnetic resonance imaging Like NMR spectroscopy, MRI is a method that exploits the magnetic properties of the atomic nucleus. However, where the homogeneity of the static magnetic field is critical for the measurement of useful NMR spectra, it is the controlled use of imposed inhomogeneities which allows spatial information about the distribution of spins to be further extracted from signals into magnetic resonance images. The spatial encoding of the signals is produced by creating variable, spatially controlled linear magnetic field gradients in all three dimensions, x, y, z. Consequently, while gradients are applied, the precession frequency of the nuclei in a sample is now also a function of their position within the sample, leading to an extension of Equation (2): ␯⫽

␥ ⭈ (Bo ⫹ G ⭈ r ) 2⭈␲

(8)

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where G is the added gradient field which is a function of spatial position, r. The signals from different parts of a sample are thus different and spatial information expressed as an image can be recovered from the signals emitted by the whole sample. Signals are detected from subvolumes within the larger sample, termed voxels. Each voxel is the three-dimensional volume from which the signal is detected and displayed as a two-dimensional pixel in the image. These pixels are arranged in slices, representing a two-dimensional image. Multiple slices can be arranged together to form a three-dimensional volume image. The intensity of the signal from each voxel of sample is determined by: spin density (how many detectable nuclei are present within the voxel), T1 relaxation, T2 and T *2 relaxation and movement (e.g. diffusion and flow). The contrast in MRI images is provided by variation in these parameters in different parts of the sample. Accordingly, it is the dependence of these parameters on the level, mobility and binding of water in the sample that makes MRI useful in food science. 2.6.1 MRI pulse sequences

In principle, it is possible to perform the same experiments with MRI as with NMR spectroscopy, with only field gradients and short unselective RF pulses. Many images are acquired using combinations of RF pulses or gradient reversal pulses to refocus the spin phases in an echo, where the signal is acquired by recording the time dependence of the echo amplitude. A spin echo pulse sequence can be designed to emphasize different patterns of contrast, e.g. T1- or T2-contrasted. Furthermore, it is often useful to use longer, lower amplitude RF pulses to excite selectively. This can be applied either as chemical selection (as used for water presaturation described above), or in the presence of a magnetic field gradient the selective RF pulse can be used to excite only those spins within a particular layer of the sample, referred to as slice selection. Choice of pulse sequence is important in determining patterns of contrast, since the ability to distinguish specific tissues varies with pulse sequences. One possibility is chemical shift imaging, where compounds whose signals appear in the image are selected by their chemical shift. Other available and often used pulse sequences include fast imaging sequences, i.e. FLASH (fast low angle shot), FAST (fast), EPI (echo planar imaging) and projection imaging. These techniques are all fast enough to follow changes on the time scale of food processing. However, due to limited space, these sequences will not be discussed here and readers who wish to gain insight into these techniques are referred to several books available (Callaghan, 1995; Hills, 1998).

2.7 NMR and MRI instruments An NMR spectrometer consists basically of a magnet surrounding the sample, an RF transmitter, receiver and a computer. For low field (also referred to as low resolution) NMR operating at the lower range of magnetic field strength (0.235–2.35 Tesla, corresponding to a 1H NMR frequency of 10–100 MHz) a permanent magnet is often used. Such an instrument is available as a benchtop system and is capable of measuring solid-liquid ratio, moisture or oil content and relaxation times in whole foods via

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time-domain decays. High field (or high resolution) NMR requires a superconducting magnet to generate the field strength necessary (7.05–14.1 Tesla, corresponding to a 1 H NMR frequency of 300–600 MHz). Performing high field NMR on either liquids or solids results, in principle, in complete chemical analysis of specific components in whole foods via a frequency domain spectrum. However, the complex data that most foods generate often limit the interpretation of the spectrum. Furthermore, in solid state high field NMR, resolution in the spectra suffers from dipole-dipole couplings and magnetic susceptibility inhomogeneity which broadens the signals. Resolution is recovered by using of magic angle spinning (MAS), where the sample is placed in a rotor and spun at thousands of hertz about an axis that makes the angle 54.7° – the magic angle – with the static magnetic field. The use of magnetic field gradients permits diffusion-edited NMR to define both bulk diffusion and molecular self-diffusion rates. The extension of the instrument with gradient coils inside the main magnet in principle also leads to an MRI system and the stronger the gradients used, the better the spatial resolution of the image. The advent of fast and functional imaging techniques has further added to MRI’s ability to define not only structure, but spatially defined processes. MRI instruments normally operate at the lower range of magnetic field strength (0.5–1.5 Tesla, corresponding to a 1H NMR frequency of 21–64 MHz); however, applications using high field MRI magnets can be found in the literature. The food sample for both NMR and MRI can be virtually anything from liquids (beverage, oil, milk) to semi-solids (cheese, gel, butter, yogurt, emulsion) and solids (milk powder, sugar, grain), depending only on how the instrument is equipped. This introduction to the basic theory of NMR and MRI has hopefully provided a suitable background for the following interpretation of various applications of NMR for monitoring thermal processes.

2.8 NMR and multivariate data analysis The feasibility of NMR in food research including thermal processing has been hampered by the lack of tools capable of utilizing the complex NMR data that most foods generate, but advanced mathematical modelling (chemometrics) has the potential of taking NMR to a new frontier within food research. The two basic algorithms used in chemometrics are principal component analysis (PCA) and partial least squares (PLS) regression. They are based on the principle of two-dimensional data analysis in which data from several samples are analysed simultaneously to extract common latent structures and individual scores or concentrations (Figure 21.4). PCA finds the main variation in a multidimensional data set by creating new linear combinations of the raw data, a method which is especially well suited to highly collinear data, as is the case in most spectroscopic or instrumental techniques (Bechmann et al., 1999; Hotelling, 1933). In short, a data matrix (samples [x] spectra) X is decomposed into a lower dimensional score matrix (T) and a loading matrix (P). In this way the information in X is projected onto a lower dimensional subspace where the loading vectors for the principal components (PC) can be considered as pure

564 Monitoring Thermal Processes by NMR Technology

X

O

Figure 21.4 Schematic representation of the principle behind two-dimensional (chemometric) data analysis. From a data table with many observed variables for each sample, important patterns are found and visualized as latent structures and related scores (concentrations).

mathematical spectra that are common to all the measured spectra. What makes the individual raw spectra different are the amounts (scores) of hidden spectra (loadings). PLS regression is the supervised version of PCA and applies to the simultaneous analysis of two sets of variables on the same objects (Wold et al., 1983; Martens and Næs, 1993). PLS is commonly used in quantitative spectroscopy to correlate the spectroscopic data (X-block – fast spectral measurements) with related physical-chemical data (Y-block – time-consuming and laborious measurements). The main purpose of the regression is to build a predictive model as a screening tool to enable the prediction of a desired characteristic (y) from a measured spectrum (x). The performance of a PLS regression is often measured in a correlation coefficient and a root mean square error of prediction (RMSEP).

3 NMR and thermal processes One of the important advantages of NMR as a technology for food processing is that it is possible to take measurements from the sample over a period of many hours. Changes in the measured data can be related to changes in the sample and, in this way, it is possible to probe the response of samples to various processing treatments. Temperature-dependent magnetic resonance parameters include the molecular selfdiffusion coefficient, relaxation times and the proton chemical shift. The following sections discuss some of the NMR and MRI approaches that have been found useful.

3.1 Rheo-NMR The rheological properties of foods are of great importance to their texture, to their processing and to their storage characteristics and characterization of the rheological properties is an essential step in the optimization of many manufacturing processes. Semi-solids, such as many foods, are both solid- and liquid-like in nature. For this reason, they show both viscous and elastic responses when exposed to deformation. The

NMR and thermal processes 565

rheological behaviour originates in molecular composition interactions and dynamics and naturally a molecular investigation is necessary fully to understand the underlying processes. Low field NMR and MRI have recently been evolved into this area, mainly due to the capacity of the RF pulse to penetrate the whole sample and the effectiveness of the method to distinguish between the liquid and solid phases, based on the varying mobility of the species reflected in the relaxation parameters. The relaxation time constants (T1 and T2) represent the relaxation rates of the different types of protons (e.g. fat, structural water, bulk water) in the sample. Rheo-NMR requires a special setup created inside the magnet within the RF coil. Several shearing and extensional flow cells have been developed for use within an NMR system with microimaging feasibility (Britton et al., 1998). The combination of flow imaging pulse sequences with other pulse sequences sensitive to relaxation times, diffusion coefficients or chemical shifts makes it, in principle, possible to probe velocity simultaneously with other parameters like reaction rate, concentration or temperature. The potential of magnetic resonance to measure shear viscosity of fluids has been investigated in detail during the last decade (Arola et al., 1997, 1999). Several studies have since employed rheo-NMR especially for polymer conformation changes like investigations of shear flow in semi-dilute solutions of polyacrylamide (Callaghan and Gil, 2000) and characterization of shear-sensitivity of hyaluronan in aqueous solution (Fischer et al., 2002). Rheological profiling of complex fluids with reference to food systems has been studied (Britton and Callaghan, 1997; Callaghan, 1999) and, recently, rheo-NMR has been used in the analysis of whole food systems as in the study of temperature dependent rheology of butter, semi-soft butter and margarine (Britton and Callaghan, 2000) and in simplified food systems as in the investigation of local molecular dynamics in a food system containing carrageenan gels and sols (Callaghan and Gil, 2001). In conclusion, the relatively few reported applications of rheo-NMR in food science only reveal a small selection of the many possible applications of NMR to investigate the complex rheology of food systems. It would appear that NMR is an ideal technology for studying both macroscopic shear rheology by providing magnetic resonance images and microscopic interfacial rheology by providing information about molecular interactions.

3.2 NMR and MRI baking Low field 1H NMR and MRI have recently been introduced in studies of bread and dough (see below). Magnetic resonance has the advantage that it enables the detection of changes in the distribution and mobility of mainly water protons crucial to the process of making dough into bread and at the other extreme to the staling of breadcrumb. In general, transverse T2 relaxation of complex food systems such as bread and dough exhibit multicomponent behaviour (usually two to four components) in which the individual components can be interpreted as representing different water regions and diffusive exchange between separate regions in the system (Belton, 1990). The investigation of water mobility and migration in dough and bread using NMR and MRI has to date yielded ambiguous results, as the number of components and

566 Monitoring Thermal Processes by NMR Technology

related time constants vary (Leung et al., 1979, 1983; D’Avignon et al., 1990; Ruan et al., 1996; Chen et al., 1997; Roudaut et al., 1998; Fukuoka et al., 2000; Engelsen et al., 2001; Kim and Cornillon, 2001). Based on water absorption values of various components, 46 per cent of the total water in dough is estimated to be associated with starch, 31 per cent with proteins and 23 per cent with pentosans. In bread, 77 per cent of the total water is estimated to be associated with starch, none with proteins and 23 per cent with pentosans (Bushuk, 1966). The major storage quality problem with bread is staling, which results in a firm and inelastic breadcrumb. Staling is generally associated with starch retrogradation, but changes in other components and their interactions with starch may also play important roles in the staling process. Whatever the mechanism, the protons in the system should become increasingly less mobile during staling. Decreasing relaxation time constants during staling, indicating an overall decrease in water mobility, has been reported in several NMR experiments (Wynne-Jones and Blanshard, 1986; Kim-Shin et al., 1991; Seow and Teo, 1996; Chen et al., 1997). Strong significant correlations between NMR relaxation data and firmness, measured by texture analysis, have also been reported (Seow and Teo, 1996; Chen et al., 1997). These correlations were based on the NMR solid-liquid phase ratio signal and relaxation time constants of protons in bread, respectively. Moreover, changes in T2 values found by MRI have also been found to be highly correlated to firmness, as measured by texture analysis (Ruan et al., 1996). In an NMR study of baking and determination of instrumental texture parameters in bread, both the kinetics of the bread baking process and staling of white breadcrumb was investigated by NMR using multivariate data analysis (chemometrics) (Engelsen et al., 2001). The T2 relaxation of protons was measured during a baking process performed inside a low field NMR spectrometer (Figure 21.5). To study the

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NMR and thermal processes 567

overall behaviour of the dough during the baking process, a PCA was performed on the FID and T2 relaxation data. By this simple setup, two major phase-transitions were detected during NMR baking: one at 55°C (beginning of gelatinization of starch) and one at 85°C (end of gelatinization). Furthermore, multi-exponential curve-fitting was used to determine the number of components in the system and throughout the entire baking process, from dough to bread, three T2 components were determined. The staling of white breadcrumb aged 0–8 days was investigated by texture analysis and NMR relaxation in the same study. PLS regression models were applied to relate the two data sets and high correlations were found. Consequently, staling can be monitored with NMR relaxation data evaluated by chemometrics (Engelsen et al., 2001). MRI, in particular, has also been applied to other aspects of bread properties. In a recent study, dough fermentation was monitored by MRI and volumetric measurements and the pore structure was observed by MRI (Goetz et al., 2003). In another study, the gas cell formation during proving of dough was monitored by MRI (van Duynhoven et al., 2003). Various stress effects (kneading temperature and moulding) were introduced and the dough volume subsequently investigated by MRI. Using MRI, moulding could be dedicated the largest negative effect. Thus far, only very simple NMR and MRI measurements have been performed to investigate dynamically the dough-to-bread process. Future NMR applications will provide a much more detailed picture of the water migration, intermolecular interactions and phase-transitions occurring during bread baking, allowing also for detailed studies of the functionality of additives and ingredients.

3.3 Cooking with NMR Conversion of living muscle into ‘ready to eat’ meat is one of the most longstanding and important food processes known to mankind. In the meat industry, much attention has been directed towards the understanding and optimization of the quality of meat as a raw material. Low field NMR has been implemented in the study of the distribution of water in muscle and has proven to be a powerful tool for identifying water components. The results have been related mainly to water-holding capacity (Bertram et al., 2001; Brøndum et al., 2000b) and sensory properties (Fjelkner-Modig and Tornberg, 1986; Brøndum et al., 2000a). A few investigations have been carried out on the effect of cooking on the states of water in meat using low field NMR (Fjelkner-Modig and Tornberg, 1986; Borisova and Oreshkin, 1992; Tornberg et al., 1993), but not with continuous or dynamic measurements. Dynamic low field NMR measurements have proven to provide valuable information about complex food items undergoing processing or storage (see previous sections). A natural extension is cooking of meat inside the NMR instrument to study the transitions in the states of water occurring at different temperatures in the process of cooking meat. This approach was applied in the two reports described in the following (Micklander et al., 2002; Bertram et al., 2004). In the study by Micklander et al. (2002), low field NMR data obtained during cooking of meat were utilized to identify important temperatures at which major changes in meat

568 Monitoring Thermal Processes by NMR Technology

46 Myosin transverse shrinkage

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Figure 21.6 Unsupervised PCA from NMR cooking of meat represented by 43 consecutive measurements (relaxation decays) obtained during a period of 4 hours. PC1, PC2 and PC3 explain 97 per cent, 2.3 per cent and 0.6 per cent of the variance in the NMR data. The approximate centre temperatures and physical event are indicated where all major changes in the scores are observed.

structure occur. As in the study of NMR baking (Engelsen et al., 2001), NMR relaxation data evaluated by chemometrics were used. It could be observed from the relaxation curves obtained throughout the process of cooking that the overall proton relaxation rate steadily increased with temperature. This was (primarily) due to the increased mobility of protons with increasing temperature. It was also revealed that the total NMR signal towards higher temperatures was reduced due to the less favourable Boltzmann distribution between the two spin-states, resulting in a decrease in the signal-to-noise relationship with increasing temperature. A PCA identified large changes in the properties of water at 46 and 66°C, but minor changes were also observed at approximately 42, 57 and 76°C. These transition temperatures can be explained by changes in the constituents of meat during cooking, namely, denaturation and shrinkage of the myofibrillar proteins and the connective tissue, which affects the water-holding capacity/distribution of water in meat (Figure 21.6). Furthermore, the characteristics of the water population present in the meat at different stages of cooking were estimated by multi-exponential curve-fitting of the relaxation curves (Figure 21.7). It was found that a new water population was developed in meat as a result of cooking (above 40°C) and a dual hypothesis was presented – the new water component can be either water expelled from the meat matrix or it can be water trapped in a myosin gel (Micklander et al., 2002). In a subsequent study, low field NMR was used to investigate the effects of curing and the Rendement Napole (RN) gene on water properties during cooking of pork (Bertram et al., 2004). Distributed exponential fitting revealed transition from a system with relatively well-separated components to a less well-defined system with a

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Figure 21.7 Contour plot of distributed exponential fitting of T2 relaxation curves from NMR cooking of meat (16–94°C). Result from discrete exponential fitting is superimposed on the plots; bi- and triexponential fitting overlap in the temperature interval 40–60°C. (Adapted from Micklander et al. (2002)).

wide distribution of relaxation times and merged components during cooking. In addition, distributed exponential fitting analysis implied changes in relaxation characteristics that were tentatively ascribed to denaturation of myosin and subsequent shrinkage of the myofibrillar structures. The results were further suggested to reflect formation of new compartments in the myofibrillar lattice during the shrinkage of the existing structures with a succeeding redistribution of water. Nitrite curing was found to affect the distribution of water as well as the progress in the different water populations during cooking. A PCA revealed two major shifts in relaxation characteristics around 43 and 56°C in uncured samples and around 43 and 63°C in cured samples and the shift around 43°C was found to be significantly affected by RN-genotype. The strong shift in water properties around 43°C is suggested to be a result of myosin denaturation and thus the results implied differences in myosin denaturation between meat from RN-carriers and meat from non-carriers. The second shift in water properties was suggested to reflect the onset of collagen shrinkage causing longitudinal shrinkage of meat and, accordingly, the shift from 56 to 63°C in the presence of salt pointed towards an effect of curing on structural alterations during cooking. From a sampling and measurement perspective, opaque and water-rich meat samples are difficult to handle. NMR would appear to be an ideal monitoring technique for heating processes in other food samples (especially starchy foods such as rice grains, noodles or potatoes) and the results are already numerous.

3.4 MRI freezing The formation of ice during the freezing of food has also been examined in several studies using MRI. Accordingly, the textural changes associated with the freezing

570 Monitoring Thermal Processes by NMR Technology

process should be reflected by changes in both the spatial distribution of the water and the value of its MRI parameters. Many questions could be solved by studying freezing processes by MRI, from illegal sale of meat as fresh, although previously frozen and thawed (Guiheneuf et al., 1997; Hall et al., 1998) to optimization of the storage of various foods (Kerr et al., 1996, 1998; Kuo et al., 2003). Relaxation data are often used in these studies. It has been found that T1 relaxation times are particularly sensitive to freeze-thawing of various meat samples, with a significant decrease in the T1 value upon freezing. This is explained by protein denaturation and aggregation in the frozen meat (Evans et al., 1998; Hall et al., 1998). Spatial changes in the distribution of water T2 relaxation times and changes of water self-diffusion coefficients measured by MRI have been used in a study of the effects of freezing on mozzarella cheeses (Kuo et al., 2003). Liquid water turning to ice during the freezing of food can also be monitored straightforwardly, since this should be seen as a reduction in the spatially located MRI signal intensity (Kerr et al., 1996, 1998). By this approach, MRI could easily serve to assess optimized freezing times and storage temperatures.

4 Future directions for process NMR Low field NMR and MRI are particularly widespread in the food industry due to their excellent capacity to measure physical and spatial information of water and fat. The research carried out on the basis of these techniques has demonstrated that one truly powerful asset of low field magnetic resonance is the ability to probe samples during ongoing processes such as baking, cooking and freezing. High field NMR is probably the most successful and versatile spectroscopic technique yet to be developed and, although its implementation as an on-line monitoring tool is severely limited by the requirement of a strong external magnetic field and the high costs of equipment, it is foreseen that this technique will also permeate the more advanced segments of the food industries for process control. The ongoing improvements in NMR hardware and data analysis tools will continue to expand new NMR applications. As described above, NMR is sensitive to both chemical and mobility changes within the sample. This leads to the many possible applications within low field NMR; however, it also introduces a problem into traditional high field liquid NMR. The signals in the spectra are widely broadened and overlap each other, leaving a poor spectral resolution. High resolution magic angle spinning (HR MAS) overcomes this problem and the recent development of HR MAS hardware for conventional high field liquid NMR spectrometers will continue the expansion of new NMR food applications, e.g. in the dairy industry. Moreover, the feasibility of especially high field NMR in food research has thus far been hindered by the lack of tools capable of utilizing the complex data that most foods generate, but advanced mathematical modelling – chemometrics – has the potential of taking high field NMR to a new frontier within food science.

Nomenclature 571

5 Conclusions NMR, which is the most successful and versatile analytical technique of our time, is capable of providing complex multivariate information concerning food samples and systems and has a great potential for monitoring thermal transformation processes in liquids, gels and suspensions. Predictive mathematical models in combination with spectroscopic sensors have an enormous potential in food research and industry to control and monitor the quality of the raw materials, the food processing and the final products. A growing interest is therefore foreseen in advanced fingerprinting methods, including high resolution NMR sensors. In this chapter, it has been shown that NMR measurements can serve as a window into monitoring thermal processes which occur inside complex and optically opaque food matrices, despite the low sensitivity of NMR, as changes can be observed in a noninvasive manner. Especially excellent is the technique for detection of changes in the distribution and mobility of water. It is to be foreseen that the next research frontier in thermal food processing will require a molecular understanding of main events leading to desirable and safe foods. The multiparametric and multifaceted NMR in combination with multivariate data analysis seems to have the potential to be a direct source of information for the molecular understanding of the complex phenomena occurring in food during thermal processing, including denaturation, mass transport phenomena, phase transitions and even the formation and release of flavoursome and toxic compounds.

Nomenclature 1D 2D Bo  D DOSY E EPI FID FLASH  g G 1 H h HR MAS I k

one dimensional two dimensional external magnetic field chemical shift or time duration of gradient pulse diffusion time diffusion coefficient diffusion ordered spectroscopy energy difference echo planar imaging free induction decay fast low angle shot gyromagnetic ratio gradient strength gradient field proton Plank’s constant high resolution magic angle spinning quantum number or signal intensity Boltzmann constant or correction factor

572 Monitoring Thermal Processes by NMR Technology

kHz m M M0 MAS MHz MRI  N NMR P PC PCA PLS ppm q r R RF RMSEP RN s 

T T T T1 T2 T2* TMS TSP WATERGATE X Y

kilohertz metre net magnetization vector equilibrium magnetization magic angle spinning megahertz magnetic resonance imaging Larmor frequency population nuclear magnetic resonance loading matrix principal components principal component analysis partial least squares parts per million correction factor spatial position constant accounting for relaxation radio frequency root mean square error of prediction Rendement Napole second shielding constant time delay temperature score matrix Tesla longitudinal or spin-lattice relaxation time transverse or spin-spin relaxation time experimental transverse or spin-spin relaxation time tetramethyl silane trimethylsilyl proprionic acid water suppression by gradient tailored excitation data matrix data matrix

References Arola DF, Barrall GA, Powell RL, McCarthy KL, McCarthy MJ (1997) Use of nuclear magnetic resonance imaging as a viscometer for process monitoring. Chemical Engineering Science, 52, 2049–2057. Arola DF, Powell RL, Barrall GA, McCarthy MJ (1999) Pointwise observations for rheological characterization using nuclear magnetic resonance imaging. Journal of Rheology, 43, 9–30.

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D’Avignon DA, Hung CC, Pagel MTL, Hart B, Bretthorst GL, Ackerman JJH (1990) 1H and 2H NMR studies of water in work-free wheat flour doughs. In NMR Applications in Biopolymers (Finley JW, Schmidt SJ, Serianni AS, eds). New York: Plenum Press, pp. 391–414. Engelsen SB, Jensen MK, Pedersen HT, Nørgaard L, Munck L (2001) NMR-baking and multivariate prediction of instrumental texture parameters in bread. Journal of Cereal Science, 33, 59–69. Evans SD, Nott KP, Kshirsagar AA, Hall LD (1998) The effect of freezing and thawing on the magnetic resonance imaging parameters of water in beef, lamb and pork meat. International Journal of Food Science and Technology, 33, 317–328. Fischer E, Callaghan PT, Heatley F, Scott JE (2002) Shear flow affects secondary and tertiary structures in hyaluronan solution as shown by rheo-NMR. Journal of Molecular Structure, 602, 303–311. Fjelkner-Modig S, Tornberg E (1986) Water distribution in Porcine M. longissimus dorsi in relation to sensory properties. Meat Science, 17, 213–231. Fukuoka M, Mihori T, Watanabe H (2000) MRI observation and mathematical model simulation of water migration in wheat flour dough during boiling. Journal of Food Science, 65, 1343–1348. Goetz J, Gross D, Koehler P (2003) On-line observation of dough fermentation by magnetic resonance imaging and volumetric measurements. European Food Research and Technology, 217, 504–511. Guiheneuf TM, Parker AD, Tessier JJ, Hall LD (1997) Authentication of the effect of freezing/thawing of pork by quantitative magnetic resonance imaging. Magnetic Resonance in Chemistry, 35, 112–118. Hall LD, Evans SD, Nott KP (1998) Measurement of textural changes of food by MRI relaxometry. Magnetic Resonance Imaging, 16, 485–492. Hills BP (1998) Magnetic Resonance Imaging in Food Science. New York: John Wiley & Sons, Inc. Hotelling H (1933) Analysis of a complex of statistical variables into principal components. Journal of Educational Psychology, 24, 417–441. Kerr WL, Kauten RJ, Ozilgen M, McCarthy MJ, Reid DS (1996) NMR imaging, calorimetric, and mathematical modeling studies of food freezing. Journal of Food Process Engineering, 19, 363–384. Kerr WL, Kauten RJ, McCarthy MJ, Reid DS (1998) Monitoring the formation of ice during food freezing by magnetic resonance imaging. Lebensmittel-Wissenschaft und Technologie, 31, 215–220. Kim YR, Cornillon P (2001) Effects of temperature and mixing time on molecular mobility in wheat dough. Lebensmittel-Wissenschaft und Technologie, 34, 417–423. Kim-Shin MS, Marí F, Rao PA, Stengle TR, Chinachoti P (1991) 17O Nuclear magnetic resonance studies of water mobility during bread staling. Journal of Agricultural and Food Chemistry, 39, 1915–1920. Kuo MI, Anderson ME, Gunasekaran S (2003) Determining effects of freezing on pasta filata and non-pasta filata Mozzarella cheeses by nuclear magnetic resonance Imaging. Journal of Dairy Science, 86, 2525–2536.

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Lauterbur PC (1973) Image formation by induced local interactions – examples employing nuclear magnetic-resonance. Nature, 242, 190–191. Leung HK, Magnuson JA, Bruinsma BL (1979) Pulsed nuclear magnetic resonance study of water mobility in flour doughs. Journal of Food Science, 44, 1408–1411. Leung HK, Magnuson JA, Bruinsma BL (1983) Water binding of wheat flour doughs and breads as studied by deuteron relaxation. Journal of Food Science, 48, 95–99. Mansfield P, Grannell PK (1973) NMR diffraction in solids. Journal of Physics C – Solid State Physics, 6, 422–426. Martens H, Næs T (1993) Multivariate Calibration. New York: Wiley. Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. The Review of Scientific Instruments, 29, 688–691. Micklander E, Peshlov B, Purslow PP, Engelsen SB (2002) NMR-cooking: Monitoring the changes in meat during cooking by low-field 1H-NMR. Trends in Food Science and Technology, 13, 341–346. Piotto M, Saudek V, Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous-solutions. Journal of Biomolecular NMR, 2, 661–665. Proctor WG, Yu FC (1950) The dependence of a nuclear resonance frequency upon chemical compound. Physical Review, 77, 717. Purcell EM, Torrey HC, Pound RV (1946) Resonance absorption by nuclear magnetic moments in a solid. Physical Review, 69, 37–38. Roudaut G, van Dusschoten D, Van As H, Hemminga MA, Le Meste M (1998) Mobility of lipids in low moisture bread as studied by NMR. Journal of Cereal Science, 28, 147–155. Ruan R, Almaer S, Huang VT, Perkins P, Chen P, Fulcher RG (1996) Relationship between firming and water mobility in starch-based food systems during storage. Cereal Chemistry, 73, 328–332. Seow CC, Teo CH (1996) Staling of starch-based products: a comparative study by firmness and pulsed NMR measurements. Starch/Stärke, 48, 90–93. Tornberg E, Andersson A, Göransson Å, von Seth G (1993) Water and fat distribution in pork in relation to sensory properties. In Pork Quality: Genetic and Metabolic factors (Poulanne E, Demeyer DI, eds). Wallingford, Oxfordshire: CAB International, pp. 239–258. van Duynhoven JPM, van Kempen GMP, van Sluis R et al. (2003) Quantitative assessment of gas cell development during the proofing of dough by magnetic resonance imaging and image analysis. Cereal Chemistry, 80, 390–395. Wold S, Martens H, Wold H (1983) The multivariate calibration problem in chemistry solved by the PLS method. Lecture Notes in Mathematics, 973, 286–293. Wynne-Jones S, Blanshard JM (1986) Hydration studies of wheat starch, amylopectin, amylose gels and bread by proton magnetic resonance. Carbohydrate Polymers, 6, 289–306.

Vacuum Cooling of Foods Liyun Zheng and Da-Wen Sun Food Refrigeration and Computerised Food Technology Group, National University of Ireland, Dublin, Ireland

Vacuum cooling is a rapid evaporative cooling technique, which is mainly achieved through evaporating part of the moisture of the product under vacuum. The advantages of vacuum cooling include shorter processing time, extended product shelf-life, improved product quality and safety. These have consequently increased its popularity among food manufacturers and research scientists. Initially, this chapter outlines the principles and equipment of vacuum cooling. Further to this, it surveys the current status of vacuum cooling in various sectors of the food processing industry, which include both well-established commercial applications and applications that still remain at the research stage. The advantages and disadvantages of this technique compared to other cooling methods are also discussed, as well as factors that affect its performance.

1 Introduction Vacuum cooling is achieved through boiling part of the moisture of the product under vacuum conditions. The major characteristic of vacuum cooling is that the product can be cooled at extremely high speed, which is unsurpassed by conventional cooling methods. Traditionally it has been used to remove field heat of leafy vegetables after harvest so as to prolong product shelf-life (Anon, 1981). In the past decade, its application has been extended to other sectors of the food industry, e.g. bakery, fishery, sauces and particulate foods processing, etc. (Everington, 1993; Shaevel, 1993; McDonald and Sun, 2000; Wang and Sun, 2001). Recent trends in the food industry also highlight a growing interest in integrating vacuum cooling into the processing procedures of some prepared consumer foods, e.g. cooked meats and ready meals, the potential of which has been shown promising and research work is still ongoing (McDonald and Sun, 2000; Wang and Sun, 2001; Zheng and Sun, 2004).

Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

22

580 Vacuum Cooling of Foods

2 Vacuum cooling principles, process and equipment 2.1 Vacuum cooling principles It is well known that substances exist in three principle phases, solid, liquid and gas. Each phase is characterized by a different molecular arrangement. In the liquid phase, molecules exist in the form of chunks. Although chunks of molecules can float over each other, the molecules within each chunk still maintain an orderly structure at fixed positions relative to each other. This orderly structure, however, no longer exists when the substance is in a gaseous state. Instead, molecules are far apart, moving around randomly and continually colliding into each other. Therefore, when any portion of a liquid evaporates, it needs to absorb heat in order to maintain this higher energy level of molecular movement. The amount of energy required is called latent heat, which must be supplied from the product itself or from the surroundings. Either of them will consequently be refrigerated. The temperature at which liquid starts to evaporate is called the liquid saturation temperature. It is dependent on the surrounding vapour pressure. Figure 22.1 shows the saturated water temperature and pressure relationship. It is clear from this diagram that at 101 kPa (1 atmospheric pressure), water boils at 100°C. However, reduction in the imposed pressure will cause water to evaporate at a lower temperature. For any product containing free water, if placed in a closed vessel where pressure is reduced through a vacuum pump, the vapour pressure difference between the water in the product and the surrounding atmosphere will cause water to evaporate and the generated vapour to escape to the surrounding atmosphere. Since the product is in a closed system, the latent heat required for evaporation therefore has to be furnished by itself through the conversion of sensible heat. Consequently, the product temperature is reduced. The cooling effect continues corresponding with the pressure reduction exerted by the vacuum pump. This process is called vacuum cooling, as cooling is achieved through boiling part of the moisture of the product under vacuum conditions. The final product temperature can be controlled precisely through the regulation of

120 Pressure (kPa)

100 80 60 40 20 0 0

20

40

60

80

Temperature (°C) Figure 22.1

Saturation pressure of water.

100

120

Vacuum cooling principles, process and equipment 581

the final vapour pressure inside the vessel, which is usually set at no less than 6.5 mbar for food products, otherwise freezing may occur and consequently damage the product (McDonald, 2001). Theoretically, vacuum cooling is a potential cooling treatment for the food industry since most food products contain significant amounts of water. However, unlike other cooling methods, it is a highly product specific process and mostly applicable to porous products only. For instance, it has been successfully used in cooling of lettuces, cabbage, mushrooms, etc, while it has been proven unsuccessful for oranges, tomatoes and apples, unless peeled. Furthermore, any product to be vacuum cooled needs to be able to afford to lose a certain amount of water without causing deterioration to its quality.

2.2 Vacuum cooling process A typical vacuum cooling process is illustrated in Figure 22.2. Food product (represented by the elliptical shape) with an initial temperature Ti is loaded into the vacuum chamber and the door is closed (Figure 22.2a). The vacuum pump is switched on. Air is being evacuated and chamber pressure P starts to decrease. At Figure 22.2b, although P is smaller than the atmospheric pressure, it is still higher than the initial working pressure Psat (which is also the saturation vapour pressure corresponding to Ti) and thus not sufficient for evaporation to occur. Consequently, temperature change to the product is still unnoticeable. When P reaches Psat, water inside the product starts to evaporate and latent heat is released (Figure 22.2c). The beginning of boiling is called flash point (Wang and Sun, 2002a). Generally, it is required that the chamber pressure is reduced to the flash point as soon as possible since prior to this, the vacuum pump merely evacuates the air and no cooling is achieved. With the generated vapour being continuously removed by the vacuum pump and/or through condensation when a condenser is installed inside the chamber, the internal pressure P is further reduced to below Psat (Figure 22.2d). This encourages water evaporation to continue and thus the cooling process. This cooling process ceases when the desirable product temperature Tf is reached (Figure 22.2e). Then, the ventilation valve is opened, air is re-admitted into the chamber and the products are taken out of the chamber and stored at the recommended temperature.

2.3 Vacuum cooling equipment Vacuum cooling installation varies in size and shape, depending on the individual application. However, the basic components are similar to those shown in Figure 22.3, which consist of the vacuum chamber, the pumping system and associated pipeworks and controls. The vacuum chamber is the place where the food product is placed and cooled. It is normally floor mounted and oriented horizontally with either rectangular or circular cross-sections. It has one or two doors that are hermetically sealed and sliding open or hinged. Usually a guide rail or roller conveyor is mounted on the bottom of the

582 Vacuum Cooling of Foods

Air Psat (Ti) ⬍ P ⬍ 1 atm

P ⫽ 1 atm

T ⫽ Ti

T ⫽ Ti

(a)

(b) Air & vapour

P ⬍ Psat (Ti)

P ⫽ Psat (Ti)

Q

Q n of T ⫽ Ti (c)

Air & vapour

T ⬍ Ti (d)

Vacuum

T ⫽ Tf (e) Figure 22.2

Illustration of vacuum cooling process.

chamber to take trolleys loaded with palletized products. The chamber is connected to the pumping system through the pumping port situated at its side. Between them there is also an isolation valve. The chamber is also fitted with a bleeding valve to allow air ingress. In addition to the above, there are other ports as well for temperature indication and pressure/temperature control. The controllers operate a valve/switch that will shut down the pumping system when a predetermined temperature is reached. The selection of pumping systems needs to consider the economic aspects, such as capital cost, energy consumption, water and steam usage, utilities and services available, etc. The basic component required is the vacuum pump, which is used to reduce the chamber pressure and generate a vacuum. The most commonly used types are the oil-sealed rotary pump or steam ejector (Malpas, 1972). The former is a mechanical pump that uses a rotor with sealing against air leakage accomplished by the use of

Vacuum cooling principles, process and equipment 583

Pressure gauge Isolation valve

Condenser

Pressure transducer

Vacuum chamber Bleeding valve

Pressure indicator/controller

Air exit Coolant in

Coolant out

Product Thermocouples

Temperature indicator/controller

Data acquisition system

Control panel Vacuum pump Figure 22.3

Drain

Schematic diagram of vacuum cooling installation.

vacuum oil. It consists of a stator with a cylindrical bore into which the rotor is fitted. The rotor is offset to the stator bore and fits closely against the stator in one position. It contains two blades that slide in diametrically opposite slots, which enables the tips of the blades to be in contact with the stator wall constantly when the rotor turns. During the operation, gas from the vacuum chamber enters the pump through the inlet valve. This air is trapped, compressed and ejected to the atmosphere through the exhaust valve. Unlike the oil-sealed rotary pump, the steam ejector requires no moving mechanical parts. Instead, it operates when steam passes through a nozzle and on discharge expands into a diffuser. As the steam ejector is connected to the chamber to be evacuated, the steam entrains gas and vapours flowing from the chamber until the desired vacuum is created. The vacuum pump is usually used in conjunction with a vapour-condensing unit for both practical and economical reasons. During vacuum cooling, the amount of vapour generated (also cooling loss) can be calculated by: mw ⫽

MP TCp LH

(1)

Assuming this vapour is an ideal gas, its volume can be obtained by using the following equation: V⫽

mw /MgRTv PV

(2)

Therefore, for instance, it can be obtained from Equations (1) and (2) that to cool 1000 kg of leafy vegetables from harvest at 35°C to a storage temperature of 5°C,

584 Vacuum Cooling of Foods

the vacuum pump will need to handle over 2700 m3 of vapour. If this cooling is accomplished in half an hour, the vacuum pump will need to have a capacity of 5400 m3/h. Since most mechanical pumps can only operate at about half of their theoretical pumping speed, a vacuum pump with a speed of over 10 000 m3/h will thus be required, which is difficult to achieve in practice. Furthermore, from calculation it can be obtained that the total electricity required for the vacuum pump for the above purpose is equal to the condensation heat required to condense this vapour back to water. However, as the ratio of refrigeration capacity to the electricity consumption is 2.5, it can therefore be seen that it is over twice as economical to use the vapour-condensing unit than the vacuum pump with regard to energy usage. With the installation of a vapourcondensing unit, the vacuum pump is usually used to evacuate air that remains and leaks into the chamber only, while vapour generated from evaporation is mostly condensed and then drained out of the chamber. Either water-cooled or mildly refrigerated condensers have been used for this water vapour condensation.

3 Applications of vacuum cooling in the food industry 3.1 Fruit and vegetables The quality of vegetables begins to deteriorate upon harvesting and continues to decline quickly thereafter. The deterioration can be caused by numerous sources, including physiological breakdown, moisture loss and pathogens, most of which are strongly related to time and temperature. Hence, it is desirable to cool the vegetables as soon as possible in order to extend their shelf-life. Air blast cooling, hydro-cooling and vacuum cooling are the most commonly used rapid cooling methods. Among them, hydro-cooling probably is the cheapest, in which the product to be cooled is immersed in or sprayed with cool water (Chen, 1988). It is usually used for small products, e.g. peas, asparagus, sweet corn, carrots and peaches, especially after blanching (Bailey, 1994). Air blast cooling is achieved through heat transfer from the centre of the product to its outer surface by conduction and then from the outer surface to the circulating air by convection. Although it has proved itself a rapid cooling method for non-leafy vegetables, its cooling rate is found to be slow when applied to leafy vegetables, since the air gap between the leaves restricts the heat transfer process due to its low thermal conductivity (Sun, 2000). Therefore, when it comes to leafy vegetables, vacuum cooling is preferable due to the fact that the cooling effect is generated within the product through boiling part of its moisture and thus less dependent on product thermal conductivity. The first commercial vacuum cooling plant for lettuce was built in the USA in 1948 (Thompson and Rumsey, 1984). Since then, vacuum cooling has become a well-established commercial method in the USA and many European countries for removing field heat of lettuce before they are distributed by refrigerated vehicles into cold storage and retail outlets. The benefits of vacuum cooling have been widely reported (Harvey, 1963;

Applications of vacuum cooling in the food industry 585

Gormley and MacCanna, 1967; Shewfelt, 1986; Frost et al., 1989; Tambunan et al., 1994; Shewfelt and Philips, 1996; Sullivan et al., 1996). It can cool lettuce from 25°C to 1°C in less than 30 min (Everington, 1993) and, if combined with cold storage at 1°C, it can increase the shelf-life of lettuce from 3–5 days at ambient temperature to 14 days (Artes and Martinez, 1995, 1996). Pumping speed was found to have a large effect on the efficiency of the vacuum cooling process. Field tests using a commercial vacuum cooler show that for a vacuum installation to cool four pallets of lettuce, each weighing 700 kg, the best result is obtained when a nominal pumping speed of 1660 m3/h is used during the first 10 min followed by 1250 m3/h, when cooling was accomplished in 32 min and the final temperature of the leaves was 0.3–1.1°C, while the head was 1.6–2.3°C, which were lower than other cases and the temperature distribution across the product was also more uniform (Haas and Gur, 1987). The efficiency of vacuum cooling is also affected by packaging type. Research shows that less physiological disorders and decay were observed in lettuce heads that were packed in closed PP film after vacuum cooling, compared to those without packaging or wrapped in perforated PP film (Artes and Martinez, 1996). If lettuce is wrapped prior to vacuum cooling, the packaging material needs to be perforated so that the generated vapour can escape to the surrounding atmosphere. Furthermore, tight wrapping and excessive wrapping should be avoided since the former decreases the ventilation area between the product, while the latter would block the passage of water vapour. Currently, in industrial application, lettuce is usually packaged in perforated PP bags before being vacuum cooled, or alternatively, wrapped in PVC film after cooling is accomplished (Haas and Gur, 1987; Artes and Martinez, 1995, 1996). Pre-cooling of mushrooms is another major traditional application of vacuum cooling. The porous structure and high moisture content of mushrooms have made this possible (Noble, 1985). Burton et al. (1987) indicated that the advantage of vacuum cooling was equivalent to a prolonged shelf-life of 24 h after 102 h storage. The influence of vacuum cooling on mushroom quality, as expressed by colour, was investigated. Results indicate that, although there was no significant difference between vacuum cooling and conventional cooling if the mushroom was stored at 5°C for 102 h after cooling, vacuum cooled mushrooms were found to have a significantly better colour than conventionally cooled ones when the storage temperature was changed to 18°C (Burton et al., 1987). However, these results are applied to high quality mushrooms only. If the mushrooms had slightly deteriorated before cooling, vacuum cooling appeared to have an adverse effect by accelerating the enzymatic browning of mushroom caps, in comparison with conventional cooling methods (Gormley, 1975). Furthermore, it was found that film over-wrap was beneficial for maintaining the appearance of vacuum cooled mushrooms, possibly by reducing the activity of the enzyme system that caused browning (Gormley, 1975). This increased enzyme activity due to vacuum cooling, however, was not observed by Burton et al. (1987). Results from Barnard (1974) also showed that only high quality mushrooms could be subjected to vacuum cooling, as discoloration of damaged or wet mushrooms was accelerated during the process. These findings are significant since they indicate that a proportion of any mushroom harvest is not suitable for vacuum cooling (Anon, 1986).

586 Vacuum Cooling of Foods

Vacuum cooling resulted in around 3.6 per cent of weight loss when the mushroom temperature was reduced from 21°C to 1°C, which was higher than for air blast chilling (2 per cent) (Sun and Wang, 2001). In addition, Frost et al. (1989) found that vacuum cooled mushrooms had more water loss as vapour during storage at 5°C. This, however, was not observed by Sun (1999a), who instead found that during storage at 1°C vacuum cooled mushrooms experienced less weight loss than air blast cooled ones. Frost et al. (1989) postulated that the higher water loss for vacuum cooled mushrooms during storage was caused by increased hyphal surface area, which might be a plausible hypothesis were it not for the fact that no apparent changes in hyphal structure were shown on SEM photos (Frost et al., 1989). Pre-wetting of mushrooms prior to vacuum cooling was demonstrated to be an effective method for increasing product yield, in that mushrooms could absorb 6 per cent of their weight of water if wetted for 5 min (Sun, 1999b). The weight loss during vacuum cooling is affected by product packaging type. Mushrooms wrapped before vacuum cooling were found to have better quality than those without packaging or wrapped after vacuum cooling (Sun and Wang, 2001). Studies have also been conducted to investigate vacuum cooling of other varieties of vegetables and fruit, including broccoli (Sun, 1999a), eggplants (Hayakawa et al., 1983), cucumber (Hayakawa et al., 1983), carrot (Hayakawa et al., 1983), peppers (Sherman and Allen, 1983), turnips (Ishii and Shinbori, 1988), strawberries (Gormley, 1975b; Anon, 1981), blackcurrants (Anon, 1981) and melons (Chambroy and Flanzy, 1980). Overall results show that vacuum cooling is an effective and efficient cooling method for these products. However, generally speaking, the cooling rate for nonleafy vegetables and fruits is lower than for leafy vegetables. Results also indicate that usually 4 per cent weight loss must be encountered for a temperature reduction from 25°C to 1°C. Pre-wetting of products prior to cooling was found to be a practical method to compensate for cooling loss (Chen, 1988; Sun, 1999b).

3.2 Bakery products For the bakery industry, rapid cooling of products after being taken out of the oven and prior to packaging is essential in order to avoid vapour condensation inside the package. As a special rapid cooling method for lettuce and mushrooms, vacuum cooling can also accelerate the cooling process for a wide range of baked products, e.g. bread rolls, crusty breads, sausage rolls, pastries, meat pies, biscotti bread, cakes and baked biscuits. It is now commercially used in Italy for some delicate bakery products, such as panetonni (fermented Italian cake) (Everington, 1993). Using vacuum cooing, panetonni can be cooled in 4 min in comparison to 24 h by air. The cooling process can be operated either in batch mode or continuously (Everington, 1993). Usually modulated vacuum cooling (MVC) (Bradshaw, 1976) is applied, by which the vacuum generation rate can be more precisely modulated. The objective of using MVC is to minimize the possible adverse effects that vacuum cooling might exert on the texture and volume of the products, since pressure can build up in areas of low vapour permeability such as the crust on bread (Bradshaw, 1976).

Applications of vacuum cooling in the food industry 587

Compared with conventional cooling methods, vacuum cooling brings several benefits to the bakery industry. The most significant advantage is the high cooling rate. For a similar cooling load, conventional cooling requires 1–3 h, while it only takes 30 s to 5 min when vacuum cooling is employed (Acker and Ball, 1977; Anon, 1978). The high cooling efficiency helps to reduce product hold up time and hence increases production throughput. In terms of product quality, shape and texture, vacuum cooled baked products are superior to air cooled ones, since less contraction and collapse occur during storage as vacuum cooled baked products have a more uniform distribution of internal temperature and moisture (Wang and Sun, 2001). Furthermore, vacuum cooling helps to extend product shelf-life since spore contamination only occurs at the end of the cooling process when air is allowed to enter the vessel. This, however, can be minimized if air is readmitted via a microbiological filter system. Although vacuum cooling has been found to cause loss of volatile aromatic components, no significant difference in taste was detected (Kratochvil, 1981; Kratochvil and Holas, 1984a, b). Vacuum cooling of bakery products can usually lead to about 1 per cent of weight loss for 10°C drop in temperature, therefore, a typical temperature reduction from 98°C to 3°C will result in a weight loss of 6.8 per cent (Acker and Ball, 1977; Everington, 1993). Since the weight loss for conventional air blast cooling is about 3–5 per cent depending on air velocity (Everington, 1993), the difference is not significant and can be further compensated by reducing baking time to increase the moisture retention inside the product (Acker and Ball, 1977).

3.3 Fishery products The major current application of vacuum cooling in the fishery industry is to reduce tuna temperature to 35–40°C following steam-cooking at 65°C. This process usually resulted in 3–4 per cent weight loss. Research also indicates that vacuum cooling could be possibly applied at sea to cool small fish such as whiting or crustaceans, e.g. shrimps, with energy that powers the pumping system supplied from waste stack gases (Carver, 1975). Rolfe (1963) shows that although vacuum cooling can be used to freeze trays of cooked haddock fillets, the cooling loss is too high, around 21 per cent of the original weight.

3.4 Sauces, soups and particulate foods Many viscous food products and components, e.g. sauces, soups, meat slurries and fruit concentrates, are difficult to cool due to the high heat transfer resistance caused by the high viscosity and low thermal conductivity. This difficulty, however, can be overcome by vacuum cooling since the cooling effect is mainly achieved through water evaporation rather than conductive or convective heat transfer. It is now a common practice in the production of frozen and chilled ready meals and its benefits to the food manufacturers are large (DiRisio, 1990; James, 1990, 1997; Shaevel, 1993; Houska et al., 1996). For instance, it was reported that it only took 5 min to vacuum cool a concentrated sugar solution (with 61 per cent dry matter) made from blueberry fruits during

588 Vacuum Cooling of Foods

the preparation of yoghurts, from 90°C to 50°C (Houska et al., 1996). Vacuum cooling can also reduce the temperature of 3785 litres of tomato sauce from 93°C to 7°C in 14 min (DiRisio, 1990). Research also shows that a large batch of meat sauce that weighed 1100 kg could be cooled from 85°C to 10°C in less than 30 min using vacuum cooling, which took more than 6 h when air blast cooling was used (James, 1997). In this type of vacuum cooling system, products are placed inside a jacketed sealed vessel, cooked under pressure and then vacuum cooled. The vessel might also be installed with scraper blades to avoid the adhesion of products to the vessel wall due to their viscous nature (Anon, 1981). Since same unit is used for both cooking and cooling, the overall process time is reduced as no delay is incurred in transferring the product between vessels (Anon, 1981). Weight loss caused by vacuum cooling is not a significant problem here, as the loss can be carefully controlled by adjusting the water content of the product (Everington, 1993).

3.5 Large cooked meat joints Vacuum cooling of large cooked meat joints has been comprehensively researched in the past few years (Desmond et al, 2000, 2002; Sun and Wang, 2000, 2003; McDonald et al., 2001; McDonald and Sun, 2001a, b). As shown in Table 22.1, cooked meat can be cooled from 70–74°C to 4°C in 1–2.5 h under vacuum conditions, in comparison to 9.4–11.7 h for air blast cooling (Desmond et al., 2000; Sun and Wang, 2000; McDonald et al., 2001; McDonald and Sun, 2001b), 12–14 h for slow air cooling (Desmond et al., 2000; Sun and Wang, 2000) and 5–14.3 h for water immersion cooling (Sun and Wang, 2000; McDonald et al., 2001). The exceptional fast cooling rate of vacuum cooling is significant to the cooked meat industry. Studies show that vacuum cooling is the only cooling method available that meets the UK and Ireland government cook-chill guidelines, which recommend that meat joints should be chilled from 74 to 10°C within 2.5 h after being removed from the cooking process in order to minimize the growth of any pathogens that have survived the cooking process (Desmond et al, 2000). Vacuum cooling, however, does have adverse effects. Cooling loss during vacuum cooling is high, around 10 per cent of original weight, while loss for slow air cooling, air blast cooling and water immersion cooling is around 6 per cent (Desmond et al., 2000; Sun and Wang, 2000), 5 per cent (Desmond et al., 2000; Sun and Wang, 2000; McDonald et al., 2001; McDonald and Sun, 2001b) and less than 2 per cent (McDonald et al., 2001; Sun and Wang, 2000) respectively, for a typical temperature reduction from 72°C to 4°C (Table 22.1). The high cooling loss consequently reduces product yield, which is undesirable since yield is directly related to profits for the manufacturers. Both McDonald et al. (2001) and Desmond et al. (2002) found that increasing the amount of brine solution injected into the raw meat helped to compensate vacuum cooling loss and hence increased product yield. At 135 per cent brine injection level, the yield of vacuum cooling was similar to that of water immersion cooling at 120 per cent injection level (McDonald et al., 2001). A higher injection level was also found to lead to a more tender and juicier product as a result of more moisture being present

Applications of vacuum cooling in the food industry 589

Table 22.1 Summary of experimental studies of vacuum cooling of cooked meat joints Meat type

Weight (kg)

Injection level (%)

Final P and time to reach it

Cooling loss (%)

Product yield (%)

Cooling time (h)

Ti

Tf

Sources

Pork ham 5–6

120

11.3

96.6

1.9

70

4

Desmond et al. (2000)

Pork

5–6

120

2.0

70

4

Sun and Wang (2000)

Beef

1.5–2.0

120–145

0.8–1.2

72

4

McDonald et al. (2001)

Beef

3.0–3.5

120–145

1.0

72

4

McDonald and Sun (2001a)

Beef

5

120

Pork

5–6

Pork

4.2–7.4

120 130 110–140

7–10 mbar, 7–10 min 6.5 mbar, ⬍15 min 6.5 mbar, ⬍2 min 6.5 mbar, ⬍2 min 6.5 mbar, 10–6 min 7–10 mbar, 7–10 min 6.5 mbar, 2.5–30 min

11.9 10.4

85.8–116

10.6–12.4

87.6–89.4

2.5–3.4

72

4

McDonald and Sun (2001b)

10.7 11.4 9.9–13.1

100.3 106.5

2.3 2.4 ⬍2.0

70

4

Desmond et al. (2002)

74

10

Sun and Wang (2003)

in the product (McDonald et al., 2001; Desmond et al, 2002). However, there was an upper limit for the amount of brine solution that could be injected to the raw meat, as a high injection level increased product saltiness and consequently decreased its acceptability. A lower pressure reduction rate (evacuation rate) was able to reduce cooling loss (McDonald and Sun, 2001b), but compromised cooling rate. Installation of a water sprayer inside the chamber was also suggested (Thompson, 1996), or alternatively, pre-wetting the samples prior to cooling (McDonald and Sun, 2000), however, this might result in cross-contamination. Resulting from the high cooling loss, the moisture content of vacuum cooled products was lower than cooling using traditional methods (McDonald et al., 2000). Significant porosity development also occurred during vacuum cooling (McDonald et al., 2002). Consequently, cooked meat cooled under vacuum had lower values of thermal conductivity, thermal diffusivity, apparent density and specific heat than conventionally cooled ones (McDonald et al., 2002). The quality of vacuum cooled cooked meats, as expressed by colour, flavour, texture and overall acceptability has also been widely investigated and compared with other cooling methods (Desmond et al., 2000, 2002; McDonald et al., 2000, 2001). Sensory analysis conducted by Desmond et al. (2000) showed that vacuum cooled products were darker and less tender and juicy than other cooling treatments, since the former had a lower moisture content and higher degree of muscle compression due to more water loss, this, however, was not observed by McDonald et al. (2000). Texture profile analysis (TPA) and Warner-Bratzler shear (WBS) (tenderness) showed that the vacuum cooled product had higher hardness and shear force values compared to those cooled using other methods (Desmond et al., 2000; McDonald et al., 2000). However, Desmond et al. (2002) indicated that neither TPA nor WBS measurement showed any significant effect of cooling methods on product hardness and tenderness. Cooked

590 Vacuum Cooling of Foods

beef cooled under vacuum was found to be significantly darker than that cooled using conventional cooling methods, possibly caused by the removal of water and air from the tissues of the cooked meat, which concentrated globin myohaemichromogen and increased light penetration (McDonald et al., 2000). Sensory analysis indicated that panellists showed a preference to vacuum cooled cooked beef due to a more natural and intense flavour (McDonald et al., 2000, 2001). Regardless of the above variations, in terms of overall product acceptability, no significant difference was found between products cooled using vacuum cooling and traditional methods (Desmond et al., 2000, 2002; McDonald, 2000, 2001). Microbiological quality of vacuum cooled products was superior to other cooling treatments, as indicated by results from mesophilic aerobic plate counts, indicating that vacuum cooled products had a lower level of microbial growth during storage (McDonald, 2001).

3.6 Ready meals The market for refrigerated ready meals has grown substantially in recent years. One of the biggest challenges for food technologists and engineers is to design a refrigeration system that will ensure the ready meals are chilled rapidly after heat treatment in order to prevent microbial spoilage. Different countries have issued their individual guidelines on cooling requirements. The strictest ones are perhaps those recommended by the UK, which requires that packs of ready meals should not exceed 100 mm in thickness and height and cooling must commence within 30 min after cooking (which allows for portioning of meals). These guidelines also state that large food portions (not exceeding 100 mm in thickness and height) must be chilled to 10°C within 2.5 h, while portions smaller than 50 mm deep must be cooled down to 0–3°C within 90 min. Research using an experimental approach combined with predictive modelling was conducted to examine the possibility of achieving these guidelines using conventional air blast chilling (Evans et al., 1996) and brine immersion chilling (Ketteringham and James, 1999). Both results showed that only a 10 mm deep tray could be chilled within the above government guidelines. This has consequently encouraged food research scientists and engineers to explore new rapid cooling methods. Due to its exceptionally fast cooling rate, vacuum cooling of ready meals has therefore attracted a growing research interest in the past few years. Vacuum cooling of cooked beef in soup, a common ready meal component, was studied with the results indicating that the cooling time required was far below government legislation (Houska et al., 2003). Experimental investigation also demonstrated the effectiveness of vacuum cooling of cooked carrot (Zhang and Sun, 2003). Research work is still ongoing in the National University of Ireland to investigate the effectiveness and efficiency of vacuum cooling of other possible ready meals components, e.g. potatoes, pastas, rice, chicken, broccoli, etc., which includes studies of cooling efficiency, cooling loss and the influence of vacuum cooling on product re-heating properties, etc. Apart from food safety concerns, vacuum cooling might also bring other advantages to ready meal manufacturers. The rapid cooling could benefit maintaining high

Mathematical modelling of the vacuum cooling process 591

product quality by minimizing the destruction of vitamins (Ketteringham and James, 2000) and avoiding over-cooking of some heat-sensitive products (James et al., 1987). It also benefits increasing product throughput, either through the reduction of cooling time (Ketteringham and James, 2000) or by minimizing the delay caused by transfer of product between different vessels since both cooking and cooling could take place in one unit (James et al., 1987). However, the use of high vacuum can pull cooked products into the vacuum-pumping system or onto the roof of the processing vessels and under these circumstances, intensive cleaning will have to be performed to remove the product from the equipment so as to prevent proliferation (James et al., 1987; McDonald and Sun, 2000; Wang and Sun, 2001). During vacuum cooling, boiling mainly occurs inside the product, which can cause expansion and rupture within tissues, which might consequently affect product texture (McDonald and Sun, 2000).

4 Mathematical modelling of the vacuum cooling process Mathematical modelling of vacuum cooling is useful since it can lead not only to a better design of vacuum cooling equipment, but also to a better understanding of the effects of the process on the physical, chemical and sensory properties of the products.

4.1 Mathematical modelling of vacuum cooling of liquid food Several models have been developed for vacuum cooling of liquid food. Most of them aim at predicting the transient product temperature and chamber pressure, however, different approaches were adopted. The earliest study was perhaps carried out by Burfoot et al. (1989), in which the water evaporation rate was assumed to be proportional to mass transfer coefficient, mass transfer area and pressure difference between the saturated vapour above the liquid surface and the bottom of the vessel. This model was further developed by Houska et al. (1996), which has enabled the prediction of timevariant chamber pressure and product temperature during the cooling process. Dostal and Petera (2003) developed a different model for vacuum cooling of liquid food by making the following assumptions: (1) thermodynamic equilibrium existed between gas and liquid phases; (2) heat and mass transfer resistance prevailed on the liquid side and the heat and mass transfer coefficient could be obtained from the film theory principle.

4.2 Mathematical modelling of vacuum cooling of cooked meats Modelling of vacuum cooling of cooked meats was carried out by Wang and Sun (2002a, b). Two sub-models were developed. In the first sub-model, the mass conservation of both air and vapour were analysed as shown in Figure 22.4. The former

592 Vacuum Cooling of Foods

Condenser

Drain

Ingress air Air

Figure 22.4

Vacuum cooler

Vapour

Product

Evaporation

Vacuum pump

Illustration of air and vapour flow during the vacuum cooling process.

includes both ingress air (air leaking from the ambient) and air evacuated by the vacuum pump, while the latter consists of vapour generated under vacuum conditions, released by the vacuum pump and condensed by the condensing unit. The second submodel mainly deals with the heat and mass transfer of the system. The heat transfer process is considered as a three-dimensional transient heat conduction problem with inner heat generation, while the mass transfer process is treated as hydrodynamic vapour movement through inner pore spaces of the solid product with inner vapour generation. The variations of the physical properties as well as the shrinkage of cooked meat during the cooling process were also incorporated in the model. The two submodels were combined and solved using finite element analysis, which has led to prediction of the transient product temperature profile and internal chamber pressure, cooling loss, etc. Results predicted from the models were in good agreement with experimental measurement. The numerical model was further used to analyse the effects of the weight, size, shape, porosity, pore distribution and pore size of cooked meat joints on the cooling rate, weight loss and temperature distribution (Wang and Sun, 2002c).

5 Advantages and disadvantages of vacuum cooling 5.1 Advantages of vacuum cooling Table 22.2 summarizes the advantages and disadvantages of vacuum cooling for various sectors of the food processing industry. The advantages of vacuum cooling are several. The main one is that vacuum cooling can reduce product temperature in an extremely short time. The cooling curves for some food products are shown in Figure 22.5. Studies have been conducted to compare the rate of vacuum cooling with conventional methods and show that the former is over 50 per cent quicker for large cooked meat joints (McDonald and Sun, 2000) and 60 times faster for vegetables (Sun and Wang, 2001). Typically, vegetables can be cooled at the order of 0.5°C/min under

Advantages and disadvantages of vacuum cooling 593

Table 22.2 Advantages and disadvantages of vacuum cooling for various food industry sectors Product type

Advantages

Disadvantages

Bakery products

Very rapid cooling system for delicate products Fast cooling rate increasing productivity Superior product quality due to occurrence of less contraction and collapse during storage More sanitary process due to the absence of moulds during cooling Weight loss minimized by reducing baking time

Loss of some volatile aromatic component Specialized modulated vacuum cooling (MVC) technology required for satisfactory results

Cooked meat joints

Rapid cooling rate increasing product safety and reducing cost Only cooling method complying with the UK and Ireland government cook-chill guidelines governing large cooked meats production Low level of microbial growth during storage

High cooling loss reducing product yield Product less juicy and tender

Fruits and vegetables

Increased product shelf-life Short cooling time resulting in quick distribution Accurate temperature control possible Low running cost Uniform cooling

Applicable mostly to leafy vegetables and mushrooms only High capital cost High weight loss

Ready meals

Allowing development of integrated systems Suitable for cooling heat-sensitive products such as cream based foods

Extensive cleaning possibly required due to high vacuum causing products entering vacuum pumping system Safety concerns on system operation over a range of positive and negative pressures

Sauces and soups and other particulate food products

Efficient cooling system for viscous products with low thermal conductivity Same unit used for both cooking and cooling and thus delay due to transfer of product between vessels avoided Weight loss easily controlled by adjusting product composition

Difficulty in operating as a continuous process Difficulty in cleaning due to sauces splattering on the roofs of processing vessels during vacuum cooling

100 90

Iceberg lettuce Bread loaf Mushroom Pork pie

Temperature (ºC)

80 70 60 50 40 30 20 10 0 0

100

200

300 Time (s)

Figure 22.5

Cooling curves for some food products.

400

500

600

594 Vacuum Cooling of Foods

vacuum, which would increase to 0.05–3°C/h if cold storage or air blast is used. Although the cooling rate for both air blast and cold storage cooling can be raised by lowering the air temperature or increasing its velocity, either of these would increase the risk of product damage due to freezing or air blast. The different cooling rates between vacuum cooling and conventional cooling methods are mainly caused by the essential differences of the cooling mechanisms. For conventional methods, cooling takes place through conductive heat transfer from the core of the product to its surface followed by convective heat transfer from the product surface to the surrounding air and usually the former is the rate-limiting step due to the inherent low thermal conductivity of most food products. In contrast, vacuum cooling is accomplished through water evaporation (Sun and Wang, 2000). Since the ratio of conductive to evaporative heat transfer is less than up to 1:16 (Sun and Wang, 2001), vacuum cooling therefore was much quicker. Due to this exceptional fast cooling rate, vacuum cooling is able to provide many benefits to the food industry, e.g. shortening product hold up time, increasing production throughput, reducing energy consumption (Chen, 1986), minimizing microbial growth for cooked meats, etc. (Wang and Sun, 2002c). Unlike conventional cooling methods, during vacuum cooling, water evaporation takes place simultaneously within the product and on its surface, consequently, the product has a uniform internal temperature distribution. If a pile of products is to be vacuum cooled, the temperature of each product could be reduced at the same rate whether on the top, at the centre or on the bottom (Malpas, 1972). For products of large dimensions, the temperature difference between the surface and core by vacuum cooling is much less than by air blast cooling (Sun and Wang, 2000) and by water immersion cooling (McDonald and Sun, 2001b). This uniform temperature is beneficial to food processing. For instance, vacuum cooled baked products usually have a superior structure to those cooled with other methods since less contraction and collapse occur due to a more homogeneous temperature distribution (Acker and Ball, 1977; Chriastel, 1978; Shipman, 1978). For either air blast or cold storage cooling, the cooling rate is dependent on the surface area of the product/produce and a large storage surface area is required if the products are to be cooled correctly. This, however, is not necessary for vacuum cooling, during which products can be more tightly packed, so long as adequate venting is provided. Consequently, vacuum cooling can reduce the storage cost (Greidanus, 1971). Furthermore, vacuum cooling is a very hygienic process since air only goes into the vacuum chamber at the end of process when the vacuum chamber is opened to release vacuum. Precise product temperature control is also possible during the vacuum cooling process since product temperature can be brought down to within 1–3°C by controlling the absolute pressure (Longmore, 1973).

5.2 Disadvantages of vacuum cooling Vacuum cooling also has its disadvantages. It is a highly product specific process, which is applicable only to moist products with a porous structure. It also requires that the amount of moisture loss should not cause significant deterioration to product quality.

Factors affecting the vacuum cooling process 595

Due to the higher water loss, vacuum cooling has a lower yield than other cooling methods, which is undesirable since yield is directly related to the money that manufacturers can obtain. Various methods have been proposed to compensate for water loss, including pre-wetting products prior to cooling (Sun, 1999b) and installation of water sprayers inside the vacuum chamber (Thompson, 1996). As opposed to conventional cooling methods whose major effect is on the product surface, during vacuum cooling, boiling occurs both inside and on the surface of the product. Consequently, vacuum cooling might have a more significant effect on the internal texture and structure of products. Furthermore, currently, most vacuum cooling processes are operated in batch mode, during which foodstuffs are placed in a vacuum chamber, the chamber is then closed and evacuated to the predetermined pressure and the product is cooled and removed out of the chamber after reaching the desirable temperature. This process can be time-consuming and inefficient.

6 Factors affecting the vacuum cooling process 6.1 Factors affecting vacuum cooling rate As previously mentioned, the major advantage of vacuum cooling over traditional methods is the exceptional fast cooling rate that it offers. Aspects of vacuum cooling operation, equipment and the product itself will affect the cooling rate. During vacuum cooling, the mass transfer process takes place in two steps: initial water evaporation which takes place either on the surface or inside the macro-pores of the products; followed by diffusion of water vapour through the pore spaces to the product surface and subsequently to the surrounding atmosphere (Wang and Sun, 2002b). Therefore, vacuum cooling is largely dependent on product porosity and pore distribution, in which high porosity and uniform pore distribution will lead to a more rapid cooling process. It has been shown that minced beef that developed a porosity between 9.8 and 11.8 per cent following cooking could be vacuum cooled to 4°C in 21–15 min, while cooked whole muscle beef with a porosity between 1.44 and 2.6 per cent required a vacuum cooling time between 100 and 154 min (McDonald and Sun, 2001a). Product packaging will also affect vacuum cooling rate if packaging affects the evacuation of water vapour from the product. For instance, exposed lettuce heads or those wrapped in perforated packaging were cooled more quickly than those in cartons or impermeable packaging (Harvey, 1963). In addition, packaging type can affect the cooling process through its influence on the porous structure of the product. McDonald et al. (2001) found that cooling time for cooked meat packed in cellulose casing was over 400 times slower than those packed in elastic netting, which was possibly because the rigid structure of the former might have restricted the development of the product porosity by preventing the occurrence of product compression and decompression that usually could occur as a result of pressure changes and water loss. The weight, size and shape of the product have a negligible effect upon the vacuum cooling rate (Burfoot et al., 1990; Wang and Sun, 2002c; Sun and Wang, 2003), which indeed is one of the advantages of vacuum cooling over traditional methods.

596 Vacuum Cooling of Foods

Not just the product itself, but also the conditions at which the vacuum cooling process are operated affect the vacuum cooling rate, in particular the vacuum chamber pressure and the condenser temperature. Usually the influence of the former is expressed by the nominal speed of the vacuum pump, or pressure reduction rate. Generally, use of a high pumping speed offers a means of reducing the product to its final temperature within a shorter time. For instance, field tests using commercial vacuum coolers to cool iceberg lettuce wrapped in PVC stretch film showed that if a nominal pumping speed of 630 m3/h was used, the total cooling process required 78 min, but however only took 32 min if a nominal pumping speed of 1660 m3/h was used during the first 10 min followed by a speed of 1250 m3/h (Haas and Gur, 1987). Faster cooling rate increases production throughput, therefore, for the former case, only 24 pallets could be cooled within 8 h, compared to 52 pallets for the latter (Haas and Gur, 1987). During their investigation of vacuum cooling of large cooked meat joints, Sun and Wang (2003) indicated that a pressure reduction rate smaller than 67 mbar/min appeared to have an effect on vacuum cooling, as increasing pressure reduction rate reduced cooling time (Sun and Wang, 2003). The vacuum cooling rate is also affected by the condenser temperature. Mathematical analysis reveals that a lower condenser operating temperature increases condensing load and consequently leads to a faster cooling process (Wang and Sun, 2003). However, it cannot go below 0°C otherwise water will freeze on the condenser surface.

6.2 Factors affecting product/produce temperature distribution In addition to cooling rate, porosity and pore distribution also have a significant effect upon the internal temperature distribution of the product/produce during vacuum cooling. Product/produce with homogeneous pore distribution has a uniform internal temperature distribution, while if the pores distribute unevenly, areas with high porosity will have a temperature lower than those having a low porosity or without pores due to that high porosity facilitates the occurrence of vacuum cooling (Wang and Sun, 2002c).

6.3 Factors affecting vacuum cooling loss Cooling loss during vacuum cooling is high. The percentage weight loss per unit temperature reduction can be calculated by the following: ␻⫽

mw /Mp ⌬T

⫻ 100% ⫽

Cp LH

⫻ 100%

(3)

Equation (3) indicates that the percentage weight loss per unit temperature reduction depends on the product specific heat, which is related to the initial moisture content of the product. Since water has a high specific heat value, products with a high initial moisture content therefore require more sensible heat removal and consequently more

Nomenclature 597

Table 22.3 Moisture, specific heat, percentage weight loss per degree Celsius of temperature reduction per unit of percentage weight loss for some food products

Water Leafy vegetables Cooked meats Bakery products

Moisture (%)

Cp (kJ/kg K)

␻ (% weight loss/°C)

␩ (°C/% weight loss)

100 90 74 35

4.2 3.9 3.5 2.6

0.167 0.154 0.143 0.105

6.0 6.5 7.0 9.5

LH ⫽ 2500 kJ/kg (20°C)

water to be evaporated. For instance, as shown in Table 22.3, leafy vegetables have a higher moisture content than bakery products, consequently, for per degree Celsius temperature reduction, its cooling loss is higher.

7 Conclusions Vacuum cooling is a rapid cooling technique, which is achieved through boiling part of the moisture of the product under vacuum conditions. Any product can be vacuum cooled provided that it is porous and can afford to lose a proportion of water without an adverse effect on its quality. As a well-established commercial method for the cooling of leafy vegetables, mushrooms and some delicate bakery products, etc., it has also been employed to cool viscous and particulate food products, e.g. sauces, meat slurries, fruit concentrates, which usually are difficult to cool using traditional methods due to their high viscosity and low thermal conductivity. Increased competitiveness together with safety concerns has led to a comprehensive research study of vacuum cooling of cooked meat joints and ready meals in the past few years, with results indicating that its potential is promising and the benefits are large. However, vacuum cooling results in a high cooling loss, which decreases product yield and affects product quality, thus further research effort is still needed regarding new methods for compensating this high cooling loss and its subsequent effect on product yield and quality. In general, as an innovative cooling technique, the benefits that vacuum cooling brings to the food industry are significant, in particular in terms of reducing production cost and improving product quality and safety. As research work continues on increasing the practical applications of vacuum cooling and improving the quality of the vacuum cooled product, it can be envisaged that its usage in the food processing industry will be more competitive and widespread in the future.

Nomenclature Cp LH

specific (J/kg K) latent heat (kJ/kg)

598 Vacuum Cooling of Foods

M Mp mw P R T V T

mole mass (kg/mol) product weight (kg) cooling loss (kg) pressure (Pa) universal gas constant temperature (°C) vapour volume (m3) temperature difference (°C)

Greek letters  percentage weight loss per degree Celsius temperature reduction ␩ temperature reduction per unit of percentage weight loss Subscripts f final g gas i initial sat saturated v vapour w water

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Houska M, Podloucky S, Zitny R et al. (1996) Mathematical model of the vacuum cooling of liquids. Journal of Food Engineering, 29, 339–348. Houska M, Sun D-W, Landfeld A, Zhang Z (2003) Experimental study of vacuum cooling of cooked beef in soup. Journal of Food Engineering, 59, 105–110. Ishii K, Shinbori F (1988) Effects of outer leaf trimming, precooling methods and delay in precooling on changes in quality of turnips. Journal of Japanese Society of Horticultural Science, 57, 544–548. James SJ (1990) Cooling systems for ready meals and cooked products. In Process Engineering in the Food Industry 2: Convenience Foods and Quality Assurance (Field RW, Howell JA, eds). London: Elsevier Applied Science, pp. 88–97. James SJ (1997) Secondary chilling of meat and meat products. In Meat Refrigeration – Why and How? Bristol: University of Bristol, pp. 1–4. James SJ, Burfoot D, Bailey C (1987) The engineering aspects of ready meal production. In Process Engineering in the Food Industry: Development and Opportunities (Field RW, Howell JA, eds). London: Elsevier Applied Science, pp. 43–58. Ketteringham L, James S (1999) Immersion chilling of trays of cooked products. Journal of Food Engineering, 40, 259–267. Ketteringham L, James S (2000) The use of high thermal conductivity inserts to improve the cooling of cooked foods. Journal of Food Engineering, 45, 49–53. Kratochvil J (1981) Effect of vacuum cooling on bread aroma. In Proceedings of the 5th Symposium on Aroma Substances in Foods. Prague, pp. 143–149. Kratochvil J, Holas J (1984a) Effect of vacuum cooling on the content of aroma substances and sensory properties of break. Sobornik UVTIZ, Potravinarske Vedy, 2 (4), 241–251. Kratochvil J, Holas J (1984b) Effect of vacuum cooling on aroma of break. Getreide Meh und Brot, 38, 173–177. Longmore AP (1973) The pros and cons of vacuum cooling. Food Industries of South Africa, 26, 6–7, 9, 11. Malpas EW (1972) Vacuum equipment for evaporative cooling. Process Biochemistry, October, 15–17. McDonald (2001) Effects of vacuum cooling on processing time, mass loss, physical structure and quality of large cooked beef products. PhD thesis, University College Dublin, Ireland. McDonald K, Sun D-W (2000) Vacuum cooling technology for the food processing industry: a review. Journal of Food Engineering, 45, 55–65. McDonald K, Sun D-W (2001a) The formation of pores and their effects in a cooked beef product on the efficiency of vacuum cooling. Journal of Food Engineering, 47, 175–183. McDonald K, Sun D-W (2001b) Effect of evacuation rate on the vacuum cooling process of a cooked beef product. Journal of Food Engineering, 48, 195–202. McDonald K, Sun D-W, Kenny T (2000) Comparison of the quality of cooked beef products cooled by vacuum and by conventional cooling. Lebensmittel-Wissenschaft undTechnologie, 33, 21–29. McDonald K, Sun D-W, Kenny T (2001) The effect of injection level on the quality of a rapid vacuum cooled cooked beef product. Journal of Food Engineering, 47, 139–147.

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McDonald K, Sun D-W, James L (2002) Effect of vacuum cooling on the thermophysical properties of a cooked beef product. Journal of Food Engineering, 52, 167–176. Noble R (1985) A review of vacuum cooling of mushrooms. Mushroom Journal, 149, 168–170. Rolfe EJ (1963) The freeze-drying of fish and meat. In Freeze-Drying of Foodstuffs (Cotson D, Smith DB, eds). Manchester: Columbia Press, pp. 119–131. Shaevel ML (1993) Manufacture of frozen prepared meals. In Frozen Food Technology (Mallett CP, ed.). Glasgow: Blackie Academic and Professional, pp. 270–302. Sherman M, Allen JJ (1983) Impact of post-harvest handling procedures on soft rot decay of bell peppers. Proceedings of the Florida State Horticultural Society, 96, 320–322. Shewfelt RL (1986) Postharvest treatment for extending the shelf life of fruits and vegetables. Food Technology, 5, 70–78, 80. Shewfelt RL, Phillips RD (1996) Seven principles for better quality of refrigerated fruits and vegetables. In Refrigeration Science and Technology Proceedings. New Developments in Refrigeration for Food Safety and Quality. Lexington, Kentucky: pp. 231–236. Shipman FP (1978) Cake technology: interaction between product development and marketing. Cereal Foods World, 23, 130–133. Sullivan GH, Davenport LR, Julina JW (1996) Precooling: key factors for assuring quality in new fresh market vegetables crops. In Progress in New Crops (Janick J, ed.). Arlington: ASHS Press, pp. 521–524. Sun D-W (1999a) Comparison of rapid vacuum cooling of leafy and non-leafy vegetables. ASAE Paper No. 996117. St Joseph: ASAE. Sun D-W (1999b) Effect of pre-wetting on weight loss and cooling times of vegetables during vacuum cooling. ASAE Paper No. 996119. St Joseph: ASAE. Sun D-W (2000) Experimental research on vacuum rapid cooling of vegetables. In Advances in the Refrigeration Systems, Food Technologies and Cold Chain. Paris: International Institute of Refrigeration, pp. 342–347. Sun D-W, Wang LJ (2000) Heat transfer characteristics of cooked meats using different cooling methods. International Journal of Refrigeration, 23, 508–516. Sun D-W, Wang LJ (2001) Vacuum Cooling. In Advances in Food Refrigeration (Sun D-W, ed.). Leatherhead: Leatherhead Publishing, pp. 264–304. Sun D-W, Wang LJ (2003) Experimental investigation of performance of vacuum cooling for commercial large cooked meat joints. Journal of Food Engineering, 61 (4), 527–532. Tambunan AH, Morishima H, Kawagoe Y (1994) Measurement of evaporation coefficient of water during vacuum cooling of lettuce. In Developments in Food Engineering (Yano T, Matsumo R, Nakamura K, eds). Glasgow: Blackie Academic & Professional, pp. 328–330. Thompson AK (1996) Postharvest Technology of Fruit and Vegetables. London: Blackwell Science. Thompson JT, Rumsey TR (1984) Determining product temperature in a vacuum cooler. ASAE Paper No. 84-6543. St Joseph: ASAE. Wang LJ, Sun D-W (2001) Rapid cooling of porous and moisture foods by using vacuum cooling technology. Trends in Food Science & Technology, 12, 174–184.

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Wang LJ, Sun D-W (2002a) Modelling vacuum cooling process of cooked meat – part 1: analysis of vacuum cooling system. International Journal of Refrigeration, 25, 854–861. Wang LJ, Sun D-W (2002b) Modelling vacuum cooling process of cooked meat – part 2: mass and heat transfer of cooked meat under vacuum pressure. International Journal of Refrigeration, 25, 862–871. Wang LJ, Sun D-W (2002c) Numerical analysis of the three-dimensional mass and heat transfer with inner moisture evaporation in porous cooked meat joints during vacuum cooling. Transactions of the ASAE, 45 (6), 107–115. Zhang ZH, Sun D-W (2003) Temperature and weight loss profiles of vacuum cooling of sliced cooked carrot. In Proceedings of the 21st IIR International Congress of Refrigeration. Washington: IIR (International Institute of Refrigeration) Paper no. ICR0470. Zheng L, Sun D-W (2004) Vacuum cooling for the food industry – a review of recent research advances. Trends in Food Science & Technology, 15 (12), 555–568.

Ultrasonic Assistance of Food Freezing Liyun Zheng and Da-Wen Sun Food Refrigeration and Computerised Food Technology Group, National University of Ireland, Dublin, Ireland

Although the application of power ultrasound to food freezing is a relatively new subject, recent research advances show that its potential is promising and its benefits are wide-ranged. The beneficial use of the sound energy is realized through the various effects generated by the ultrasound upon the medium in which the ultrasound transmits. Among these effects, cavitation is perhaps the most significant, which can lead not only to the production of gas bubbles but also to the occurrence of microstreaming. The former can promote ice nucleation, while the latter is able to accelerate the heat and mass transfer process accompanying the freezing process. Similar to other dense and incompressible materials, ice crystals will fracture when subjecting to the alternating acoustic stress, which can consequently lead to products of smaller crystal size distribution, which is indeed one of the most important aspects that many freezing processes target. Resulting from these acoustic effects, power ultrasound is demonstrated to be able to perform several functions in assisting food freezing. It can be used to initiate ice nucleation and to control crystal size distribution in the frozen product during solidification of liquid food. If applied to the process of freezing fresh foodstuffs, power ultrasound can shorten the freezing process and thus lead to a product of better quality. Application of power ultrasound to the process of freezing liquid food can also prevent incrustation on the freezing surface. For the future development of this technology, several problems still remain to be explored. More fundamental research is still needed in order to identify factors that affect the ability of power ultrasound in performing the above functions. Considerable research effort is also required with regard to the development of adequate industrial equipment.

1 Introduction The use of ultrasound within the food industry has been a subject for research and development for many years. The sound range applied can be mainly classified into two distinct categories: high frequency low energy diagnostic ultrasound in the MHz range and low frequency high energy power ultrasound. Diagnostic ultrasound is mainly used as an analytical technique for quality assurance, process control and non-destructive inspection, which has been applied to determine food concentration, viscosity, composition, etc., to measure flow rate and flow level and to inspect egg shells and food packages (Floros and Liang, 1994; McClements, 1995; Mason et al., 1996; Mason, 1998). Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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Application of power ultrasound in the food processing industry, however, is relatively new and had not been broadly and profoundly explored until recently. The majority of studies are restricted to the sound range between 20 and 40 kHz. A wide variety of areas have been identified as having great potential for future development, including dehydration, filtration, fat crystallization, degassing, flavour extraction, oxidation, meat tenderization, homogenization, sterilization, etc. (Floros and Liang, 1994; McClements, 1995; Mason et al., 1996; Mason, 1998; Gennaro et al., 1999). Freezing is one of the most important unit operations in food processing and is used not only to manufacture products that are consumed in a frozen state, e.g. ice cream, frozen yoghurts, sorbets, etc, but also as a major means of preserving fresh foodstuffs. Application of power ultrasound to food freezing, however, has not been sufficiently exploited, although its potential appears to be promising. The beneficial use of the sound is realized through the mechanical and physical effects that it generates upon the medium in which it transmits. This chapter is intended to better inform scientists and engineers working in the field of food freezing about a new and important area for future research and development. It will comprehensively discuss the major functions that power ultrasound can perform in assisting food freezing and the basic mechanisms involved. In addition to these, basics of power ultrasound generation and equipment will also be surveyed together with some proposals for conjoining an ultrasonic device to some existing food freezing processes.

2 Power ultrasound generation and equipment 2.1 Basic components required for power ultrasound generation Power ultrasound can be generated in different ways, by electrical power, by liquid movement, by gas jet, or by other means. In this chapter, the main focus will be on the electricity driven ultrasonic system, which is most commonly used in the food processing industry. The basic component consists of the power generator that supplies electricity at the desired ultrasonic frequency to the transducer and the transducer, which functions by converting the electrical power to mechanical vibrations. 2.1.1 Power generator

The major function of the power generator is to convert a standard electrical frequency (typically 5–60 Hz) into the high alternating frequency (over 20 kHz) required in ultrasonic transmission through a series of oscillating, amplifying and matching circuits. In earlier days, the generator was designed at a fixed frequency, which was the exact resonant frequency of the transducer. However, due to manufacturing techniques, it is difficult for each transducer to have exactly the same resonant frequency. Therefore, for a multi-transducer system, only those transducers with their resonant frequency that is the closest to the generator can operate at their maximum efficiency.

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This will consequently lead to problems such as hot spots, standing waves, etc. (Mason, 1998). The sweep frequency technology developed in recent years, in which the frequency output of the generator is modulated around a central frequency (the central frequency may itself be adjustable as well), has managed to solve these problems (Fuchs, 1999). By sweeping the frequency just slightly above and below the central frequency, each transducer can be operated at their resonant frequency and thus maximum efficiency is achieved (Fuchs, 1999).

2.1.2 Ultrasound transducers

There are two major types of ultrasound transducers: magnetostrictive transducers and piezoelectric ones. The former is constructed from magnetostrictive materials. In its simplest form, it is a solenoid with the coil assembled as a laminate of many layers of magnetostrictive materials and the core is a metal strip made of copper (Figure 23.1). When a current is applied to the coil, a magnetic field is generated which causes a reduction in the dimensions of the core. Switching off the current causes the core to return to its original shape. The rapid repeated on/off of the current generates the mechanical vibrations, which are delivered to the medium through the diaphragm. It is important that the transducer is tightly bonded to the diaphragm since any air gap between the transducer and diaphragm will lose acoustic energy since air presents a very low acoustic impedance and high acoustic absorption (Gallego-Juarez, 1998). Usually epoxy resin is used to bond them together. However, it is easy for the transducer to detach from the diaphragm made of stainless steel, therefore they are welded together instead, as the solid joint will never loosen. The major advantage of the magnetostrictive transducer is in providing a large driving force because the system is of

Mechanical vibration

Output face

Metal strip attached to output diaphragm

Electrical coil

Oscillating magnetic field

Figure 23.1

Magnetostrictive transducer (adapted from Fuchs, 1999).

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Mechanical vibration

Output surface

Output section

Piezoelectric elements

Power

Electrode

Back section

Electrical insulator Pre-compression bolt Figure 23.2

Piezoelectric transducer (adapted from Fuchs, 1999).

an extremely robust construction (Mason, 1998). However, about 40 per cent of the electrical energy will be lost as heat and thus external cooling is required. Moreover, its maximum operating frequency is restricted to 100 kHz (Mason, 1998). Piezoelectric transducers are the most commonly used type of transducer. In their construction, piezoelectric ceramics are utilized, which will expand and contract in an alternating electric field, leading to pressure waves transmitting through the medium. For transducer applications, the characteristic constants of the ceramic material are required to remain stable with respect to time, mechanical stress, electric field and temperature. Therefore, only materials that have low dielectric and mechanical losses, such as lead-zicronate-titanate, niobates and barium titanate, can be used (Keil and Swamy, 1999). Since ceramic materials are brittle, it is a normal practice to ‘sandwich’ them between blocks of metals. This structure not only protects the ceramic from breakage but also prevents it from being overheated as the metal blocks can act as heat sinks. There are various designs of piezoelectric transducers. Figure 23.2 shows one specific type, which consists of two discs of piezoelectric ceramics sandwiched between two identical metal blocks. The piezoelectric ceramics are used in pairs in order to obtain additive mechanical motions. The two discs are polarized in opposite directions and separated by an electrode connected to the power. The sections are pre-stressed by means of a bolt. Compared to magnetostrictive transducers, the piezoelectric transducers offer higher electromechanical conversion (Hamonic and Decarpigny, 1988). They are over 95 per cent electrically efficient and can be operated over the whole ultrasonic range (Hamonic and Decarpigny, 1988). Furthermore, they are small in size, light in weight and inexpensive. However, they are less durable, since the piezoelectric material will deteriorate over time and thus the

Power ultrasound generation and equipment 607

Tank Process fluid

Ultrasonic transducer Generator Figure 23.3

Ultrasonic bath (adapted from Mason, 1998).

vibratory energy produced will become less and less (Thompson and Doraiswamy, 1999).

2.2 Some common power ultrasonic systems There are hundreds of types of ultrasonic systems currently in use, which vary according to generator design, transducer types and the way that ultrasound is delivered to the process. A few examples are given in the following sections. 2.2.1 Ultrasonic bath

Basically, the ultrasonic bath (Figure 23.3) is a tank that contains a process medium with transducers bonded to its base. Additional parts can be attached onto to it to make it multi-functional. For instance, the tank can be designed as a jacketed vessel with refrigerant flowing inside the jacket; electronic timers can be installed to control lengths of ultrasonic treatment and off-periods; controlled heating can also be applied as well as variable acoustic power output. The majority of ultrasonic baths are operated at around 40 kHz (Mason, 1998). Also, they are of rather low power in order to avoid cavitation damage to the tank walls (Mason, 1998). 2.2.2 Ultrasonic probe system

The power ultrasonic system can also be designed as a probe (Figure 23.4), in which one or several shaped metal horns are attached to the transducer so that high power intensity can be obtained. The horn must be designed to resonate at the same frequency as the transducer that drives it. It is important to obtain the correct amplitude of movement of the horn tips, which is dependent on the shape and dimensions. For a uniform cylinder horn, there is no amplitude gain. Instead, the horn just simply acts as an extender for the transference of acoustic energy. In some cases multiple horns are required in order to obtain some amplification; stages with the intermediate horns are usually called boosters (Keil and Swamy, 1999).

608 Ultrasonic Assistance of Food Freezing

2.2.3 Air-borne power ultrasonic system

Air has a low density, therefore it presents a very low specific acoustic impedance and high acoustic absorption. Consequently, in order to obtain air-borne power ultrasound, the transducers must be able to generate very efficient ultrasound transmission. The stepped plate transducer, designed by Gallego-Juraez et al. (1978), is the most suitable. It consists of a circular flexible vibration plate of stepped shape driven at its centre by a piezoelectric vibrator (Gallego-Juraez, 1988). The extensive surface of the

Transducer housing

Transducer

Horn

Replaceable cap

Figure 23.4

Ultrasonic probe (adapted from Mason, 1998).

Stepped plate transducer

Radiating surface

Power generator

Piezoelectric transducer

Figure 23.5

Mechanical amplifier

Air-borne power ultrasonic system (adapted from Gallego-Juarez, 1998).

Acoustic effects on the food freezing process 609

plate increases the radiation resistance and offers a good impedance matching with the medium. With the aid of adequate modifications of the plate surface any acoustic field configuration can be obtained. The overall set-up of the air-borne power ultrasonic system is shown in Figure 23.5.

3 Acoustic effects on the food freezing process One of the major characteristics of power ultrasound is its ability to produce different effects when transmitting through different media. Since foodstuffs are usually complex materials that contain multiple components in different phases and, furthermore, freezing is a phase-change process, it is thus important to understand the motions that arise when acoustic energy is transmitted through solid, liquid and gas, respectively. Knowledge about some fundamental aspects of freezing is also essential. With the above information available, it is then possible to see the various effects that power ultrasound generates when applied to the food freezing process in their right perspectives.

3.1 Acoustic effects on liquid, gas and solid 3.1.1 Acoustic effects on liquid

The most significant effect associated with the transmission of power ultrasound inside a liquid is cavitation, which refers to the motions of microbubbles in a liquid medium affected by an ultrasonic field, such as growth, shrinkage and collapse and the consequences of these physical perturbations (Prosperetti, 1984a, b; Young, 1989; Gong and Hart, 1998; Ashokkumar and Grieser, 1999). Cavitation (Figure 23.6) can be divided into two categories: stable or transient cavitations, depending on whether the bubbles break or not (Earnshaw, 1998). Water and most liquids are non-elastic materials, thus they could transit ultrasound continuously as long as the amplitude of the sound is relatively low. However, as the amplitude increases and exceeds a certain level, the magnitude of the negative pressure in the areas of rarefaction will eventually become sufficient to cause the liquid to fracture, which leads to the formation of bubbles or cavities. During the negative pressure portion of the sound wave, bubbles (including bubbles that are inherently present in the liquid) will grow rapidly and create a vacuum, which can cause gases dissolved in the liquid to start to diffuse into them. As the rarefaction portion of the sound wave passes, the negative pressure is reduced and when atmospheric pressure is reached, the bubbles will start to shrink under surface tension. Upon the commencement of the compression cycle of the sound wave and while the positive pressure lasts, gas that diffused into the bubbles will be expelled into the fluid. The diffusion of gas out of the bubbles will not take place until after the bubbles are compressed. However, once the bubble is compressed, its boundary surface area available for diffusion is decreased, therefore, the amount of gas that is expelled is less than the amount that is taken up during the rarefaction cycle. Consequently, these bubbles will grow bigger over each

610 Ultrasonic Assistance of Food Freezing

Acoustic pressure




Time 

Stable cavitation Creation

Bubble grows under negative pressure

Bubble collapse

Transient cavitation

Figure 23.6

Bubble contracts under positive pressure

Stable and transient cavitation.

ultrasound cycle, but will not reach the critical size for collapse. This process is called stable cavitation, which usually occurs at low acoustic pressure. The motions of the bubbles during stable cavitation can lead to several acoustic effects upon the medium. Microstreaming is one of them, which occurs when the oscillating bubbles produce a vigorous circulatory motion, which sets up strong eddy currents in the fluid surrounding them (Scheba et al., 1991). The diffusion of dissolved gases into and out of the bubbles also creates microcurrents around them, which can consequently spread into the liquid (Hughes and Nyborg, 1962). Computational fluid dynamics (CFD) simulation revealed that an average acoustic velocity of 3 mm/s could be obtained with 500 kHz ultrasound (Laborde et al., 2000). Microstreaming has been demonstrated to have many practical applications, for instance to induce cell membrane distortion. If carried into a region where microstreaming occurs, a cell can be damaged as a result of the unequal distribution of forces from the fluid due to the significant velocity gradient from the bubble surface to the fluid (Hughes and Nyborg, 1962). The turbulence (violent agitation) that microstreaming provides can also be used to enhance heat and mass transfer in many processes, such as to disperse particles (Ensminger, 1988), or to accelerate diffusion in many systems where mixing is not easy to achieve using ordinary mechanical agitation. Because of this, power ultrasound is shown to be able to accelerate a wide range of processes employed in the food industry, e.g. dehydration, filtration and extraction (McClements, 1995; Simal et al., 1998; Tarleton and Wakeman, 1998). Degassing is another significant application of stable cavitation, which is already being used commercially in the fermentation industry (Gallego-Juraez, 1998). The bubbles dissolved in the product act as nuclei for

Acoustic effects on the food freezing process 611

cavitation, which continue to grow during the rarefaction cycle of the sound waves by attracting other smaller bubbles and eventually become sufficiently buoyant to float to the liquid surface (Mason, 1998). At high acoustic pressure, the cavitation bubbles expand much more rapidly. After several ultrasound cycles, they will reach a critical size at which the oscillation frequency of the bubble matches the applied frequency of the sound waves. Under this circumstance, phase coupling between bubble oscillation and the sound wave causes the bubble to collapse violently during one compression cycle (see Figure 23.6). This process is called transient cavitation. Just prior to implosion, there is a tremendous amount of energy stored inside the bubbles. Therefore, when bubbles collapse, high pressure (up to 100 MPa) and high temperature (up to 5000 K) are momentarily produced (Earnshaw, 1998). The localized high temperature and pressure can destroy cell membranes and remove particles from a hard surface. It can also cause water to break down into H+ and OH species and lead to the production of hydrogen peroxide, which has excellent bactericidal properties (Ahmed and Russell, 1975; Suslick, 1988; Earnshaw, 1998). The extent to which cavitation occurs is affected by a number of parameters, acoustic power, ultrasound amplitude and frequency, nature and concentration of gases dissolved in the liquid, pressure, temperature, vessel size and geometry (Hagenson and Doraiswamy, 1998; Thompson and Doraiswamy, 1999). The frequency of ultrasound has a significant effect on cavitation since it determines the maximum bubble size (Earnshaw, 1998). At low frequencies, the bubbles produced are bigger in size and when they collapse more energies are released. At high frequencies, the bubble formation becomes increasingly difficult and when frequencies are above 2.5 MHz, cavitation does not occur. The amplitude of the ultrasound is another important parameter that affects the intensity of cavitation. High amplitude is required if high cavitation intensity is generated. However, when very high acoustic power is applied, a cloud of cavitation bubbles could be generated which is undesirable for energy transmission (Li, 2001). Cavitation is also affected by liquid viscosity since viscous fluid tends to disrupt ultrasound diffusion and thus reduces the degree of cavitation (Earnshaw, 1998). 3.1.2 Acoustic effects on gas

When applied to gas, power ultrasound can produce extreme turbulence at a gas/solid and gas/liquid interface (Hughes and Nyborg, 1962), which helps to reduce the diffusion boundary layer and can considerably increase convective mass transfer. The compression and rarefaction of the sound wave can generate pressure variations at the interface. Although its magnitude is very low, its effect is huge as the rate of oscillation is very rapid (Floros and Liang, 1994). This effect has been applied to accelerate the drying process, since during the negative pressure cycle, the moisture is removed and will not re-enter during the positive pressure cycle (Gallego-Juarez, 1998). 3.1.3 Acoustic effects on solid

When displaced in an ultrasonic field, due to the compression and rarefaction of the sound waves, solid material will experience a rapid series of alternative contractions

612 Ultrasonic Assistance of Food Freezing

and expansions in a similar way to a sponge being squeezed and released repeatedly. If the materials are compressible, during each contraction a minute quantity of water can be released, consequently, noticeable migration of water takes place (Ensminger, 1988). The sponge effect is able to lead to acceleration of the dehydration process (Gallego-Juarez, 1998). For dense materials that are practically incompressible, when subjected to the alternating acoustic stress, the sponge effect does not occur. Instead, ‘fracture’ occurs, which can lead to the formation of microscopic channels in directions normal to wave propagation during rarefaction cycle of the sound wave or parallel to wave propagation during the compression cycle (Muralidhara et al., 1985). Acoustic stress can also facilitate dewatering of dense materials by maintaining existing channels for water movement (Muralidhara et al., 1985).

3.2 Acoustic effects on the freezing process Water-based materials to be frozen may exist totally as flowing fluid or as products in which the water is effectively immobilized by the structure of the product. One typical example for the former case is the partial freezing of orange juice in a scraped surface heat exchanger, in which the fruit juice initially at a liquid state is discharged as iceconcentrate slurry. In the latter case, the foodstuffs usually consist of an aqueous phase and a matrix of insoluble solids such as protein, carbohydrates and perhaps fat as well and the freezing process involves conversion of the water in the aqueous phase into ice crystals. For either situation, the basic components of the system to be frozen can always be simply pictured as ice crystals distributed across the unfrozen aqueous phase. Therefore, when it is subjected to the action of the acoustic energy, the compression and rarefaction of the sound waves can induce the occurrence of cavitation in the aqueous phase. Cavitation will lead to production of gas bubbles, which will continue to grow under the rarefaction portion of the sound wave and serve as a nucleus for ice nucleation (Mason et al., 1996). Experiments with concentrated sucrose solution have shown that the nucleus number can be increased with the application of power ultrasound (Suslick, 1988). Food freezing is also a simultaneous heat and mass transfer process. Both ice nucleation and crystal growth release latent heat, which needs to be removed immediately in order to avoid localized reheating and subsequent melting. Along with crystal growth, non-water components are rejected at the freezing front, which need to be transported away otherwise future crystal growth will be retarded. Meanwhile, water molecules need to be transported to the freezing interface to facilitate further continuous crystal growth. As one of the most significant acoustic phenomena associated with cavitation, and with its ability to provide violent agitation in the aqueous phase (Sastry et al., 1989; Lima and Sastry, 1990), microstreaming can therefore increase the heat and mass transfer rate at the freezing interface, which consequently leads to the acceleration of the freezing process (Li and Sun, 2002). Similar to other dense and incompressible materials, ice crystals also fracture under the alternating acoustic stress, which was demonstrated by the study carried out by Acton and Morris (1992), who observed that when a pulse of ultrasound of approximately

Major functions of power ultrasound in assisting food freezing 613

0.14 With ultrasound

Fractional volume

0.12

Without ultrasound

0.1 0.08 0.06 0.04 0.02 0 20

35

50

65

80

95

110

125

140

155

170

Average diameter (␮m) Figure 23.7 Acoustic effect on crystal size distribution in frozen sucrose solution (adapted from Acton and Morris, 1992).

3 seconds was applied to a freezing sucrose solution every 30 seconds for a duration of 10 minutes, the front of the dendrite ice formed on the cold surface was clearly seen to fracture and the ice fragments were dispersed into the unfrozen bulk liquid. Fragmentation of ice crystals under acoustic stress can lead to crystal size reduction in the final product. It was found that ice crystals inside a frozen sucrose solution that was treated with power ultrasound during freezing have a smaller diameter than those without treatment. As shown in Figure 23.7, in the former, 32 per cent of the water exists in crystals with a diameter of 50 ␮m or larger while for the latter the amount is 77 per cent (Acton and Morris, 1992).

4 Major functions of power ultrasound in assisting food freezing In this section, it will be discussed how the above acoustic effects can be applied to solve some of the existing problems in food freezing as well as the significance that power ultrasound can have to some freezing processes with regard to increasing their efficiency and improving product quality.

4.1 Initiation of ice nucleation If power ultrasound is applied to the very initial stage of freezing or to a supercooled solution as a single pulse or a few short pulses, the major acoustic effect is to initiate nucleation. This process has already been widely used for some time (Fennema, 1973). Compared to other ice nucleation methods, for instance the use of some types of chemicals such as silver iodide, amino acids, ice nucleating bacteria, or seed crystals, power ultrasound offers several advantages. It is a very efficient treatment since

614 Ultrasonic Assistance of Food Freezing

one or two pulses of ultrasound can fulfil the requirement. Also, the initial nucleation temperature of the liquid can be dictated. Unlike nucleating agents, power ultrasound does not need to be in direct contact with the product to be frozen. Furthermore, it is not chemically invasive and thus is not likely to encounter legislative difficulties (Acton and Morris, 1992).

4.2 Acceleration of the freezing process Power ultrasound has also been demonstrated to be able to accelerate the food freezing process. The beneficial use of the sound is mainly realized through its ability to enhance the heat and mass transfer process accompanying freezing (Li and Sun, 2002). During immersion freezing of potatoes slices, Li and Sun (2002) applied power ultrasound intermittently when the potatoes temperature was reduced from 0 to 7°C, with each acoustic treatment lasting 30 s and the total number of treatments varied. The purpose of the intermittent treatment is to avoid the rise of refrigerant temperature, because continuous application of power ultrasound can lead to prolonged thermal effects upon the refrigerant. Li and Sun (2002) recorded the product temperature-time curves during the freezing process. From these curves, the characteristic freezing rate could be calculated, which was defined as the freezing time required for the product temperature to be reduced from 1 to 7°C when the maximum amount of ice crystals are formed. The results (Li and Sun, 2002) indicated that power ultrasound could lead to a noticeable increase in freezing rate. As an example, the freezing curve for potato samples under a power level of 15.85 W with a total acoustic treatment time of 2 min was compared to one without ultrasonic treatment (Figure 23.8). It can be calculated from the curves in Figure 23.8 that the application of power ultrasound reduced the characteristic freezing time from 8.7 min to 6.9 min. Mason et al. (1996) also attributed the influence of power ultrasound on the freezing process to a similar effect of increasing the freezing rate.

10 Without ultrasound

Temperature (°C)

5

With ultrasound 0 5 10 15 20 25 0

5

10

15

20

Time (min) Figure 23.8 Acoustic effect on the freezing rate during immersion freezing of potato slices (adapted from Li and Sun, 2002).

Major functions of power ultrasound in assisting food freezing 615

4.3 Control of the crystal size distribution in the frozen product Control of the crystal size distribution in the final product is usually one of the most important aspects for many freezing operations. Whether a large or small crystal size is preferable is mainly dependent on the purpose of freezing. For ice cream processing, it is preferred that the ice crystals are as small as possible in order to achieve a creamy and smooth product. However, if freezing is used to concentrate liquid food products, a large crystal size distribution is preferable as it can facilitate the future separation of the ice crystals from the freeze concentrate (Fellows, 2000). Freeze drying is a process in which the product is frozen, then water is removed from the frozen sample by sublimation and is currently used for the manufacture of coffee, milk, etc. It is usually desirable to produce large crystals in small numbers, since it can accelerate the subsequent sublimation process (Fellows, 2000). Power ultrasound has proved itself an effective means to modify and control crystal size distribution in the final frozen product, which is actuated by different ways. If ultrasound is applied during the crystal growth phase, fragmentation of large crystals under acoustic stress will lead to crystal size reduction. Research indicated that the influence of power ultrasound on the production of ice lollipops was considerable, in that the ice crystals formed were significantly smaller and distributed evenly across the solid product, which strongly improved the adhesion of the lollipop to the supporting wooden stick (Gareth, 1992). However, the small crystals made the product harder and difficult to bite. As power ultrasound is able to induce nucleation in many freezing operations at controlled temperature, this effect can also be used to control crystal size distribution in the frozen product. Its working principles are explained in the following. The freezing process consists of nucleation and crystal growth, both of which are dependent on the degree of supercooling of the system, however, in a different relationship, as shown in Figure 23.9 (Fennema, 1973). If power ultrasound is applied when the temperature of the system is maintained between the freezing point and point N, where crystal growth is much more dominant than nucleation, only a few nuclei will form and each will grow extensively. On the other hand, if acoustic energy is applied when the system is cooled to a temperature below point N, since the nucleation rate is much higher than the crystal growth rate, many nuclei will form. However, each will grow only to a limited extent, since the size of the complete crystals varies inversely with the number of nuclei as the total amount of ice formed is controlled by the degree of freezing, with its maximum value limited by the freezing system itself. Acton and Morris (1992) have successfully applied these principles to control ice crystal size for a freeze-drying process. They found that by irradiating sucrose solution with ultrasound for up to 5 s at 1°C of supercooling, only a few large crystals were formed, which were initially disc like and then grew into stellar dendrites. In contrast, when ultrasound was applied at 5°C of supercooling, a rapid freezing occurred with multiple nucleation sites and the crystals were smaller. After drying, the former sample was observed to have large pores, while pores inside the latter sample were much smaller. It is important to note that when the purpose is to produce large ice crystals, ultrasound can only be applied for a very short time, preferably several seconds, as continuous application of ultrasound would lead to fragmentation of the crystals.

616 Ultrasonic Assistance of Food Freezing

Rate

Heterogeneous nucleation

N

0

Growth

Temperature (°C)

50

Degree of supercooling Figure 23.9 Comparative rates of nucleation and crystal growth of water as influenced by supercooling (adapted from Fennema, 1973).

4.4 Improvement of frozen food microstructure 4.4.1 Effect of freezing on quality of frozen food

Freezing has been widely used to extend the shelf-life of many foodstuffs, due to a combination of low temperature and reduced water activity resulting from the concentration of dissolved solutes in the unfrozen water caused by the immobilization of water into ice (Fellows, 2000). However freezing causes cell damage to the products due to the formation of ice crystals, the extent of which is largely dependent on the size and location of the ice crystals inside the frozen food (Powrie, 1973; Aguilera and Stanley, 1990; Grout et al., 1991; Arthey, 1993; Cano, 1996). Most foodstuffs consist of animal and/or vegetable cells that form biological tissues. The solution of the tissues is contained between the cells (extracellular fluid) or inside the cell (intracellular fluid). Since the concentration of intracellular fluid is higher than that in the extracellular region, when the product is frozen, freezing usually occurs first in the extracellular region (Fennema, 1973). The formation of ice crystals results in an increase in concentration of the unfrozen solution locally and thus reduces its vapour pressure. If freezing continues exclusively in the extracellular region, the intracellular fluid will remain at supercooled state and its vapour will then exceed that of the extracellular fluid, which will cause more and more water to migrate to the extracellular region and deposit on the extracellular crystals. This effect is called cellular dehydration (Powrie, 1973), which can cause the shrinkage of the cell volume and subsequently collapse of cell walls. Furthermore, continuous freezing in the extracellular region can also lead to the formation of large extracellular

Major functions of power ultrasound in assisting food freezing 617

crystals, which will occupy a larger volume and execute more physical pressure on the cell wall and thus more cell deformation and damage will occur. Consequently, on thawing, cells are unable to regain their original shape and turgidity. The product will be softened and drip loss will occur as well. Therefore, in order to obtain high quality frozen food, it is preferred that the frozen product has a similar appearance to the original unfrozen shape. To achieve this, the following are essential: (1) the formed ice crystals need to be as small as possible; and (2) ice crystal distribution across the product needs to be as similar as possible to that of the water in the unfrozen product, which requires freezing to take place simultaneously in both intracellular and extracellular regions. Fast freezing has been widely proven as the most effective method so far for producing high quality frozen food (Powrie, 1973; Arthey, 1993). The higher degree of supercooling can initiate intracellular nucleation or cause extracellular crystals to penetrate through cell membranes into the intracellular region (Powrie, 1973), both of which lead to the occurrence of intracellular freezing. Also, as the degree of supercooling increases, the nucleation rate is higher which is beneficial for the formation of small crystals.

4.4.2 Influence of power ultrasound on the microstructure of frozen food

Recent research advances obtained by Sun and Li (2003) showed that application of power ultrasound to food freezing can improve the quality of the frozen product. Their experimental investigation (Sun and Li, 2003) indicates that plant tissues of ultrasound-assisted frozen potatoes exhibit a better cellular structure than those without acoustic treatment. Cryogenic scanning electron microscope (cryo-SEM) photos show that less extracellular void and cell disruption/breakage appear in the former. This might be attributable to the fast freezing induced by power ultrasound (Li and Sun, 2002). Alternatively, it might be that cavitation bubbles have initiated the occurrence of intracellular nucleation that might not usually occur as a result of an insufficient degree of supercooling (Powrie, 1973); cavitation bubbles might also increase the nucleation rate in the extracellular region. Either of these can help the product to retain its original shape and thus improve its quality. Crystal fragmentation caused by sonication is also a possible reason, which can reduce crystal size and thus the chances of cell breakage and deformation are minimized.

4.5 Preventing incrustation on a cold surface For some freezing processes, e.g. ice cream manufacture and freeze concentration of fruit juice, crystals usually form on the coldest part of the vessels, i.e. the heat exchanger surface, and have to be removed continuously by certain mechanical actions such as scraping. Otherwise, the formed crystals will build up on the cold surface, since ice has a much lower thermal conductivity than metal and the crystal layer will act as an insulating layer and significantly reduce the heat transfer rate. If power ultrasound is applied to these processes, it will bring an additional benefit as the cleaning action of cavitation can effectively prevent the encrustation of crystals on the cold surface and thus continuous heat transfer can be achieved (Mason, 1998).

618 Ultrasonic Assistance of Food Freezing

5 Factors affecting power ultrasound efficiency A variety of factors will affect the efficiency of power ultrasound and these can be classified into two groups, the sound factor and the product factor. The product factor involves parameters such as temperature and viscosity of the fluid food, its initial gas content and bubble size, the structure of the cellular food, its moisture content, water activity, water distribution, etc. The sound factor includes the acoustic power or power intensity, ultrasound frequency, ultrasonic duration, mode of ultrasound (continuous or intermittent), etc. Indeed, the desired ultrasonic conditions are in part determined by the product factor. Furthermore, the ultrasonic conditions applied can vary significantly depending on the purpose of the acoustic treatment. There have been some recommendations on some of the parameters to be applied for certain purposes. For instance, to initiate nucleation in a fluid, it is suggested that the power be greater than 2 watt/l of liquid and the preferred frequency be 20–40 kHz, while the duration be as short as possible and preferably no more than 5 s (Acton and Morris, 1992). On the other hand, if ultrasound is used for crystal refinement, it is required that the power level should be greater than 1 watt/cm2 liquid surface and continuous application for at least 10 s is recommended (Acton and Morris, 1992). In the following section, the results obtained by Li and Sun (2002) regarding the effect of acoustic power and duration upon its ability to accelerate the process of immersion freezing of potato slices is discussed in more detail.

5.1 Acoustic power level In their experimental investigation, Li and Sun (2002) applied three different levels of acoustic power of 7.34, 15.85 and 25.89 W, respectively and observed that the first power level did not result in any noticeable change in the freezing rate while the last two both increased freezing rate as shown in Figure 23.10. Furthermore, they (Li and 4 Without ultrasound 7.34 W 15.85 W 25.89 W

Temperature (ⴗC)

0 4 8 12 16 20 0

5

10

15

20

Time (min) Figure 23.10 Influence of acoustic power on efficacy of power ultrasound in accelerating the food freezing process (adapted from Li and Sun, 2002).

Factors affecting power ultrasound efficiency 619

Sun, 2002) found that product temperature declined more quickly at a power level of 25.89 W than 15.85 W at the temperature range from 0 to 7°C when ultrasound is applied, which is also the phase changing period. This is because power ultrasound can lead to acceleration of the freezing process through enhancement of heat and mass transfer due to the violent agitation created by the cavitation bubbles in the unfrozen liquid region. Results obtained by Sastry et al. (1989) showed that the heat transfer rate increase due to ultrasonic vibration is related to the amount of acoustic power. At higher power levels, as more agitation is generated, the extent of heat transfer enhancement is higher. This gives a good explanation to the above experimental results regarding to the influence of acoustic power on the freezing rate, which suggests that the power level of 7.34 W might be too small to induce the occurrence of cavitation and its effect on freezing rate is thus minimal. More turbulence is created at a power level of 25.89 W than 15.85 W and the freezing rate of products treated under the former condition is faster. However, towards the end of the phase changing period, it was observed that the temperature of the product treated with 25.89 W declined more slowly than that with 15.85 W. The reasons are not yet fully understood. Li and Sun (2002) suggested that this might be associated with the accumulated thermal effect that power ultrasound might have upon the medium, since depending on the nature of the medium, sound waves can be absorbed by the medium in which it transmits and converted into heat (Floros and Liang, 1994). Referring back to the experimental set-up in the study by Li and Sun (2002), since the transducers were installed on the bottom of the ultrasonic bath, acoustic energy can be lost to either the refrigerant or the bath materials before it reached the product. Acoustic absorption by the product itself might also occur, in particular by the cell membranes, which were considered to have a high ability to absorb sound waves (Floros and Liang, 1994). Resulting from these acoustic absorptions, the temperature of either the refrigerant or the product will be raised, neither of which benefits the freezing process. The negative effect of power ultrasound on the refrigerant can be minimized by applying a high refrigerant flow rate as it can help to maintain the refrigerant temperature constant. The lower freezing rate observed for an acoustic power of 25.89 W compared to 15.85 W during the later stage of the freezing process seems to imply that the amount of sound waves absorbed by the freezing system is proportional to the acoustic power applied.

5.2 Acoustic duration The ability of power ultrasound in accelerating the freezing process is also affected by its duration (also known as the exposure time) (Li and Sun, 2002). As shown in Figure 23.11, ultrasonic treatment of 1 min did not cause any significant change in the freezing rate. However, a noticeable increase of freezing rate was observed when the acoustic exposure time was increased to 1.5, 2 and 2.5 min. Furthermore, during the phase changing period, freezing rate for potatoes with an acoustic exposure time of 2.5 min was found to be the fastest, followed by those treated for 2 min and potatoes treated for 1.5 min had the slowest freezing rate. Towards the end of the phase changing period, the temperature reduction for potatoes treated for 2.5 min was slower than for

620 Ultrasonic Assistance of Food Freezing

5 Without ultrasound 1 min 1.5 min 2 min 2.5 min

Temperature (°C)

0

5

10

15

20 0

5

10

15

20

Time (min) Figure 23.11 Influence of acoustic exposure time on the efficacy of power ultrasound in accelerating the food freezing process (adapted from Li and Sun, 2002).

2 min. This might again be due to the accumulated thermal effect which is proportional to the acoustic duration.

6 Embodiment of applications In the earlier sections, power ultrasound has been demonstrated as a very useful method in assisting food freezing, which not only enables freezing to be conducted more efficiently and with better control than was previously possible, but also leads to products with better quality. However, apart from its ability to solve real problems, the future development of power ultrasound in assisting food freezing is also strongly linked to the availability of adequate industrial equipment, which still requires considerable research effort. Since it is not a replacement of current freezing techniques, it is preferable that the ultrasonic device is designed in such a way that it can be used in conjunction with existing freezing equipment. Overall, acoustic energy can be applied directly to the product, e.g. by direct immersion of ultrasonic probes in the process fluid, or indirectly from a transducer coupling through parts of process vessels (Acton and Morris, 1992). The particular form of ultrasonic apparatus used will vary according to the product and the type of process vessels. A few examples are discussed below.

6.1 Freezing of ice cream inside SSHE Freezing of ice cream takes place in two stages. The initial stage is in a scraped surface heat exchanger (SSHE) (also known as an ice cream freezer), which is a jacketed cylinder. A schematic diagram of the ice cream freezer is shown in Figure 23.12. Ice cream premix at an initial temperature of 4°C is pumped into the inner cylinder (also

Embodiment of applications 621

Refrigerant out B A

nt

Air

D Product in

E

Product out

C Refrigerant in Figure 23.12

A few possible locations for the attachment of ultrasonic device to the ice cream freezer.

known as freezer barrel) while refrigerant flows through the jacket. Freezing usually takes place on the inner surface of the freezer barrel with the formed ice crystals removed continuously by the scraper blades in order to avoid the build up of the ice crystal layers on the surface, which would otherwise slow down the heat removal rate and thus the freezing process. The semi-frozen product is discharged from the freezer at an outlet temperature from 4 to 9°C. The second stage of freezing is known as ‘hardening’ where the temperature of the ice cream is further reduced to 18°C in an air blast or plate freezer. Unlike most foodstuffs, ice cream is designed to be consumed in a frozen state, therefore, crystal size influences texture and taste directly. For a creamy product, it is required that ice crystals be as small as possible, preferably less than 25 ␮m. Application of power ultrasound to ice cream manufacture during freezing inside the scraped surface heat exchanger will bring several benefits. The most significant effect is to cause fragmentation of large crystals. It can also lead to enhancement of heat and mass transfer as microstreaming can create turbulence in the fluid. Furthermore, the cleaning action associated with cavitation might benefit the prevention of ice crystals from scaling on the inner wall surface of the freezer barrel. This might consequently lead to removal of the mechanical scrapers, which can not only simplify freezer design, but also reduce friction heat due to the scraping action upon the freezer wall surface. However, since commercial ice cream contains up to 50 per cent by volume of entrapped air and power ultrasound is known as a commercial method for degassing in some food manufacture processes, e.g. canning lines (Mason, 1998), application of acoustic energy is therefore likely to result in undesirable modification of the ice cream texture. To overcome this, Acton and Morris (1992) proposed increasing the initial gas content so that the proportion of air lost due to the ultrasound irradiation can be compensated, or to carry out the process under increased pressure, or to incorporate air into the partially frozen product rather than at the initial stage of the freezing process. With respect to equipment design, the ultrasonic device can be attached to the freezer at several different locations as shown in Figure 23.12 (Acton and Morris, 1992). The

622 Ultrasonic Assistance of Food Freezing

most preferabe place will be on the external surface of the freezer barrel (A). In the freezer, the region of most rapid crystal growth is near the inner surface of the freezer barrel. Such positioning of an acoustic resource will appear to generate the maximum effect of crystal fragmentation. Since crystals grow over the whole surface of the freezer barrel wall, the maximum benefit would be obtained by applying ultrasound over the whole processing surface. This arrangement can also bring an additional benefit, i.e. any heat generated by the transducers will be absorbed by the coolant flow rather than by the product. Transducers may also be mounted on the outside surface of the freezer (B), in this case ultrasound will be transmitted through the outer wall of the refrigerant jacket, the refrigerant and finally the barrel wall. The major advantage of this configuration is that transducers could be attached to the freezer very easily, however, significant acoustic power may be lost to the vessels walls and the refrigerant. The transducers can also be sealed through the outer wall of the freezer jacket and directly attached to the inner wall (C). Alternatively, they might be mounted on the rotor (D), or the ultrasound vibration can be produced via a vibrating plate positioned in the bulk of the liquid (E). The latter, however, requires special care due to the movement of the rotor and the heat generated by the transducers within the liquid needs to be considered as well.

6.2 Manufacture of moulded frozen products Power ultrasound can also benefit the manufacture of moulded frozen products, such as sorbets, ice lollipops, etc. as sonication can reduce crystal size. The acoustic vibration can be applied through coupling transducers to the mould walls. In some cases, freezing is achieved by partial immersion of the moulds on a conveyor belt through a refrigerant. Under such circumstances, ultrasound can be transmitted through the refrigerant, with the mechanical vibrations either generated by ultrasonic probes directly positioned inside the refrigerant or transducers mounted to the walls of the refrigerant tank. As suggested by Acton and Morris (1992), the power required needs to be greater than 5 W/l of refrigerant.

6.3 Freezing and frozen storage of fresh foodstuffs With its ability to accelerate the freezing process and improve frozen food quality, power ultrasound has proved itself very useful in freeze preservation and frozen storage of fresh foodstuffs. Currently, different freezing methods are employed in the food freezing industry, including immersion freezing, air blast freezing, contact freezing and cold storage freezing (Fellows, 2000). Associated with these freezing methods, different types of freezers have been used. Immersion freezing (immersion freezer is usually used) is the process where packaged food is passed through a bath of refrigerant on a submerged mesh conveyor. Similar to the process of mould freezing, ultrasound can be applied through the refrigerant and generated by any means. Air blast freezing is used most widely in the current food industry and is operated either in batch mode or continuously. Equipment for the continuous process consists of trolleys stacked with food or of conveyor belts, which carry the food through an insulated tunnel. Therefore,

Conclusions 623

Generator

Radiating surface

Product

Conveyor belt

Figure 23.13 Schematic illustration of ultrasound-assisted food freezing on a conveyor belt freezer inside a blast room.

technical problems might arise regarding how to attach an ultrasonic device to these moving parts. One possibility is to adopt the air-borne power ultrasound technology that is already in use for the beverage industry for defoaming in the canning lines (Gallego-Juraez, 1998), since air-borne power ultrasound does not require direct contact of the ultrasonic device to the freezing equipment. An example of applying the airborne power ultrasound to freezing of fresh foodstuffs on a conveyor belt freezer inside a blast room is given in Figure 23.13. A plate freezer is usually utilized for contact surface freezing (Fellows, 2000). The transducers can be directly welded to the underneath of the contact surface. This arrangement also brings an additional benefit that heat generated by the transducer can be carried away by the refrigerant. Cold storage freezing is the process where food is frozen in stationary air. Instead of being a commercial freezing method, it is used more often for storage of foodstuffs frozen by other methods or for ice cream hardening due to its very low freezing rate. During frozen storage, ice recrystallization occurs very often due to temperature fluctuation, which will increase crystal size and consequently cause loss of product quality (Fellows, 2000). This negative effect can be reduced by acoustic treatment resulting from its ability to fracture large crystals. The acoustic energy can either be air borne or applied through transducer coupling with product holders.

7 Conclusions In this chapter, the various effects that power ultrasound generates when it is applied to the specific field of food freezing are comprehensively reviewed. As has been

624 Ultrasonic Assistance of Food Freezing

shown, cavitation in the unfrozen liquid phase is one of the most significant phenomena provoked by the action of the acoustic energy. Cavitation results in the occurrence of microstreaming, which is able to enhance the heat and mass transfer accompanying the freezing process. Cavitation also benefits initiating the occurrence of nucleation and increasing the rate of nucleation as the gas bubbles produced can act as nucleating agents. Crystal fragmentation is another significant acoustic phenomenon, which can lead to crystal size reduction and, indeed, is one of the most important aspects that many freezing processes target. Resulting from these acoustic effects, power ultrasound has proved itself an effective method in assisting food freezing and its benefits are wide-ranging. It can be utilized to induce nucleation and to control crystal size distribution in the frozen products during solidification of fluid food. If it is applied to the process of freezing fresh foodstuffs, it not only can increase the freezing rate, but also improve the quality of the frozen products. Application of power ultrasound can also benefit the prevention of incrustation on the freezing surface. The ability of power ultrasound in performing these functions is affected by a wide variety of parameters, such as the duration, intensity or frequency of ultrasonic waves, etc. More fundamental research is still needed to establish their relationship with acoustic efficiency. The future of power ultrasound in assisting food freezing is also linked to the development of adequate industrial equipment. In fact, the commercial application of a new technology depends on, in addition to its ability to solve real problems, the design of equipment that is easy to operate and is cost effective. In this chapter, some methods are also reviewed on how to integrate ultrasonic devices to some commercial freezing equipment. Most of them remain proposals only and considerable research effort is still required to verify their feasibility and practicality. In general, research up to date indicates that, although commercial development of this technology is still not available, the potential of power ultrasound to aid food freezing is promising, research hurdles do not appear to be insurmountable and its benefits to manufacturers seem great.

References Acton E, Morris GJ (1992) Method and apparatus for the control of solidification in liquids. WO 99/20420, USA Patent Application. Aguilera LM, Stanley DW (1990) Microstructural Principles of Food Processing & Engineering. London: Elsevier Science Publishers Ltd. Ahmed FIK, Russell C (1975) Synergism between ultrasonic waves and hydrogen peroxide in the killing of microorganisms. Journal of Applied Bacteriology, 39, 31–40. Arthey D (1993) Freezing of vegetables and fruits. In Frozen Food Technology (Mallett CP, ed.). Glasgow: Blackie Academic & Professional, pp. 237–269. Ashokkumar M, Grieser F (1999) Ultrasound assisted chemical process. Reviews in Chemical Engineering, 15 (1), 41–83. Cano MP (1996) Vegetables. In Freezing Effects on Food Quality (Jeremiah LE, ed.). New York: Marcel Dekker, Inc., pp. 247–298.

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Earnshaw RG (1998) Ultrasound: a new opportunity for food preservation. In Ultrasound in Food Processing (Povey MJW, Mason TJ, eds). Glasgow: Blackie Academic & Professional, pp. 183–192. Ensminger D (1988) Acoustic and electroacoustic methods of dewatering and drying. Drying Technology, 6, 473–499. Fellows P (2000) Food Processing Technology – Principles and Practice, 2nd edn. Cambridge: Woodhead Publishing. Fennema O (1973) Nature of the freezing process. In Low Temperature Preservation of Food and Living Matter (Fennema RO, Powrie DW, Marth EH, eds). New York: Marcel Dekker Inc., pp. 151–227. Floros JD, Liang HH (1994) Acoustically assisted diffusion through membranes. Food Technology, December, 79–84. Fuchs FJ (1999) Ultrasonic cleaning: fundamental theory and application. New York: Applications Engineering, Blackstone-Ney Ultrasonics Inc. Gallego-Juarez JA (1988) High power ultrasonic transducers for use in gases and interphases. In Power Sonic and Ultrasonic Transducers Design (Hamonic B, Decarpigny JN, eds). Heidelberg: Springer-Verlag, pp. 175–184. Gallego-Juarez JA (1998) Some applications of air-bone power ultrasound to food processing. In Ultrasound in Food Processing (Povey MJW, Mason TJ, eds). Glasgow: Blackie Academic & Professional, pp.127–143. Gallego-Juarez JA, Rodriguez-Corral G, Gaete-Garreton L (1978) An ultrasonic transducer for high-power application in gases. Ultrasonics, 16, 267–271. Gareth J (ed.) (1992) Current Trends in Sonochemistry. Cambridge: The Royal Society of Chemistry. Gennaro LD, Cavella S, Romano R, Masi P (1999) The use of ultrasound in food technology I: inactivation of peroxidase by thermosonication. Journal of Food Engineering, 39, 401–407. Gong CL, Hart DP (1998) Ultrasound induced cavitation and sonochemical yields. Journal of American Acoustical Society, 104, 2675–2682. Grout BWW, Morris GJM, McLellan MR (1991) The freezing of fruits and vegetables. In Food Freezing: Today and Tomorrow (Bald WB, ed.). Berlin: Springer-Verlag, pp. 113–123. Hagenson LC, Doraiswamy LK (1998) Comparison of the effects of ultrasound and mechanical agitation on a reacting solid-liquid system. Chemical Engineering Science, 53, 131–148. Hamonic B, Decarpigny JN (eds) (1988) Power Sonic and Ultrasonic Transducer Design, Heidelberg: Springer-Verlag. Hughes DE, Nyborg WL (1962) Cell disruption by ultrasound. Science, 138, 108–114. Keil FJ, Swamy KM (1999) Reactors for sonochemical engineering – present status. Reviews in Chemical Engineering, 15 (2), 85–155. Laborde JL, Hita A, Caltagirone JP, Gerard A (2000) Fluid dynamics phenomena induced by power ultrasounds. Ultrasonics, 38, 297–300. Li B (2001) Immersion freezing of potato assisted by power ultrasound to improve freezing efficiency and microstructure. Masters Thesis, University College Dublin, Ireland.

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Li B, Sun D-W (2002) Effect of power ultrasound on freezing rate during immersion freezing. Journal of Food Engineering, 55 (3), 277–282. Lima M, Sastry SK (1990) Influence of fluid rheological properties and particle location on ultrasound-assisted heat transfer between liquid and particles. Journal of Food Science, 55, 1112–1115. Mason TJ (1998) Power ultrasound in food processing – the way forward. In Ultrasound in Food Processing (Povey MJW, Mason TJ, eds). Glasgow: Blackie Academic & Professional, pp. 104–124. Mason TJ, Paniwnyk L, Lorimer JP (1996) The use of ultrasound in food technology. Ultrasonics Sonochemistry, 3, S253–S256. McClements J (1995) Advances in the application of ultrasound in food analysis and processing. Trends in Food Science and Technology, 6, 293–299. Muralidhara HS, Ensminger D, Putnam A (1985) Acoustic dewatering and drying (low and high frequency). Drying Technology, 3, 529. Powrie WD (1973) Characteristics of food myosystems and their behaviour during freezepreservation. In Low-temperature Preservation of Foods and Living Matter (Fennema OR, Powrie WD, Marth EH, eds). New York: Marcel Dekker, Inc., pp. 354–380. Prosperetti A (1984a) Bubble phenomena in sound fields: Part one. Ultrasonics, 22, 69–77. Prosperetti A (1984b) Bubble phenomena in sound fields: Part two. Ultrasonics, 22, 115–124. Sastry SK, Shen GQ, Blaisdell JL (1989) Effect of ultrasonic vibration on fluid-to-particle convective heat transfer coefficients. Journal of Food Science, 54, 229–230. Scheba G, Weige RB, O’Brien JR (1991) Quantitative assessment of germicidal efficiency of ultrasonic energy. Applied Environmental Microbiology, 57, 2079–2084. Simal S, Benekito J, Sanchez ES, Rossello C (1998) Use of ultrasound to increase mass transport rates during osmotic dehydration. Journal of Food Engineering, 36, 323–336. Sun D-W, Li B (2003) Microstructural change of potato tissues frozen by ultrasoundassisted immersion freezing. Journal of Food Engineering, 57, 337–345. Suslick KS (1988) Chemical, biological and physical effects. In Ultrasound (Suslick KS, ed.). New York: VCH, pp. 123–163. Tarleton ES, Wakeman RJ (1998) Ultrasonically assisted separation processes. In Ultrasound in Food Processing (Povey MJW, Mason TJ, eds). London: Blackie Academic & Professional, pp. 193–218. Thompson LH, Doraiswamy LK (1999) Sonochemistry: science and engineering. Industry Engineering and Chemistry Research, 38, 1215–1249. Young FR (1989) Cavitation. New York: McGraw-Hill.

High-Pressure Freezing Pedro D Sanz and Laura Otero Instituto del Frío (CSIC), Department of Engineering, Madrid, Spain

In the last decade, high-pressure freezing has drawn the attention of many food researchers, mainly due to its potential for improving the kinetics of freezing and the characteristics of the ice crystals formed. This chapter examines the process in depth. Different types of highpressure freezing processes are described and compared with conventional freezing in atmospheric conditions. The effects on the quality (microstructure, texture, drip losses, colour, microbial inactivation, etc.) of different kinds of food are also discussed. Special attention has been paid to modelling of these new processes. Much research is still required, but future prospects are promising.

1 Introduction Freezing is an excellent food preserving method that delays or prevents microbial, chemical and physical alterations by reducing water activity (solidification to ice), microbial growth and reaction velocities in enzymatic systems (temperature reduction imposes suboptimal conditions). To be suitable, a freezing method must preserve all the organoleptic characteristics of the fresh product (flavour, colour, aroma, etc.) and the nutritional value (Martí and Aguilera, 1991). However, ice crystals can severely damage the tissues, affect texture and cause major drip loss during thawing. Ice forms in two stages: formation of ice nuclei, followed by growth of these nuclei. The distribution of ice crystal size throughout a sample depends on both the number of nuclei (or seeds) formed in the earlier phase and the rate of crystal growth, which influences the shape and the size of the final crystals. In the first stage of freezing, the temperature of the product must fall well below the solid-liquid equilibrium point to produce nucleation. This is an activated process driven by supercooling (the difference between the actual temperature and that of the solid-liquid equilibrium). The greater the degree of supercooling, the larger is the number of nuclei created thus, the rate of ice nucleation increases roughly tenfold for every degree of supercooling (Burke et al., 1975). Crystal growth takes place only once nucleation has occurred through addition of water molecules to the nuclei already formed and it mainly depends on the freezing rate. In slow freezing, where the sample temperature remains close to the solid-liquid equilibrium curve for a long time, the rate of nucleation is low and only a few nuclei will be formed, leading to large final ice crystals. On the other hand, in a system where freezing rates are higher, many nuclei will be formed, limiting the final crystal Emerging technologies for food processing ISBN: 0-12-676757-2

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628 High-Pressure Freezing

size. In traditional freezing methods, when a food product comes into contact with the refrigerating medium, ice nucleation only occurs in the region next to the refrigerated border and is controlled by the magnitude of supercooling reached in this zone (Bevilacqua and Zaritzky, 1980). In the inner regions of the product, the supercooling required to produce ice nucleation is not achieved because of the thermal gradients and the result is the growth of large ice crystals. The optimum freezing rate for food products is highly dependent on the food system. Slow freezing generally results in extensive mechanical damage and reduces the maximum attainable food quality. Then again, ultra-rapid freezing may cause mechanical cracking (Kalichevsky et al., 1995; Otero et al., 2000a), particularly in large samples with high water content and low porosity (Kim and Hung, 1994). Improvement of known freezing methods and the development of new techniques are important research objectives for the food industry (Li and Sun, 2001). High-pressure freezing has been studied in depth over the last ten years thanks to its potential for improving the kinetics of freezing and the characteristics of the ice crystals thus formed (Sanz, 2004).

2 High-pressure freezing Figure 24.1 shows the phase diagram of water. Ice I, the common ice at atmospheric pressure, is stable up to 210 MPa. Above this pressure level, different ice polymorphs are thermodynamically stable according to the pressure/temperature coordinates (Bridgman, 1912). The freezing point of water decreases with pressure up to 210 MPa. The opposite was observed above this level for ice types other than ice I (see Figure 24.1). According to Clausius-Clapeyron equation: dT
V  Tk  dP
H

(1)

where T is temperature and P is pressure, the negative slope of the liquid/ice I equilibrium line implies that the signs of the volume change, ⌬V, and the latent heat ⌬H in the above equation are different. In the case of liquid-solid phase transition, ⌬H is negative irrespective of the modification of the solid state. The variation of volume therefore must be positive for ice I. Liquid water is denser than ice I, which presents a hexagonal structure with much empty space. This increase in volume upon freezing is mainly responsible for damage to biological systems when frozen. On the other hand, the volume increment in liquid-ice III, liquid-ice V or liquid-ice VI phase changes is negative; also, the phase change enthalpy increment is negative and the slope of the melting curve is positive. We may therefore expect less damage to these ice polymorphs upon freezing. In principle, no solid (ordered) phase can exist in the liquid (disordered) domain, but the inverse is possible as a metastable state. This phenomenon, called supercooling

High-pressure freezing 629

80 Liquid water

Temperature (°C)

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III

I IV V

Ice VI

II ⫺80

⫺120

Ice VIII Ice IX

⫺160

0

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Pressure (MPa) Figure 24.1

Phase diagram of water.

or undercooling, seems to be enhanced by pressure. The homogeneous nucleation temperature of water decreases even more than the melting temperature with pressure up to 210 MPa. The metastable range is thus significantly enlarged with the application of pressure. The minimum temperature at which it is possible to find supercooled water is therefore reduced from ⫺40°C at atmospheric pressure to ⫺92°C at about 210 MPa (Lüdermann, 1994).

2.1 Types of high-pressure freezing processes According to the phase diagram of water, three different types of high-pressure freezing processes can be distinguished in terms of the way in which phase transition occurs: 1 high-pressure assisted freezing (HPAF) 2 high-pressure shift freezing (HPSF) 3 high-pressure induced freezing (HPIF) where pressure-assisted means phase transition under constant pressure; pressureshift means phase transition due to a pressure release and pressure-induced means phase transition initiated by a pressure change and continued at constant pressure. This terminology was first suggested by Knorr et al. (1998), who presented different idealized freezing paths on the phase diagram of water. Before that, terms such as pressure-assisted freezing, pressure-supported freezing or freezing under pressure were used in a general way to denote any kind of freezing process operated with help, support or assistance of pressure.

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High-pressure freezing processes operate in pressure-resistant vessels with thermally isolated thermostatic circuits to reach temperatures below 0°C. Packed foods are immersed in the pressure/cooling medium and frozen. The apparatus must be equipped with thermocouples to monitor the evolution of temperature during the process in both the product and the pressure medium and also at least with a pressure gauge to measure pressure in the circuit. Different pressure/cooling media have been employed as reported in the literature. These are either pure substances or mixtures: silicon oil (Luscher et al., 2005), propylene glycol (Teramoto and Fuchigami, 2000), glycol/water: 62/38, v/v (Kalichevsky et al., 2000), ethylene glycol/water: 75/25, v/v (Otero and Sanz, 2000), ethanol/water: 50/50,v/v (Chevalier et al., 2001b), ethanol/glycol: 20/80, v/v (Schlüter et al., 1998), castor oil/ethanol: 15/85, v/v (Johnston, 2000) or propanediol/water: 55/45, v/v (Lévy et al., 1999) among others. When choosing a pressure medium, it is necessary to take into account the freezing point of the fluid under pressure, its viscosity and other thermophysical properties (heat capacity, thermal expansion coefficient and specific volume) that influence pressure-induced temperature changes. 2.1.1 High-pressure assisted freezing

2.1.1.1 Description of the process Figure 24.2 shows a high-pressure assisted freezing process. Phase transition occurs under constant pressure, higher than atmospheric pressure, while the temperature is lowered to below the corresponding freezing point. In this way, ice I or other known ice polymorphs can be obtained. According to Urrutia Benet et al. (2004), the term highpressure assisted freezing with no extra specifications must be used exclusively to refer to ice I. When a higher ice modification is frozen, this should be explicitly indicated. Different degrees of supercooling have been reported to initiate nucleation under pressure in different products. However, the supercooling that takes place experimentally depends on many factors like the product, its size, the container in which it is located, the cooling rate, the pressure level and others. Even when all these factors are controlled, results can be non-repetitive (Heneghan et al., 2002) due to the stochastic nature of the nucleation phenomenon. Knorr et al. (1998) required only negligible supercooling to freeze potato tissue at 70 MPa/⫺17°C. However, greater supercooling was found at 100 MPa/⫺19°C (about 5°C) and 140 MPa/⫺23°C (about 7°C). In recent experiments, this same group reported negligible supercooling in freezing experiments at 140 MPa and 209 MPa in potato tissue, maintaining the freezing medium at 25°C lower than the corresponding freezing point (Schlüter et al., 2004). For other kinds of ice, greater supercooling seems to be needed to initiate nucleation. Knorr’s group concluded that the formation of ice III in potato tissue was only possible after minimum supercooling of 15°C (Schlüter and Knorr, 2002; Luscher et al., 2005). In general terms, the higher the pressure, the greater the degree of supercooling is needed (Schlüter et al., 2004). Fuchigami et al. (2002) needed negligible supercooling to freeze tofu at 0.1 and 100 MPa (pressure medium at ⫺20°C), but about 9°C of supercooling was needed to obtain ice under 686 MPa. Molina-García et al. (2004) also needed high degrees of supercooling to obtain ice VI in water and meat samples at 700 MPa (13  3°C and

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30 20

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B

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10

A

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Liquid water

10 20

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Ice I

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E D

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Pressure (MPa) Figure 24.2 Different high-pressure assisted freezing processes producing different ice polymorphs: ice I (ABCDE) or ice III (ABCDE) (taken from Otero and Sanz, 2003).

10  3°C respectively). This degree of supercooling was approximately twice that needed for ice I freezing at atmospheric pressure in identical experimental conditions. Therefore, supercooling is essential when designing freezing experiments under pressure. Also, experimental temperature-pressure values need to be precisely monitored to ensure that freezing has actually occurred. Pressure increases or decreases (related to volume changes), in addition to sudden temperature changes, have been effectively used as indicators of phase transition events (Knorr et al., 1998; Molina-García et al., 2004). Without these measurements, there is no way to be certain that a phase change has really occurred. In this connection, earlier experiments from Fuchigami’s group in carrots (Fuchigami et al., 1996, 1997a, b), tofu (Fuchigami and Teramoto, 1996, 1997; Fuchigami et al., 1998b) and agar gel (Fuchigami and Teramoto, 1998) present some uncertainty. Cooling of the sample proceeds from surface to centre and the process is governed by thermal gradients as in classical freezing processes at atmospheric pressure. Thus, ice nucleation only occurs in the outer zone of the product that is in direct contact with the cooling medium; the resulting ice crystals are large, needle-shaped, radially oriented and present a marked size gradient from sample surface to centre (Fuchigami and Teramoto, 1997; Lévy et al., 1999). Once the freezing plateau is complete and the sample reaches the final temperature, the pressure is released. If ice I has formed, the sample temperature will drop after expansion; if not, this is a clear sign that freezing was not complete under pressure. If another ice polymorph was formed, there will be a solid-solid phase transition to ice I upon pressure release to atmospheric conditions (Fuchigami et al., 1997a, 2002; Cheftel et al., 2000; Fuchigami and Teramoto, 2003). Latent heat of water decreases with pressure up to 210 MPa (Hobbs, 1974). In this connection, Knorr et al. (1998) reported reductions of the freezing time with increasing pressure (0.1–210 MPa) compared to atmospheric conditions. When comparing

632 High-Pressure Freezing

freezing times, it is essential that the ⌬T between the sample and the cooling medium remain constant, since the freezing point also decreases with pressure up to 210 MPa (see Figure 24.1). If the cooling medium temperature is kept constant, the ⌬T between it and the sample will be smaller when pressure is increased (freezing point decreases) and the freezing times will increase (Levy et al., 1999). 2.1.1.2 Quality of pressure-assisted frozen foods Observations of the structure of different high-pressure assisted frozen products (ice I or other ice polymorphs) show that crystal size always grows from the sample surface to the centre as a result of the thermal gradients established during the phase transition (Levy et al., 1999). If thermal gradients between the freezing medium and the sample are constant (taking into account the freezing point depression with pressure), less damage to sample structure is expected from ice polymorphs other than ice I, mainly because of their corresponding decrease in volume during the phase transition. However, these types of ice will revert to ice I during pressure release at below 0°C, thus cancelling out the expected advantages. In this connection, Fuchigami and Teramoto (2003) found no important improvements in texture and structure of high-pressure assisted frozen gellan gum gels to ice V (600–686 MPa and ⫺20°C) with respect to those frozen at atmospheric pressure at ⫺20°C. Various authors have designed freezing/thawing experiments under constant pressure to avoid passing through the ice I phase in the expansion (Fuchigami and Teramoto, 1998; Fuchigami et al., 1998b; Molina-García et al., 2004; Luscher et al., 2005). Highpressure assisted frozen samples can be stored at constant pressure and then thawed under the same constant pressure (by raising the temperature) when desired. Experiments of this kind make it possible to study the real effect of a certain ice polymorph while excluding the added effect of the solid-solid phase transition to ice I. For example, Molina-García et al. (2004) froze meat samples to ice VI at 700 MPa/0°C and fixed them under pressure (with Carnoy fixing agent). After 14 h in the frozen state, the temperature was raised; the sample was thawed at 700 MPa and then pressure was released to atmospheric conditions. Ice VI freeze-substitution microscopy showed no traces of ice on muscle fibres compared with the extensive damage caused by ice I freezing (Figure 24.3). Luscher et al. (2005) have proved that freezing to ice III (at 320 MPa) produced less damage to potato tissue than freezing to ice I (at 0.1 MPa or 200 MPa) or to ice V (at 400 MPa). These authors (Luscher et al., 2005) maintained the same thermal gradient between the cooling medium and the sample in all cases, taking into account pressure-induced depression of the freezing point. Post-freezing damage in cellular membranes seems to be influenced by two factors: the duration of the phase transition and the volume change of water during it. Luscher et al. (2005) have shown that ice I is the most harmful ice for cellular membranes of potato tissue. There was more damage at 200 MPa (volume increase of 0.13 cm3/g) than at atmospheric pressure (volume increase of 0.09 cm3/g). Ice V at 400 MPa (volume increase of ⫺0.07 cm3/g) caused less damage and ice III at 320 MPa (volume increase of ⫺0.03 cm3/g) was found the least destructive. Thus, a phase change with a volume decrease is less destructive for cellular membranes than

High-pressure freezing 633

(a)

(b)

(c)

(d)

Figure 24.3 Freeze-substitution micrographs of pork meat (loin). (a) Fresh. (b) Frozen to ice VI (at 700 MPa and 0°C). (c) Frozen to ice I (at 0.1 Pa and ⫺18°C). (d) Treated at 700 MPa and 25°C. The bar indicates 100 m (taken from Molina-García et al., 2004).

a phase change with a volume increase, but a larger negative change in volume is more destructive than a smaller negative change. The same authors (Luscher et al., 2005) found that with freezing to ice III/thawing under 320 MPa, the potato texture was quite close to the untreated control and there was slightly less browning compared to conventional freezing and thawing. High-pressure assisted freezing would appear to be of limited interest to the food industry, since frozen storage and thawing under pressure are required to preserve the advantages of ice polymorphs denser than ice I by avoiding solid-solid phase transitions to ice I in the expansion. This would only be practicable in the case of high-value biological samples. 2.1.2 High-pressure shift freezing

2.1.2.1 Description of the process Figure 24.4 shows a high-pressure shift freezing process. The sample is cooled under pressure below 0°C, but kept in the liquid state according to the phase diagram of water superimposed in Figure 24.4. Once the desired temperature is reached in the product, pressure is released either slowly over several minutes (Fuchigami et al., 1997b; Levy et al., 1999) or quickly in a matter of seconds (Kanda et al., 1992; Otero et al., 1998; Levy et al., 1999), inducing uniform supercooling throughout the sample

634 High-Pressure Freezing

30

Temperature (°C)

20 10

A

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Liquid water

E 10 20

2 D

Ice I

30

C

1

40 50 0

50

100 150 Pressure (MPa)

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Figure 24.4 High-pressure shift freezing processes. ABCDE: rapid expansion; ABC12E: slow expansion (taken from Otero and Sanz, 2003).

due to the isostatic nature of pressure. The faster the pressure release, the lower is the nucleation pressure and the greater the degree of supercooling caused (Thiebaud et al., 2002). This supercooling, under pressure or in atmospheric conditions, produces quasi-instantaneous and uniform nucleation throughout the sample (whatever its form and size), which raises the sample temperature to the corresponding freezing point due to the release of latent heat. A percentage of water is instantaneously frozen. The higher the pressure and the lower the temperature before expansion, the more ice is formed and hence the shorter the plateau time is for a given cooling temperature (Otero and Sanz, 2000). Freezing is then completed at atmospheric pressure. It is generally accepted that no further nucleation occurs at this stage. Total freezing times in high-pressure shift freezing experiments are longer than in conventional freezing at atmospheric pressure, since the cooling step before nucleation must be taken into account, even though this is not actual freezing (see Figure 24.4). On the other hand, the phase transition time is much shorter due to the percentage of water instantaneously frozen after expansion. Various researchers have reported major reductions of the freezing plateau with respect to conventional freezing in HPSF experiments: 28 per cent in -lactoglubulin gels from 207 MPa/20°C (Barry et al., 1998); 20 per cent in oil-in-water emulsions from 207 MPa/18°C (Levy et al., 1999) and 14.3 per cent and 15.8 per cent in meat samples from 100 MPa/11°C and 200 MPa/12°C, respectively (Massaux et al., 1998). These reductions must be attributed not only to the instantaneous freezing of water after expansion, but also to the temperature drop in the pressure medium after pressure release. Pressure media that undergo large temperature changes with pressure may therefore be useful in HPSF processes, since the corresponding temperature drop after expansion aids the removal of latent heat from the sample (Kalichevsky et al., 2000). 2.1.2.2 Quality of high-pressure shift frozen foods Observations of ice crystal imprints, either by cryoscanning electron microscopy or by light microscopy after thawing, show small ice crystals of granular shape, without

High-pressure freezing 635

specific orientation, dispersed throughout the sample, demonstrating that ice nucleation occurs throughout the product and not only on the surface in the expansion (Martino et al., 1998; Lévy et al., 1999). As a result, there is none of the damage to tissues and cell structures typically caused by large crystals in conventional freezing in atmospheric conditions, there is less drip loss and the resulting textures are generally better. After expansion, however, freezing is completed at atmospheric pressure and thermal gradients are established during the crystal growth step. In this phase, the removal of latent heat is slower and there is a risk of recrystallization in the central parts of the sample. Using image analysis, Thiebaud et al. (2002) demonstrated that each ice crystal cluster present in a pressure shift frozen oil-in-water emulsion derived from the aggregation of smaller ice crystals, probably during ice crystal growth at atmospheric pressure. Micrographs from different products show that ice crystal diameters grow from the surface to the centre of the sample (Fuchigami and Teramoto, 1997; Martino et al., 1998; Lévy et al., 1999). Therefore, latent heat must be quickly removed if the initial advantages of the nucleation step are to be maintained. As noted, the depressurization rate influences the extent of supercooling and consequently nucleation. Microstructural studies in oil-in-water emulsions frozen by slow pressure release showed ice crystals with intermediate characteristics between those frozen by fast pressure release and under constant pressure (Lévy et al., 1999). Fast expansions would therefore seem to be better than slow ones from the standpoint of quality. High-pressure shift freezing has been applied both to model systems and to real foods. Kalichevsky et al. (2000) compared the effects of pressure-shift and conventional freezing on different food models. Microscopic examination showed that pressure-shift freezing (200 MPa/⫺15°C) produced smaller, more uniform ice crystal damage than conventional freezing at ⫺30°C. Their results also suggest that the freeze-thaw behaviour of food gels can be categorized into two general types: (1) gels which have a reduced gel strength as a result of mechanical damage to the gel microstructure caused by ice crystal formation (for example, agar and gelatin) and (2) gels which have an enhanced gel strength as a result of molecular structural changes that take place in the frozen stage (for example, gels of -lactoglobulin protein isolate and of ovalbumin). Results from Fuchigami and Teramoto (1998) in agar gel also confirm the above observations. Agar gels pressure-shift frozen at 200–400 MPa/20°C/45 min improved their texture and structure compared to gels frozen at constant pressure (0.1, 100, 600 or 700 MPa), but reduced their strength compared to the control. When agar gels were pressurized at 200–400 MPa/20°C/45 min and then heated under pressure for 70 min, the texture of the gels was the same as the control, since no freezing took place. Also, the gel network was better preserved in pressure-shift frozen gels of -lactoglobulin than in gels frozen in atmospheric conditions, but rigidity was higher (Barry et al., 1998). Examination of high-pressure shift frozen plant tissues like potatoes (Koch et al., 1996; Luscher et al., 2005), carrots (Fuchigami et al., 1997b), whole eggplants (Otero et al., 1998) or whole peaches and mangoes (Otero et al., 2000a) indicated that the microstructure of the tissues was better after pressure-shift freezing than after conventional freezing in atmospheric conditions. The shorter phase transition time in HPSF processes, combined with the formation of smaller ice crystals, caused less damage to

636 High-Pressure Freezing

the membranes (related to the turgor pressure) and the framework of the cell walls than during conventional freezing, although ice I was formed (Luscher et al., 2005). Textural damage was also minimized in most high-pressure shift frozen plant tissues (Koch et al., 1996; Fuchigami et al., 1997a; Otero et al., 1998). However, it is worth noting that the texture of leafy vegetables largely depends on the turgor pressure and the state of their voluminous gas-filled vacuoles. Fuchigami et al. (1998a) studied the effect of pressure freezing in Chinese cabbage. Histological examination showed that high-pressure shift freezing from 200–400 MPa/⫺20°C produced less damage than pressure-assisted freezing. However, there was a pronounced increase of softness in all pressure frozen samples, which became flexible and the crispness of the raw product was lost. Drip losses have also been studied in potato (Koch et al., 1996; Luscher et al., 2005) and eggplants (Otero et al., 1998). Drip losses were significantly lower in pressureshift frozen products than in samples frozen at atmospheric pressure. Subsequent frozen storage at ⫺18°C increased drip loss in HPSF potatoes due to Ostwaldripening (Koch et al., 1996). As for colour, Luscher et al. (2005) detected considerably less browning in high-pressure shift frozen (250 MPa and ⫺27°C) potato samples than in samples frozen to ice I and ice III by high-pressure assisted freezing or in conventionally frozen samples. Conventional freezing in animal tissues mainly produces deterioration in texture, colour and flavour, which can be attributed to protein denaturation. One of the factors in this protein denaturation is the size of the ice crystals formed. Large ice crystals produce cellular disruption that may induce interaction among enzymes, lipids and proteins and lead to protein denaturation and lipid degradation. Moreover, drip losses are important, inducing losses of moisture that affect the quality of the frozen product. Pressure-shift freezing in animal tissues like pork muscle (Otero et al., 1997a; Martino et al., 1998), Norway lobster (Chevalier et al., 2000a) and turbot (Chevalier et al., 2001b) produced small, rounded ice crystals evenly distributed throughout the sample. After 75 days of frozen storage at a constant temperature (⫺20  1°C), no significant gradual accretion of ice crystals was observed in turbot fillets (Chevalier et al., 2001b). HPSF also reduced drip losses in turbot fillets more than air-blast freezing did (Chevalier et al., 2001b). On the other hand, drip losses after centrifugation were higher in pressure-shift frozen than in raw and conventionally-frozen beef and pork muscle samples (Fernández-Martín et al., 2000). Also, pressure-shift frozen samples were tougher. Toughness results were similar in cooked Norway lobsters pressure-shift frozen at 200 MPa/18°C (Chevalier et al., 2000a) and turbot fillets pressure-shift frozen at 140 MPa/14°C (Chevalier et al., 2000b) immediately after freezing (no storage period). When protein extractability was considered as an indicator of protein denaturation, no significant differences in water-soluble protein extractability (related to sarcoplasmic proteins) were observed among samples immediately after the treatment. Conversely, salt-soluble protein extractability (related to myofibrillar proteins) decreased in all frozen samples and, most of all, in high-pressure shift frozen samples. SDS-PAGE also showed that pressure-shift freezing induced modifications in sarcoplasmic and myofibrillar protein profiles (Chevalier et al., 2000b). DSC thermograms in beef and pork muscles (Fernández-Martín et al., 2000) and in turbot fillets

High-pressure freezing 637

(Chevalier et al., 2001b) suggested that myosin and actin were significantly affected by pressure-shift freezing. Connective proteins remained practically unaltered. Pressure induces denaturation of the main myofibrillar proteins (actin and myosin) and the loss of extractability can be attributed to cross-linking of proteins mainly by electrostatic and disulphide bonds (Ikeuchi et al., 1992). The extractability of salt-soluble proteins in turbot fillets decreased with storage in air-blast frozen samples; no significant changes were observed in pressure shift frozen samples. Also, the toughness of the conventionally frozen samples showed a significant tendency to increase during frozen storage while remaining practically constant in high-pressure shift frozen samples. Thus, toughness of air-blast frozen turbot fillets after 75 days of storage was significantly higher than that of pressure-shift frozen samples (Chevalier et al., 2001b). Transmission electron microscopy images from high-pressure shift frozen beef and pork muscles revealed major ultrastructural damage. Also, there was substantial destruction of sarcomeres (broken A-band with extinction of M-line and H-zone and loss of defined filamentous structure (myosin dissociation); broken I-bands and thickening of Z-line by collapse of the I-band and deposit of dense material) and marked fragmentation of myofibrils. As regards colour, pressure-shift freezing produced whitening of thawed beef and pork muscles (Fernández-Martín et al., 2000) and of turbot fillets (Chevalier et al., 2000b) lending them the appearance of cooked muscle (overall increase of L* and b* values and a decrease in a*). Air-blast freezing produced colour alterations to a lesser extent. Goutefongea et al. (1995) attributed the shift towards lighter tones to pressureinduced coagulation (with resulting loss of solubility) of sarcoplasmic and/or myofibrillar proteins. Frozen storage did not particularly modify colour parameters of pressure-frozen turbot fillets, which were stable during storage. It is important to note that the appearance of fish fillets frozen either by air-blast and by pressure release was similar in the frozen and cooked state (Chevalier et al., 2001b). When freezing animal tissues, it is important to bear in mind that pressure considerably influences protein denaturation, even at 200 MPa and that modifications of proteins may cancel out the benefit of the formation of smaller ice crystals during pressure-shift freezing. 2.1.3 High-pressure induced freezing

This process was first described by Urrutia Benet et al. (2004) very recently and there are no further references to experimentation in the literature. Figure 24.5 shows a schematic of a high-pressure induced freezing process. The sample is cooled under pressure and the phase change is induced by a subsequent pressure increase. As there are no experimental data yet, the effects of this process on food quality cannot be commented on.

2.2 Use of additives There are only a few studies on the effect of additives or solutes on the kinetics of pressure freezing and the resulting ice crystals. Several solutes or macromolecules are

638 High-Pressure Freezing

10 5

Temperature (°C)

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100

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Pressure (MPa) High-pressure induced freezing process.

known to affect ice nucleation and crystal growth at atmospheric pressure, especially sugars and hydrocolloids. Sugars inhibit ice crystal growth; they also modify freezing and glass transition temperatures and the amount of frozen water at a given temperature. Polysaccharides such as gelatin, carrageenan, locust bean gum or alginate are commonly used to control ice recrystallization during frozen storage in ice creams and other desserts. These stabilizers have little or no effect either on heterogeneous nucleation or on freezing temperatures and they do not change the amount of freezable water or the glass transition temperature. Thiebaud et al. (2002) studied the effects of fructose (10, 18 or 25 per cent w/w), sodium alginate (0.25 or 0.5 per cent w/w) or both (18 per cent fructose ⫹0.25 per cent sodium alginate) in high-pressure shift frozen (200 MPa/⫺18°C) oil-in-water emulsions. The presence of fructose decreased the extent of supercooling (due to freezing point depression) and the duration of the freezing plateau (lower water content). Ice crystal clusters were more numerous, smaller and more spherical, resulting from a smaller number of nuclei per crystal cluster when the fructose concentration increased. The presence of sodium alginate, with or without fructose, did not significantly affect the freezing kinetics and did not reduce the size of ice crystal clusters when compared to the control emulsion. Fuchigami et al. (2002) also studied the effect of 0, 2.5 or 5 per cent trehalose in pressure frozen tofu (high-pressure shift freezing at 200, 340, 400, 500 and 600 MPa/ ⫺20°C and pressure-assisted freezing at 100 and 686 MPa/⫺20°C). The addition of trehalose was effective in decreasing the size of ice crystals, in preventing tofu from becoming too firm and in improving the mouth-feel (texture) of pressure-frozen tofu. The addition of sucrose (0, 5, 10 and 20 per cent) was also effective in improving the quality of pressure- (assisted- and shift-) frozen agar gels (Fuchigami and Teramoto, 1998) and gellan gum gel (Fuchigami and Teramoto, 2003). However, the most important

Modelling high-pressure freezing processes 639

factor influencing the size of the ice crystals was the type of process. High-pressure shift freezing processes consistently produced the best results.

2.3 Microbial and enzymatic inactivation Conventional freezing and subsequent frozen storage of foods induce no great inactivation of most detrimental microorganisms. High pressure can be an effective mode of microbial inactivation if appropriate process conditions are chosen. Some references in the literature indicate that pressurization at subzero temperatures may be more effective for microbial inactivation (Takahashi, 1992; Hashizume et al., 1995). Nevertheless, studies on the effects of pressure freezing on microbial inactivation of food are scarce. Recently, Picart et al. (2004) reported on inactivation of Pseudomonas fluorescens, Micrococcus luteus and Listeria innocua inoculated in smoked salmon after different pressure treatments. Good results were obtained for pressure shift freezing with slow expansion (cooling at 207 MPa for 23 min and pressure release in 18 min from 207 MPa/⫺21°C followed by further freezing to ⫺25°C at 0.1 MPa). The authors (Picart et al., 2004) reported reductions of 4.6, 2.5 and 2 log cycles for Psedomonas fluorescens, Micrococcus luteus and Listeria innocua, respectively. Results at higher pressures and temperatures (300 MPa and ⫹5/⫺5°C) were similar. It therefore appears that high-pressure shift freezing (207 MPa/⫺21°C) is able to inactivate various microorganisms in a real and complex food system. However, much more research is required with other microorganisms and foods. There are only a few studies in the literature on enzymatic inactivation after highpressure freezing. Indrawati et al. (1998) studied inactivation in enzyme solutions after high-pressure shift freezing (200–400 MPa, ⫺22/⫺10°C) and assisted freezing (350 MPa/⫺22°C and 400 MPa/⫺22°C). They found that polyphenoloxidase was not inactivated and pectin methylesterase was only inactivated slightly. -amylases from Bacillus subtilis and peroxidase were slightly and reversibly inactivated and lipoxygenase was irreversibly inactivated (60 per cent inactivation at 200 MPa). Recent experiments (not yet published) in high-pressure shift frozen potatoes (120, 150, 180, 210 MPa/⫺8, ⫺12, ⫺16 and ⫺20°C) indicated that peroxidase and polyphenoloxidase were not completely inactivated and that peroxidase was more resistant to treatment. High-pressure shift frozen potatoes should therefore be blanched before HPSF to inactivate the enzymes to maintain consumer acceptability throughout storage. As noted by Cheftel et al. (2000), further studies of enzyme inactivation are required to elucidate the possible synergistic or antagonistic affects of pressure, cold temperature and ice crystal formation on protein denaturation.

3 Modelling high-pressure freezing processes Modelling food freezing processes at atmospheric pressure is a difficult task because biological structures are heterogeneous and water is not totally available for freezing. Initial freezing temperatures are variable and may be lower than 0°C depending on

640 High-Pressure Freezing

sample composition; also, water solidifies within a range of freezing temperature rather than at a freezing point, due to freeze concentration of solutes (Franke, 2000). Moreover, in selecting thermal properties, identification of values for foods is difficult because these change as the freezing process progresses, as does the convective heat transfer coefficient, which has a significant influence on the freezing time (Hung, 1990). Modelling the process is complicated considerably when the phase change takes place under pressure or by fast pressure release (Otero and Sanz, 2003). High-pressure freezing processes are driven by both pressure and temperature. The pressure dependent physical properties of the substances involved and the convective motion of the pressure medium significantly influence the heat transfer (Hartmann et al., 2003). Moreover, there are temperature variations produced by pressure changes, especially in high-pressure shift freezing processes, where there is considerable supercooling after expansion. There is also a need for in-depth research on such phenomena as the sudden ice nucleation that characterizes HPSF processes.

3.1 Thermophysical properties under pressure One of the main difficulties when modelling heat transfer in high-pressure freezing processes is the lack of appropriate thermophysical properties of the processed materials. The determination of these properties under pressure through conventional techniques is hampered by practical problems and, as a result, the literature currently available lacks enough data for food and its components, even for the main component, i.e. water. Water is of especial interest because all raw foods contain mainly water. In 1995, the International Association for the Properties of Water and Steam (IAPWS) adopted a new formulation for water thermodynamic properties ‘for general and scientific use’ (IAPWS, 1996). The National Institute of Standards (NIST) has incorporated this formulation in the computer program NIST Standard Reference Database 10: NIST/ASME Steam Properties, a pay software implementation. A limited set of properties based on this formulation is also freely available from NIST on the Web at the NIST WebBook (Lemmon et al., 2001). Nevertheless, this formulation is only valid in the stable fluid region of water from the melting-pressure curve to 1000°C at pressures up to 1000 MPa and so does not include data for supercooled water. Otero et al. (2002b) have published a review on some interrelated thermophysical properties of liquid water and ice I (thermal expansion coefficient , isothermal compressibility coefficient , specific volume V and specific isobaric heat capacity cp) in a pressure/temperature range of interest for high-pressure food processing, including supercooled water. The corresponding calculation routines are publicly available through the Web (Otero et al., 2002b). However, real processes involve real products, which are never pure water. The behaviour of the properties of real foods with pressure and temperature variations evidently cannot be described by the equations governing pure water. Food comprises other phases, where different types of bonding determine other thermophysical properties. Also, substances dissolved or suspended in the actual water phase alter its properties. The validity of these properties is even affected by the physical structuring and micro-compartmentalization characterizing many food products and derived from

Modelling high-pressure freezing processes 641

cellular structure, polymers networks, or emulsion phenomena. Nevertheless, only some experimental determinations have been described in a few products under pressure: density in apple sauce and tomato paste (Denys et al., 2000a); thermal expansion coefficient in apple sauce, tomato paste and agar gel (Denys et al., 2000a, b); thermal conductivity in tomato paste and apple pulp (Denys and Hendrickx, 1999); and latent heat in tylose (Denys et al., 2000c) and MgSO4 aqueous solutions (Chourot et al., 2000). Most authors built their freezing models using simplifying approximations to estimate thermal properties of food under pressure. Thus, Denys et al. (1997) shifted thermal data at atmospheric pressure according to the prevalent pressure and Schlüter et al. (1998, 2004) obtained data under pressure regressively from the freezing curves. A lot of further research is therefore required. Experimental determinations of thermophysical properties under pressure, including phase transition data and latent heat, must be made in both food and pressurizing fluids since these properties are essential to improve models.

3.2 Temperature variation after an adiabatic pressure change Another factor that must be taken into account when modelling high-pressure freezing processes is the temperature variation produced by a pressure change. Pressurization/depressurization always induces a temperature increase/decrease due to the work of compression/expansion in both the food and the pressurizing fluid. When modelling, this temperature variation produced by an adiabatic pressure change can be calculated by means of the following well-known thermodynamic equation: T  V 

dT  k dP cp

(2)

dT/dP (K/Pa) depends on the sample composition and its physical state through V (m3/kg), (1/K) and cp (J/kg  K), but these three parameters depend on temperature and pressure, so the calculation of dT/dP is complex. It is particularly important to bear in mind the different thermophysical properties of the sample and the pressurizing fluid that will induce different temperature increments after a pressure change. For example, the temperature of an ethanol/water mixture decreased from 14°C to 24.4°C after an expansion from 140 MPa to atmospheric pressure, while the temperature of the sample increased to its normal freezing point due to ice nucleation (Chevalier et al., 2000b). This large temperature drop in the pressure medium must be taken into account when modelling since it will influence the subsequent temperature evolution in the sample.

3.3 Convective phenomena Hartmann (2002) and Hartmann and Delgado (2002, 2003) demonstrated the importance of the convective motion in the pressure medium in pressure processes. They studied the spatial and temporal evolution of the temperature and fluid velocity fields

642 High-Pressure Freezing

and its importance in the inactivation of enzymes and microorganisms. They proved that the uniformity of a high-pressure effect can be disturbed by convective and conductive heat and mass transport conditions that are affected by parameters such as the compression rate, the size of the pressure chamber or the fluid viscosity. During the freezing process, important thermal gradients are established between the sample, the pressure medium and the vessel. Moreover, after compression and expansion, temperature is modified in the sample and the pressure medium in a different way. Thus, convective phenomena are induced and must be taken into account when modelling. They are especially important when the sample volume/vessel volume ratio is low. However, convective phenomena are not only induced in the pressure medium. Kowalczyk et al. (2004) have built a model for water freezing processes under pressure taking into account both conductive and convective heat transfer inside the liquid sample. The system of equations consists of the conservation laws of mass, momentum and energy. It also contains the equation of phase change and the equation of state for water and ice. The model does not take supercooling and nucleation phenomena into account, but it does give fairly good results.

3.4 Modelling high-pressure assisted freezing processes High-pressure assisted freezing takes place under pressure, but in essence the freezing process is the same as in atmospheric conditions. It is governed by thermal gradients between the cooling medium and the sample as occurs in classical freezing. So, any model developed for a classical freezing process at atmospheric pressure should be valid to reproduce the process under pressure if appropriate thermophysical properties are considered. The model might take into account the temperature/pressure dependent thermophysical properties of the product and the pressure medium. The latent heat and freezing point of the sample also vary with pressure and must be considered. In addition, it is essential to include in the model the temperature change that the system (sample and pressurizing fluid) undergoes after an adiabatic compression or expansion. Schlüter et al. (1998) modelled thermal exchanges in potato cylinders subjected to high-pressure assisted freezing. They employed a finite difference numerical procedure (assuming radial symmetry and one dimensional heat conduction). A heat balance was implemented at each volume element to adjust the temperature evolution with time. The moving freezing front was modelled by introducing temperature dependent apparent specific heat and thermal conductivity values in the vicinity of the freezing point into the heat balances of the finite difference scheme. Due to the lack of values for thermal properties of potato under pressure, the authors (Schlüter et al., 1998) employed modified values of these properties at atmospheric pressure (regressively derived from the freezing curves) to fit the experimental freezing curves.

3.5 Modelling high-pressure shift freezing processes Some authors have made attempts to model high-pressure shift freezing processes. The main difficulty that they have found is how to model the phenomenon of uniform

Modelling high-pressure freezing processes 643

Table 24.1 Various approaches used to calculate the amount of ice instantaneously produced after adiabatic expansion in high-pressure shift freezing processes by different authorsa Supercooling phenomena

Equation

Not taken into account

  dV  2  Tk  Vi     dP   mi   P  L s T     V (1 mi )   w  P 

Reference

  V  cp  T    i   (Vw  Vi )  i 2 k  (Vw  Vi )2   T  L  p 

 2  Tk  L T

 V   w  T

(3)

(Chizhov and Nagornov, 1991)(Oteroetal.,1997b)

   (V  V )  cpw  Tk  (V  V )2   w i w i  2 L  p 

Taken into account

mw  cpw 
Tsup  L  mi

(4)

(Le Bail et al., 1997)

Taken into account

mi  L  mi  cpi 
T  (1 mi )  cpw 
T   

(5)

(Barry et al., 1998)

Taken into account

cpw 
T  (1 mi ) cpi 
T  mi  mi  mi  L

(6)

(Otero and Sanz, 2000)

a

mi denotes mass of ice and mw denotes mass of liquid water.

nucleation induced by pressure release (Levy et al., 1999) and calculate the amount of ice instantaneously formed just after expansion. The first models considered that the sample extended over its melting curve in the adiabatic expansion (no supercooling is considered), although in practice it has been proved that metastable conditions are always reached after the pressure release (Sanz et al., 1997). Otero et al. (1997b) built a model based on an analysis of the variation of the thermodynamic properties of ice/water along its melting curve (Table 24.1; Chizhov and Nagornov, 1991). These authors (Otero et al., 1997b) obtained 36 per cent instantaneous ice after an expansion from 210 MPa down to atmospheric pressure. Recently, Chevalier et al. (2001a), following this same outline, calculated the ice fraction formed in water samples after the pressure release, with varying initial conditions. These authors (Chevalier et al., 2001a) also evaluated experimentally the ice fraction formed by isothermal calorimetry. Their experimental results were consistently higher (between 13 and 23 per cent) than the numerically calculated values, probably due to the small size of the samples employed. Therefore, the experimental ice ratio could be increased by handling the samples prior to calorimetry determination. Table 24.1 shows various approaches used by different authors to calculate the amount of ice (mi) produced after expansion, taking into account the supercooling (⌬T) reached (Le Bail et al., 1997; Barry et al, 1999; Otero and Sanz, 2000). All of them are more or less sophisticated heat balances which assume that the latent heat (L) released by nucleation is equal to the sensible heat absorbed by the sample in transition from metastable conditions to its freezing point at the nucleation pressure. Table 24.2 shows the amount of ice produced after expansions from different coordinates over the melting curve of water. The theoretical supercooling reached after expansion was calculated according to Equation (2) and incorporated Equation (6) in

644 High-Pressure Freezing

Table 24.2 Amount of ice instantaneously formed after adiabatic expansions from different pressure/temperature coordinates Initial temperature (°C)

⫺22.0 ⫺20.7 ⫺19.4 ⫺18.1 ⫺16.9 ⫺15.6 ⫺14.4 ⫺13.2 ⫺12.1 ⫺11.0 ⫺9.9 ⫺8.8

From melting curve

From extreme pressure conditions (according to the phase diagram of water)

Pressure (MPa)

Ice formed (%)

Pressure (MPa)

Ice formed (%)

210 200 190 180 170 160 150 140 130 120 110 100

24.8 23.5 22.1 20.2 19.4 18.1 16.8 15.5 14.2 13.0 11.7 10.5

210 232.3 262.7 303.2 351.9 368.3 384.9 401.6 418.4 435.2 452.1 469.0

24.8 23.7 23.3 23.1 23.08 22.66 21.87 21.13 20.79 20.12 19.47 19.20

Table 24.1, on the assumption that nucleation occurred at atmospheric pressure. Results differ slightly from those presented by Otero and Sanz (2000), since an improvement made by Otero et al. (2000b) has been introduced in the calculus of the theoretical supercooling. However, for a given temperature, maximum supercooling is reached after expansion from the maximum pressure possible in liquid state according the phase diagram of water. This maximum pressure was calculated for different temperatures in Table 24.2 (Wagner et al., 1994) and the amount of ice from these new pressure/temperature coordinates was calculated again. It is interesting to note that, at least in theory, large amounts of frozen water could be obtained after expansions from relatively high temperatures if pressure is increased. Recent experiments (Otero and Sanz, 2006) confirm these theoretical results. Moreover, Schlüter et al. (2004) have suggested the possibility of carrying out expansions from metastable zones to increase the amount of frozen water after the pressure release, taking advantage of the considerable supercooling needed to initiate nucleation under pressure. Sanz and Otero (2000) applied a mathematical model in three steps (precooling, phase change and tempering) to give a comprehensive idea of high-pressure shift freezing processes and compare them to traditional freezing in atmospheric conditions. They employed a large cylindrical sample of agar gel containing 99 per cent water. Precooling and tempering stages were solved using the transient state heat transfer equations for finite cylinders presented by Chung and Merritt (1991) and thermophysical properties of water under pressure (Otero et al., 2002b) for the precooling stage. To determine the time needed to complete the phase change stage, a modified Plank’s equation (De Michelis and Calvelo, 1983) was used taking into account the amount of ice instantaneously produced after the adiabatic expansion (see Table 24.1). Once the amount of ice instantaneously produced after the expansion is known, it is easy to calculate the time for the freezing plateau corresponding to the remaining liquid water

Modelling high-pressure freezing processes 645

using the modified Plank’s equation presented by De Michelis and Calvelo (1983). The complete model by Sanz and Otero (2000) satisfactorily reproduced experimental freezing processes, both at atmospheric pressure and by high-pressure shift freezing, in large cylindrical samples (high sample volume/vessel volume ratio). Its main purpose was to serve as a comprehensive tool for comparing both processes. Nevertheless, complex situations (for example, complex geometries, or slow expansions where supercooling and nucleation occur under pressure before reaching atmospheric pressure, or freezing small samples where convective phenomena in the pressure medium gain importance, etc.) and more accurate predictions would require the use of numerical methods. Denys et al. (1997) employed a numerical solution for two-dimensional heat transfer to predict heat conduction during high-pressure shift freezing of a tylose cylinder using an explicit finite difference scheme. The method is based on an energy balance that assumes that the sensible heat required for the temperature increase in the expansion is subtracted from the total enthalpy of the sample. This model also took into account the temperature decrease of the high-pressure medium during adiabatic expansion. To deal with the problem of phase change, these authors (Denys et al., 1997) considered temperature-dependent apparent volumetric specific heat and thermal conductivity. The approach followed, to deal with the influence of temperature and pressure in the thermal values, consisted in ‘shifting’ these data according to the prevalent pressure. Clearly, this approach assumes a non-real ‘volumetric latent heat’ independent of pressure. Nevertheless, the authors (Denys et al., 1997) obtained good agreement between experimental and predicted temperature profiles due to the fact that in the experiments considered the phase transition occurred at 0.1 MPa. Recently, Schlüter et al. (2004) used a numerical, one-dimensional explicit finite difference scheme to model high-pressure assisted (ice I and ice III) and shift-freezing processes in potato cylinders. The model reproduces nucleation temperatures, freezing times and phase transition times for different processes and pressures rather well, but it gives no in-depth explanations of nucleation temperature prediction in highpressure assisted freezing and nucleation phenomena after expansion in high-pressure shift freezing.

3.6 Future perspectives: thermal control of high-pressure freezing processes High-pressure freezing models developed up to now focus on the freezing process per se and usually only consider the sample and the pressure medium. However, industrial application of these new methods requires control and optimization of the complete system. Otero et al. (2002a) developed a macroscopic model that takes into account all the thermal exchanges in a complete high-pressure system (sample, pressurizing fluid, steel pressure vessel and thermoregulating equipment) really to ascertain the impact of the different components and hence be able to control the temperature of a given process. Future models should consider the whole high-pressure system and take into account all the thermal exchanges involved, including those between

646 High-Pressure Freezing

sample-pressurizing fluid and steel mass of the vessel. Models of this kind would be accurate tools for achieving real thermal control and would make it possible to choose the best conditions for a given pressure treatment.

4 Conclusions The advantages of high-pressure freezing processes for product quality as compared to conventional methods stem from two main parameters: reduced duration of the phase transition and less mechanical stress during formation of ice crystals. When freezing at constant pressure (atmospheric conditions and high-pressure assisted freezing), nucleation occurs only near the sample surface, in direct contact with the cooling medium. When the latent heat of crystallization is removed, ice crystals grow from the surface to the centre of the product. The final ice crystals are needle-shaped and radially oriented. High-pressure assisted freezing to ice I offers some advantages because the latent heat of the water decreases with pressure. However, the freezing point also decreases and so the temperature of the cooling medium must be lowered to keep the ⌬T between it and the sample constant. Highpressure assisted freezing to dense forms of ice produces a quality improvement; however, to preserve that improvement thawing must take place under pressure to avoid solid-solid phase transition to ice I during expansion. Transferring this technology to an industrial scale is therefore a challenge, since continuous storage under high pressure at low temperature seems to be a cost-intensive technology. High-pressure shift freezing entails homogeneous supercooling throughout the sample after the expansion that induces uniform nuclei formation in the product. When the latent heat is removed, nuclei grow to form small ice crystals, which are granular shaped and without specific orientation. Phase transition times are always shorter than in atmospheric conditions and important quality improvements have been reported for many products. Successful application of high-pressure shift freezing is strongly dependent on the food matrix involved and the process parameters applied. It seems that vegetable tissues are more susceptible to benefits than muscle tissues, especially at pressure levels above 200 MPa where protein denaturation is important. At the same time, an enhancement of product safety can be expected as a result of assumed synergetic inactivation effects on enzymes and microorganisms during highpressure processes at subzero temperatures. However, a lot of research work is still needed. More high-pressure assisted freezing studies are required in different products, with precise pressure/temperature monitoring. The real effects of each ice polymorph, while avoiding the solid-solid phase transition to ice I, are yet to be determined. Special attention must be paid to ice VI, which can be maintained under pressure at ambient temperature. Certain parameters in the amount of ice instantaneously formed after the expansion in HPSF processes need to be extensively studied, as do the effects of additives on the shape, size and stability of ice crystals during storage. Particularly in the subzero temperature range at high-hydrostatic pressure, more data on inactivation kinetics of enzymes and

References 647

microorganisms are required to quantify the effects of combination processes relevant for food production. Moreover, special efforts are needed to determine thermophysical properties of different foods and pressure mediums in modelling pressure freezing processes. Complete modelling of high-pressure systems is required to achieve real control and optimization of both freezing processes and equipment.

Nomenclature cp specific isobaric heat capacity (J/kg  K)
H enthalpy change (J/kg) L latent heat (J/kg) m mass fraction P pressure (Pa) T temperature (°C) temperature (K) Tk
Tsup supercooling extent (°C) V specific volume (m3/kg) Greek symbols

thermal expansion coefficient (K1) Subindexes i: Ice w: Liquid water

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Hung Y (1990) Prediction of cooling and freezing times. Food Technology, 44 (5), 137–153. Ikeuchi Y, Tanji H, Kim K, Suzuki A (1992) Mechanism of heat-induced gelation of pressurized actomyosin: pressure-induced changes in actin and myosin in actomyosin. Journal of Agricultural and Food Chemistry, 40, 1756–1761. Indrawati, Van Loey AM, Denys S, Hendrickx ME (1998) Enzyme sensibility towards high pressure at low temperature. Food Biotechnology, 12, 263–277. International Association for the Properties of Water and Steam (IAPWS) (1996) Release on the IAPWS Formulation 1995 for the thermodynamic properties of ordinary water substance for general and scientific use. Fredericia (Denmark), 8–14 September 1996. Copies of this and other IAPWS releases can be obtained from the Executive Secretary of IAPWS, Dr RB. Dooley, Electric Power Research Institute, 3412 Hillview Avenue, Palo Alto, CA 94304, USA. Johnston DE (2000) The effects of freezing at high pressure on the rheology of Cheddar and Mozzarella cheeses. Milchwissenschaft, 55 (10), 559–562. Kalichevsky MT, Knorr D, Lillford PJ (1995) Potential food applications of high-pressure effects on ice-water transition. Trends in Food Science & Technology, 6, 253–259. Kalichevsky MT, Ablett S, Lillford P, Knorr D (2000) Effects of pressure-shift freezing and conventional freezing on model food gels. International Journal of Food Science and Technology, 35, 163–172. Kanda Y, Aoki M, Kosugi T (1992) Freezing of tofu (soybean curd) by pressure-shift freezing and its structure. Nippon Shokuhin Kogyo Gakkaishi, 39 (7), 608–614. Kim NK, Hung YC (1994) Freeze-cracking in foods as affected by physical properties. Journal of Food Science, 59 (3), 669–674. Knorr D, Schlueter O, Heinz V (1998) Impact of high hydrostatic pressure on phase transition of foods. Food Technology, 52 (9), 42–45. Koch H, Seyderhelm I, Wille P, Kalichevsky MT, Knorr D (1996) Pressure-shift freezing and its influence on texture, colour, microstructure and rehydration behaviour of potato cubes. Nahrung, 40 (2), 125–131. Kowalczyk W, Hartmann Ch, Delgado A (2004) Modelling and numerical simulation of convection driven high pressure induced phase changes. International Journal of Heat and Mass Transfer, 47 (5), 1079–1089. Le Bail A, Chourot JM, Barillot P, Lebas JM (1997) Congélation-décongélation a haute pression. Revue Generale du Froid, 972, 51–56. Lemmon EW, McLinden MO, Friend DG (2001) Thermophysical properties of fluid systems. In NIST Chemistry WebBook, NIST Standard Reference Database Number 69 (Linstrom PJ, Mallard WG, eds). Gaithersburg: National Institute of Standards and Technology (http://webbook.nist.gov) Levy J, Dumay E, Kolodziejczyk E, Cheftel JC (1999) Freezing kinetics of a model oil-in-water emulsion under high pressure or by pressure release. Impact on ice crystals and oil droplets. Lebensmittel- Wissenschaft und- Technologie, 32, 396–405. Li B, Sun DW (2001) Novel methods for rapid freezing and thawing of foods – a review. Journal of Food Engineering, 54, 175–182. Lüdermann HD (1994) Water solutions at high pressures and low temperatures. Polish Journal of Chemistry, 68, 1–22.

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Luscher C, Schlüter O, Knorr D (2005). High pressure-low temperature processing of foods: Impact on cell membranes, texture, color and visual appearance of potato tissue. Innovative Food Science and Emerging Technologies (in press). Martí J, Aguilera JM (1991). Efecto de la velocidad de congelación en las características mecánicas y microestructurales del arándano y de la mora silvestre. Revista Agroquímica y Tecnología de Alimentos, 31 (4), 493–504. Martino MN, Otero L, Sanz PD, Zaritzky NE (1998) Size and location of ice crystals in pork frozen by high pressure assisted freezing as compared to classical methods. Meat Science, 50 (3), 303–313. Massaux C, Berá F, Steyer B, Sindic M, Deroanne C (1998) High hydrostatic pressure freezing and thawing of pork meat: quality preservation, processing times and highpressure treatments advantages. In Abstracts of High-Pressure Bioscience & Biotechnology; IVth joint meeting of Japanese and European seminars on highpressure bioscience and biotechnology, Heidelberg, Germany, p. 86. Molina-García AD, Otero L, Martino MN et al. (2004) Ice VI freezing of meat: supercooling and ultrastructural studies. Meat Science, 66, 709–718. Otero L, Sanz PD (2000) High-pressure shift freezing. Part 1. Amount of ice instantaneously formed in the process. Biotechnology Progress, 16, 1030–1036. Otero L, Sanz PD (2003) Modelling heat transfer in high pressure food processing: a review. Innonvative Food Science and Emerging Technologies, 4, 121–134. Otero L, Sanz PD (2006) High-pressure-shift freezing: main factors implied in the phase transition time. Journal of Food Engineering (in press). Otero L, Martino MN, Sanz PD, Zaritzky NE (1997a) Histological analysis of ice crystals in pork frozen by liquid N2 and high-pressure-assisted freezing. Scanning, 19 (3), 241–242. Otero L, Sanz PD, de Elvira C, Carrasco JA (1997b) Modelling thermodynamic properties of water in the high-pressure assisted freezing process. In High-Pressure Research in the Biosciences and Biotechnology (Heremans K, ed.). Leuven: Leuven University Press, pp. 347–350. Otero L, Solas MT, Sanz PD, de Elvira C, Carrasco JA (1998) Contrasting effects of highpressure-assisted freezing with conventional air freezing on eggplants tissue microstructure. Lebensmittel-Untersuchung und Forschung (A), 206 (5), 338–342. Otero L, Martino MN, Zaritzky NE, Solas MT, Sanz, PD (2000a) Preservation of microstructure throughout the volume of peach and mango frozen by high-pressure-shift freezing. Journal of Food Science, 65 (3), 466–470. Otero L, Molina-García AD, Sanz PD (2000b) Thermal effect in foods during quasiadiabatic pressure treatments. Innovative Food Science & Emerging Technologies, 1, 119–126. Otero L, Molina A, Ramos A, Sanz PD (2002a) A model for a real thermal control in highpressure treatment of foods. Biotechnology Progress, 18, 904–908. Otero L, Molina-García AD, Sanz PD (2002b) Some interrelated thermophysical properties of liquid water and ice I: a user-friendly modelling review for food high-pressure processing. Critical Reviews in Food Science and Nutrition, 42 (4), 339–352. Picart L, Dumay E, Guiraaud JP, Cheftel JC (2004) Microbial inactivation by pressure-shift freezing: effects on smoked salmon mince inoculated with Pseudomonas fluorescens,

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Controlling the Freezing Process with Antifreeze Proteins Brent Wathen and Zongchao Jia Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada

Antifreeze proteins (AFPs), a diverse collection of small proteins found in many cold-climate organisms ranging from fish to bacteria, have the unusual ability to influence ice growth by interacting directly with ice surfaces. At low concentrations, these proteins are able to inhibit ice re-crystallization, a process by which larger ice crystals are formed at the expense of smaller ones. At high concentrations, these proteins are able to produce complete ice growth inhibition over a temperature range that is AFP-dependent, a feat that effectively lowers the freezing points of AFP-laden fluids. Although the specific molecular details are still being investigated, it is generally believed that AFPs influence ice growth by binding to the surfaces of ice crystals, thereby causing an increase in the local ice surface curvature and hence the surface-area-to-volume ratio of ice embryos beyond their ability to grow. The lack of a detailed molecular understanding of AFP activity has not stopped research into the practical applications of these proteins. Their use in food processing has long been anticipated and their effectiveness at improving food quality and nutritional value has been investigated. The addition of AFPs to meats and other cellular products has decreased cellular damage resulting from the freezing and thawing process, while the introduction of these proteins into frozen foods such as ice cream has helped to reduce ice crystal formation that would otherwise alter food texture. Because AFPs operate extracellularly, the most practical method of introducing AFPs into food products is by direct mechanical means, including mixing, injecting, soaking or vacuum-infiltration. Efforts have also been made to introduce AFP genes directly into selected organisms, though, to date, this technique has had only limited success.

1 Introduction It hardly seems possible to overstate the importance of water for life as we know it. Indeed, the maxim, ‘Where there is water, there is life’, seems almost an irrefutable truism. This thinking underlies our present attempts to discover traces of water on our nearest neighbouring planet: the discovery of water on Mars is considered a necessary prerequisite for finding life there and perhaps confirming that we are not alone in the universe. In fact, it is debatable whether or not we would even recognize life on planets Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

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completely devoid of water altogether. On our own planet, water covers roughly three quarters of the earth’s surface and it is generally accepted from a scientific perspective that life here on this watery world began in the oceans, from which eventually emerged the whole plethora of creatures now inhabiting our biosphere. But why is it that water is so important for life? Its chemical composition, one oxygen atom connected to two hydrogen atoms, seems almost too simple for such a grand role in biology, while its great abundance on earth gives the impression that it is too common to hold much value. Such impressions, however, discount the proposition that from the simple comes the sublime, a phrase very fitting for water. It is the primary substance of all cellular life – that is, all terrestrial life – as cells are composed

␦
105° ␦ (a)

␦


(b)

c-axis Basal plane

c-axis

(c) Secondary prism plane

Figure 25.1 The water molecule. (a) The non-linear connections between the oxygen and hydrogen atoms in a water molecule make the molecule polar. (b) A representation of water molecules in solution, where they spontaneously arrange themselves to promote the formation of hydrogen bonds. (c) Hexagon ice Ih. The free movement of water molecules in solution is replaced by a rigid, hexagonal structure of water molecules in the most common form of ice.

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of between 70 and 95 per cent water (Campbell, 1987). As the major constituent of all cells, water provides the medium in which almost all biochemical reactions take place. Moreover, water itself is involved in many of these reactions. Although a water molecule as a whole is uncharged, the non-linear connections between its hydrogen and oxygen atoms (Figure 25.1a) render it polar, or locally charged. This polarity gives liquid water a strong cohesiveness, as the water molecules align themselves spontaneously to form a special type of bonding, termed hydrogen bonding, between the partially positive and negative components of neighbouring waters (Figure 25.1b). Its high specific heat capacity allows it to absorb much of the energy given off during metabolic processes that might otherwise boil a cell. Furthermore, its polar nature allows water to solvate other polar molecules, including a host of biologically important molecules such as fuels, building blocks and information carriers, thereby allowing them to coexist in high concentrations within cells where they can diffuse and find each other as needed to sustain life (Stryer, 1995). From this partial list of properties alone it is clear that water plays a fundamental role in sustaining life.

2 Antifreeze proteins 2.1 Ice and freeze survival Ice, the solid form of water, can exist in several crystalline structures. At normal atmospheric pressures between the temperatures of 0 and 60°C, only the hexagonal form of ice, ice Ih, is stable (Figure 25.1c). In this crystal form, the water molecules arrange themselves so as to establish a hydrogen-bonding network wherein each water molecule forms four hydrogen bonds with its nearest neighbouring waters. This water arrangement gives rise to oriented crystal growth, with three symmetric a-axes lying in one plane and a lone c-axis lying perpendicular to this plane. Three common ice planes have their own names. The planes normal to the a- and c-axes are termed the secondary prism and basal planes of ice respectively, while the planes lying at right angles to both of these planes are termed primary prism planes. Because the water molecules of ice Ih are oriented, each plane on the surface of an ice Ih crystal contains a different configuration of water molecules and hydrogen bonding opportunities. These differences in surface characteristics result in different rates of ice growth in different directions, termed anisotropic growth, with growth occurring much faster in both the primary and secondary prism plane directions than in the basal plane direction (Maruyama et al., 1999). Growth rates are also a function of temperature. For example, ice crystals growing at temperatures just below 0°C adopt circular-disc morphology (Maruyama et al., 1999) (Figure 25.2a). At lower temperatures, ice growth is more dendritic. Biologically, ice poses serious challenges for organisms living in cold climates. When liquid water undergoes a phase transition to solid ice, its suitability for sustaining life decreases dramatically. Most obviously, it no longer provides a fluid medium to facilitate cellular activities and, consequently, almost all biochemical reactions come to a halt. Additionally, because solutes are generally forced ahead of growing

656 Controlling the Freezing Process with Antifreeze Proteins

(a)

(b)

c-axis

(c)

c-axis

c-axis

Figure 25.2 Ice crystal morphology. (a) Shown is an ice crystal growing at low degrees of super-cooling, looking down the c-axis (basal plane). (b) An ice crystal grown in the presence of fish type I AFP from winter flounder. (c) An ice crystal grown in the presence of the insect AFP from Tenebrio molitor.

ice surfaces (Cheng and DeVries, 1991), the freezing process causes a lethal increase in the concentration of electrolytes and other solutes in body fluids that do not freeze. Furthermore, freezing temperatures can result in damaging phase changes to other cell components, including cell membranes and proteins (Ramlov, 2000). Indeed, unmitigated freezing leads to death of the organism. Life, however, is nothing if not persistent. Over time, organisms living in cold climates have developed a number of strategies for survival. These strategies can be divided into two basic categories. The strategies in the first category, freeze-tolerance, primarily involve controlling the freezing process so as to ensure that freezing only occurs outside of cells. Organisms accomplish this by actively promoting the freezing of their extracellular body fluids through the use of ice nucleators (substances that induce the formation of ice). Once ice is formed extracellularly, the resulting osmotic pressure will draw water out from cells, with the net result being a reduction in the freeing point within cells. Typically, species employing this type of freeze-tolerance will have elevated levels of cryoprotectants such as glycerol or glucose. These cryoprotectants both help to modulate cell dehydration and may help to stabilize cell membranes and proteins against damage from freezing temperatures and physical stresses (Wharton et al., 2000). There are even some known cases of organisms that can tolerate intracellular freezing, though this is much less common (Lee and Costanzo, 1998). The second category of survival strategies is freeze-avoidance. In climates that experience seasonal reductions in temperatures, organisms with mobility may adopt migration as a means of freeze-avoidance. Others that remain during colder months may rely on thick insulating coats and metabolic processes to maintain body heat and may resort to hibernation to further conserve energy. On a molecular level, several freeze-avoidance mechanisms have been discovered. Freeze-avoidance organisms typically rid themselves of all ice nucleators at the onset of colder weather, both by halting their own production of such substances and by ejecting any ice nucleators from their gut that may have been ingested during feeding. Moreover, freeze-avoidance organisms often decrease the freezing point of their bodily fluids by increasing their concentrations of small solutes such as glucose and glycerol. These solutes effectively

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reduce the vapour pressure of water, a feat that results in the depression of the freezing point. Such freezing point depression is termed colligative. In addition, freeze-avoidance organisms often try to maintain their fluids in a supercooled state. Although we generally consider 0°C as the freezing point for water, this is technically not true. Often, in fact, it is necessary to reduce water temperature considerably below 0°C before ice formation occurs. The reason for this is that the initial formation of a tiny ice embryo, known as nucleation, is not an energetically favourable process and will not occur spontaneously until the temperature has dropped to around 40°C (Hew and Yang, 1992). After an initial ice embryo has been created, however, subsequent ice growth will proceed spontaneously providing the temperature remains below 0°C. Many substances are known to act as nucleators by providing surfaces on which ice can grow, including silver iodide and many inorganic clays (Hew and Yang, 1992). As already noted, such nucleators can factor into a freeze-tolerant organism’s survival strategy. For freeze-avoidance organisms, however, this need for a nucleation event to initiate ice growth opens a window of opportunity. The challenge for these organisms is to remain in a supercooled state without freezing by avoiding ice nucleation events. As mentioned, a primary task for these organisms is to rid their bodies of all potential ice nucleators at the onset of cold weather. Any nucleators that might subsequently be ingested, including even the tiniest ice crystals, must likewise be eliminated. These organisms have evolved safeguards to protect all porous openings from the inadvertent uptake of ice nucleators. For example, the urethra of Antarctic fish, a potential avenue for the inward propagation of ice, is terminated by a strong muscular sphincter coupled with a substantial amount of mucus to help keep ice particles at bay (Cheng and DeVries, 1991). Other polar fish have adapted to live in a supercooled state in deep waters where the water pressure inhibits ice nucleation altogether (Raymond and DeVries, 1977). While these strategies for freeze-tolerance and freeze-avoidance clearly show the ingenuity that permeates the biological world, our understanding of how organisms have evolved to adapt to cold climates is far from complete and continues to be an active area of research. One area that has garnered much attention is the investigation of a class of fascinating proteins that are produced by many cold-climate organisms to supplement their freeze-tolerance or freeze-avoidance survival strategies. These proteins, known collectively as antifreeze proteins (AFPs), have been found in a broad spectrum of cold-climate species, ranging from fish and plants, to insects and bacteria. The diversity of the species that produce these proteins is also matched by the diversity present in the proteins themselves, which together form a dazzling array of protein shapes and functions that have puzzled researchers for a number of years. What these proteins share in common is an unusual ability to influence ice growth by interacting directly with the surface of ice crystals, something that few other proteins can do. This ability has made AFPs an effective component in the survival strategies for many cold-climate organisms. With regard to freeze-avoidance organisms, in many cases ice nucleation cannot be completely eliminated. To counter this lethal situation, many freeze-avoidance organisms produce AFPs that can effectively block the growth of ice embryos in their bodily fluids by attaching to ice crystal surfaces (Jia and Davies, 2002). This has the effect of depressing the freezing point of these

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supercooled fluids. AFPs in freeze-tolerant organisms, on the other hand, appear to play a different role altogether. In general, this set of AFPs cannot depress the freezing point of bodily fluids to any significant degree. Instead, they are thought to play an important role in the freezing and thawing process by interacting with extracellular ice crystals to inhibit ice re-crystallization, a damaging process by which larger ice crystals are formed from the merging of smaller ones (Jia and Davies, 2002). Although the specific mechanism(s) by which AFPs influence ice growth remains an active area of research, much has been learned about AFPs since their discovery in the early 1970s. Herein, we present an overview of AFP research and discuss the promise they hold for the food processing industry.

2.2 The discovery of AFPs The discovery of AFPs in the blood of Antarctic fish in the early 1970s helped to explain a previously baffling phenomenon regarding fish living in both the Arctic and Antarctic oceans. Marine physiologists were aware that the high concentrations of electrolytes in these fish, most notably sodium chloride, were responsible for colligatively depressing their freezing points by between 0.6 and 0.7°C (Hew and Yang, 1992). However, because of their higher salt concentrations, the oceans at the poles remain year-round at about 1.9°C, which is only marginally above the freezing point of seawater. Moreover, these oceans become ice-covered, both in shallow sections and near shores, during winter seasons. Supercooled fish in these oceans, then, appear to face the constant threat of freezing from contact with this ice or other nucleators. How was it that they were not freezing? An analysis of the blood from Antarctic fish confirmed that the colligative effects of sodium chloride and other electrolytes only accounted for approximately 40–50 per cent of the freezing point depression that occurs in these fish (Duman and DeVries, 1975). The remainder of the depression was shown to be associated with small blood proteins (Cheng and DeVries, 1991), a finding that quickly provoked great interest among researchers. This enthusiasm was buoyed by the thought that an understanding of how these proteins function might lead to the possibility of creating synthetic analogues, perhaps ones that could produce even higher freezing point depressions, that would be of use in preservation technologies and cryo-biology (Madura et al., 2000). Over the next decade, efforts were made to better characterize these proteins. Numerous varieties were found in different species, some of which turned out to be glycosolated (linked to sugar molecules). All shared the property of reducing the freezing points of bodily fluids non-colligatively, without affecting the melting points of these fluids, though the extent of the reduction differed from protein to protein. This gap between freezing and melting points, termed the thermal hysteresis (TH) gap, was taken as a measure of the effectiveness of these proteins to act as an antifreeze and these proteins became known as antifreeze proteins (AFPs). Along with characterizing AFPs, research efforts have studied how these proteins are incorporated into the freeze-avoidance strategy of cold-climate fish. Most AFPs are synthesized in the liver and then secreted into the circulatory system (Hudson

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et al., 1979). These proteins have been found to constitute as much as 3 per cent of the blood protein in many cold-climate fish (Cheng and DeVries, 1991), indicating just how important they are for freeze-avoidance. Once in the circulatory system, AFPs are carried to bodily fluids throughout the fish where they provide extracellular antifreeze protection. Indeed, to date no antifreeze activity has been detected within cells themselves, suggesting that these proteins do not re-enter cells following secretion from the liver (Ahlgren et al., 1988). Only the discovery of a curious AFP synthesized in the skin cells of winter flounder has provided some indication that AFPs might play some non-antifreeze role within cells (Ewart et al., 1999). This particular AFP lacks the pre- and prosequences used by cells to decide which proteins are to be exported to extracellular fluids. What the possible functions of an intracellular AFP might be, however, remains unclear, though it is speculated that it may play a role in cell membrane stabilization at temperatures above 0°C (Ewart et al., 1999). The details of some of the strategies adopted by the Antarctic nototheniid fish to keep its fluids from freezing are illustrative of the complexities involved in coldclimate survival. Some of these fluids, including the pericardial, peritoneal and extradural fluids, obtain AFPs by diffusion from the circulatory system and so achieve antifreeze protection directly (Ahlgren et al., 1988). The intestines, which are particularly prone to freezing due to the high amount of ice-laden polar water that these fish must take in to maintain their salt and water balance, likewise receive AFPs from the blood. In this case, AFPs arrive intermixed with bile in the anterior end of the intestine following gallbladder evacuation (Cheng and DeVries, 1991). Amazingly, the intestines of these fish have evolved so as to leave the AFPs in tact, neither digesting nor reabsorbing them (Cheng and DeVries, 1991). During digestion, when salt, water and other nutrients are absorbed by the fish, the AFP concentration increases, so much so that by the time fluids reach the distal end of the intestine, it has a freezing point of 2.2°C, below that of sea water (O’Grady et al., 1983). In contrast, some fluids in these fish have no discernable amounts of AFPs. The endolymph, for example, contains no proteins whatsoever. Its deep-seated location in the fish, however, coupled by the fact that all of its surrounding tissues are protected by AFPs, makes ice propagation in this fluid unlikely (Cheng and DeVries, 1991). Other antifreeze-free fluids are not so fortuitously placed. Urine, for one, must be secreted and so by necessity comes in direct contact with seawater. This poses a serious dilemma for cold-water fish. On one hand, they cannot afford to have their urine freeze internally, while on the other hand it is energetically very costly to protect this fluid from freezing through the use of AFPs because these proteins would simply end up being expelled from their bodies with their urine. To solve this dilemma, cold-water fish have evolved (i) a number of mechanisms to ensure that AFPs do not enter their urine, and (ii) a strong muscular sphincter at the end of their urethra, well covered by mucus, to block inward ice propagation (Cheng and DeVries, 1991). Similarly, physical barriers have evolved to protect ocular fluids from freezing (Turner et al., 1985). Since the early studies on cold-water fish, AFPs have been found in many coldclimate species. As a group, these proteins show considerable diversity in their effects on ice. As mentioned, AFPs from freeze-avoidance organisms directly inhibit ice growth, whereas AFPs from freeze-tolerant organisms appear instead to mitigate the

660 Controlling the Freezing Process with Antifreeze Proteins

freezing and thawing process by minimizing the amount of ice re-crystallization that occurs. The amount of antifreeze protection in freeze-avoidance organisms is AFPdependent, with cold-climate insects receiving considerably more antifreeze protection from their AFPs than cold-water fish do from theirs (Jia and Davies, 2002). Furthermore, evidence suggests that this protection is concentration dependent and when insect and fish AFPs are compared at equal concentrations, the former offer tenfold more antifreeze activity than the latter (Liou et al., 2000). In addition to freezing point depression and the inhibition of ice re-crystallization, these proteins directly affect the morphology of ice crystals. Once again, the specific AFP-induced ice morphology expressed is AFP-dependent. Ice crystals grown in the presence of AFPs from cold-water fish typically adopt a hexagonal bi-pyramid morphology (Hew and Yang, 1992) (see Figure 25.2b), whereas ice crystals grown in the presence of AFPs from insects adopt lemon-shaped morphologies (Liou et al., 2000) (see Figure 25.2c). The nature of AFP-ice interactions has been studied for some time. Early studies demonstrated that AFPs interact differently with ice than other solutes do. When ice that is grown in a solution containing low concentrations of AFPs and other proteins is subsequently melted, only AFPs are found in the resulting melt in any significant concentration (Raymond and DeVries, 1977). Furthermore, AFPs affect the freezing process in a manner different from other solutes. The presence of solutes during the freezing process generally results in a broad temperature range over which freezing occurs, suggesting a slower phase transition from water to ice that enables the solutes to be excluded from the growing ice (Hew and Yang, 1992). In contrast, freezing occurs at precise temperatures in either pure water or aqueous solutions containing AFPs (Hew and Yang 1992). These and other results (Brown et al., 1985; Knight et al., 1991) have established that AFPs, unlike most other solutes, become incorporated into growing ice crystals and imply that some form of AFP-ice binding occurs. Such AFP-ice interactions have been shown to leave the ice Ih crystal structure unaltered (Raymond and DeVries, 1977). Moreover, ice-etching experiments have been instrumental for investigating the interactions between ice Ih and different types of AFPs (Knight et al., 1991). In these experiments, a single pure ice crystal is grown slowly in a dilute solution containing AFPs. Over time, AFPs bind to the particular planes of ice for which they have affinity; afterwards, these planes are clearly visible. These experiments have demonstrated that AFPs do not bind at random across the entire surface of an ice crystal, instead, each AFP shows binding affinity for a particular plane of ice (Figure 25.3). Whether this binding between AFPs and ice is reversible has been the subject of debate in the scientific community. Binding was generally considered reversible because of the dependence observed between antifreeze protection afforded by an AFP and its concentration in solution. Recent data suggest, however, that the degree of freezing point depression may not be AFP concentration-dependent after all, but rather dependent on AFP diffusion rates (Marshall et al., 2004). Lower concentrations of AFPs have been shown to achieve the same freezing point depression as higher ones if the cooling rate is sufficiently slow, allowing more time for AFP diffusion to occur. The question of whether AFP-ice binding is reversible or not remains unanswered. Regardless of the reversibility of the AFP-ice bond, general agreement exists as to the basic mechanism by which AFPs achieve ice growth inhibition. By binding to the surface

Antifreeze proteins 661

1 cm (a)

Prism plane view

1 cm

c-axis (b)

c-axis

Prism plane view

Figure 25.3 Results from ice-etching experiments. Shown here are the ice-etching results for (a) the fish type I AFP from winter flounder and (b) the fish type I AFP from shorthorn sculpin. The multiple planes visible in each etch are in fact symmetrical copies of the particular plane to which each of these AFPs has affinity.

Bound AFPs Water

0°C Ice

Figure 25.4 AFP mechanism for ice crystal grown inhibition – the ‘Kelvin’ effect. Ice growth becomes restricted to areas between bound AFPs, the net result of which is to increase the local surface curvature. Given sufficient concentrations of AFPs, the surface curvature is increased sufficiently to alter the surfacearea-to-volume ratio of the ice crystal so that subsequent growth is no longer energetically favourable.

of an ice crystal, AFPs interfere with normal surface propagation by restricting subsequent ice growth to surface regions between bound AFPs (Raymond and DeVries, 1977). Such restricted growth increases the surface curvature of the ice crystal and, given a sufficient concentration of bound AFPs, effectively increasing the surface-area-to-volume ratio beyond the maximal level required for spontaneous ice growth. This mechanism, known as the ‘Kelvin’ effect or the button-mattress theory, is illustrated in Figure 25.4. Because the surface-area-to-volume ratio at which spontaneous growth will occur increases as temperature decreases, the growth inhibition effects of an AFP remain until the temperature is decreased to the point where the exhibited surface-area-to-volume ratio again induces spontaneous ice growth. At this point, burst growth occurs and ice overruns the bound AFPs, trapping them inside the growing ice crystal. While this explanation of AFP activity is widely accepted, it contains few specific details of how particular AFPs bind to ice and achieve their unique level of antifreeze

662 Controlling the Freezing Process with Antifreeze Proteins

protection. It does not, for example, explain the hyperactivity of insect AFPs compared to fish AFPs. Nor does it provide any indication of what characteristics separate AFPs from other proteins. To begin to answer these types of detailed questions, more specific structural information about these curious proteins is required.

2.3 AFP structures and evolution The last 50 or so years have seen an unprecedented explosion in our understanding of biochemical molecules and processes. Perhaps the single most important discovery of the last century – in a century full of incredible discoveries – was the unravelling of the mystery of deoxy-ribonucleic acid, DNA, and its central role in all life on earth. Since its discovery in the 1950s (Watson and Crick, 1953), it has come to be thought of as the ‘blue print’ for life and many of the basic mechanisms by which it influences the unfolding of life at the molecular level are largely understood today. The entire ‘blueprints’ for a number of species have recently been decoded, the most notable among them being none other than that for human beings. If DNA is considered to be the ‘blueprint’ for life, then proteins can be thought of as its building blocks. In a complex process involving considerable molecular machinery for both synthesis and regulation, proteins are produced from the instructions encoded in the coding regions of DNA, termed genes, to perform an incredible myriad of cellular functions, ranging from catalysis to storage, from immune protection to the control of growth and differentiation. Though all proteins are composed of strings of amino acids, or residues, it is the particular sequence of these amino acids that gives each protein its specific characteristics and which gives the set of proteins as a whole such a bewildering array of functions and roles in the biological world. Once synthesized, each protein undergoes a spontaneous folding process that sees its particular linear string of amino acids assume a precise three-dimensional structure that is (relatively) stable in a wide range of environments. It is these final threedimensional structures that give rise to the inherent power of proteins as biological molecules, for it is their shape and surface characteristics that direct the types of molecular interactions in which they will participate and so determine their function(s). Because the structure-function relationship lies at the heart of the power of proteins, much scientific effort has been expended to determine exact three-dimensional representations of proteins. Two principal techniques have been developed that can ‘solve’ protein structures: nuclear magnetic resonance (NMR) and X-ray crystallography. NMR relies on a phenomenon that occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. This technique is generally used with small proteins that are maintained in solution at high concentrations. Crystallography, on the other hand, requires proteins in a solid crystal form, from which it can reveal atomic details by examining X-ray diffraction patterns. These techniques have been remarkably successful, with well over 10 000 protein structures determined to date. Almost all structures contain sections made up of ␣-helices, ␤-strands and/or ␤-turns, three of the most stable localized arrangements of amino acids (Figure 25.5). This collection of structural data has been

Antifreeze proteins 663

(a)

(b)

(c)

(d) Fish AFPs

(e)

(f) Insect AFPs

Figure 25.5 Representative structures for four fish AFPs (a–d) and two insect AFPs (E–F). (a) Type I AFP from winter flounder (PDB code: 1WFA). (b) Type II AFP from sea raven (PDB code: 2AFP). (c) Proposed structure for type IV AFP from longhorn sculpin. (d) Type III AFP from ocean pout (1MSI). (e) Tenebrio molitor AFP (PDB code: 1EZG). (f ) Spruce budworm AFP (PDB code: 1EWW). The diversity in AFP structures is evident, with two fish AFPs being largely ␣-helical (a, c), two being globular (b, d) and the two insect AFPs being ␤-helical, though of different handedness.

instrumental in uncovering specific protein folds that are responsible for many biological functions and, coupled with genetic information from a variety of species, has enabled researchers to study the genetic evolution that has driven protein development. Over time, proteins evolve as the DNA responsible for their synthesis undergoes mutations. Looking backwards from the present set of genetic and structural data, it is often possible to trace the history of protein development, a procedure that frequently provides insight into the structure-function relationship. Two specific types of genetic evolution exist. Under divergent evolution, a common ancestral gene mutates over time to produce a diverse set of descendant genes, causing a proliferation from one protein to many. While the proteins produced by such descendants may still retain similar structures and functions, each specific protein will have structural differences

664 Controlling the Freezing Process with Antifreeze Proteins

from its other family members that make it appropriately suited for its particular environment. In contrast to divergent evolution, convergent evolution works in the opposite direction, from many to one. In this case, environmental pressures cause different, unrelated ancestral genes to give rise to descendant genes whose corresponding proteins share common structural features and functionality. Like almost all other biochemical fields, AFP research has benefited tremendously from protein structural data. To date, structures have been determined for a number of fish (Yang et al., 1988; Jia et al., 1996; Gronwald et al., 1998) and insect AFPs (Graether et al., 2000; Liou et al., 2000; Leinala et al., 2000a, b) (Figure 25.5) and models have been proposed for others based on their primary sequence similarities to proteins whose structures are known (Davies and Sykes, 1997). As a group, AFPs are remarkable in their structural diversity. Fish AFPs alone have been classified into five types, all with different structural characteristics. The two insect AFP structures, both of which are ␤-helical (though one is left-handed and the other is right-handed), do not share common structural features with any of the fish AFPs and, while no plant AFP structures have been determined to date, their amino acid sequences indicate that each of these proteins is likely structurally unique. This structural diversity suggests that each type of AFP evolved both recently and independently, well after present species had reached their current form (Ewart et al., 1999). It is likely that species underwent tremendous evolutionary pressures to avoid freezing during geologically recent glacial periods. If we consider just fish AFPs alone, the multitude of completely different AFP structures in species of the same family suggests very recent evolution indeed. From an analysis of the record of the earth’s past climate, it has been suggested that these various fish AFPs evolved independently to counter the threat of freezing in icy water 1–2 and 10–20 million years ago in the northern and southern hemispheres respectively (Jia and Davies, 2002). A closer look at the known AFP structures reveals just how diverse this set of proteins really is (see Figure 25.5). Fish type I AFP is a single rod-like ␣-helical protein made up predominantly of alanine residues. The most studied type I AFP comes from winter flounder. Like several other types of AFPs, the flounder type I AFP has a repetitive nature that results in a regular alignment of another residue, threonine, on the same side of the protein every third turn of the helix (Yang et al., 1988). These threonine residues have long been suspected to play a fundamental role in ice interactions. In contrast, the type I AFP from shorthorn sculpin, though itself also an ␣-helical protein comprised largely of alanine residues, does not contain the obvious repeating elements of winter flounder (Hew et al., 1985). In fact, its residue sequence is not particularly similar to that of the type I AFP from winter flounder, suggesting that these two type I AFPs might be the result of convergent evolution from different ancestral proteins. Another type of fish AFP, AFGP, is also a highly repetitive, alaninerich ␣-helical protein (DeVries et al., 1971). This fish AFP, however, is glycosolated, having sugar molecules connected to each threonine residue in its sequence. A third fish AFP, type IV, is again largely ␣-helical in nature. Although the actual structure has not been determined, based on similar proteins whose structures have been determined this protein is almost certainly a bundle of four ␣-helices (Davies and Sykes,

Antifreeze proteins 665

1997) and not just the single ␣-helices of type I AFP and AFGP. Clearly, although these fish AFPs share an ␣-helical nature, they have many differences. The other two fish AFPs, types II and III, share nothing in common with each other or any of the ␣-helical fish AFPs (see Figure 25.5). Both of these proteins are globular rather than rod-like and neither protein appears to have any regularity in its surface-exposed residues. Fish type III AFP does not show any similarities to any other protein, antifreeze or otherwise, discovered thus far and so its evolution is unclear. Presumably the evolutionary pressures resulting from a cooling environment caused this protein to diverge from some ancestral protein, perhaps one whose other descendants have not yet been discovered. Fish type II AFPs, on the other hand, have amino acid sequences that are very similar to those from the carbohydrate recognition domain (CRD) of another family of proteins, the calcium-dependent C-type lectins (Ewart et al., 1992). Moreover, their three-dimensional shapes are also similar. C-type CRDs are found in a variety of proteins, including those with immunological, metabolic and structural roles (Drickamer and Taylor, 1993). The C-type lectins form a large family of proteins with highly conserved domains, making it difficult to determine which member(s) of this family may have given rise to fish type II AFPs. Of the three known fish type II AFPs, two (one from Atlantic herring and the other from rainbow smelt) have retained the calcium dependency found in C-type lectins and so cannot provide antifreeze protection in the absence of calcium (Ewart et al., 1996). A third type II AFP from sea raven, sharing only 40 per cent identity in its amino acid sequence with the other two type II AFPs, has evolved to function without calcium (Ewart et al., 1999). These differences in type II AFPs are not unexpected, as herring and smelt are much more closely related to each other than to sea raven. These proteins provide an excellent example of divergent evolution. Finally, the two insect AFPs that have had their structures determined, one from the beetle Tenebrio molitor (Liou et al., 2000) and the other from the spruce budworm moth (Graether et al., 2000), do little to simplify the AFP picture. Oddly, both are ␤-helices, a relatively rare fold among proteins. At the amino acid sequence level, however, the two proteins are completely different. These two helices have opposite handedness, a different number of residues per turn of their helices and completely different patterns of internal bonding. However, given all these differences, the two proteins have a remarkably similar repetitive pattern of amino acids on one side of their surfaces, suggesting that convergent evolution may be responsible for their development. Similar to the type I AFP from winter flounder, these proteins have regularly spaced threonines on this conserved surface which are thought to play an important role in ice-binding. With regards to their ancestry, neither of these proteins shares any similarity at the amino acid level with any other protein discovered to date, making it difficult to determine their lineage. As mentioned earlier, these two proteins are hyperactive antifreezes as compared to the fish AFPs, offering a tenfold increase in antifreeze protection in vitro. As a group, then, AFPs show remarkable structural diversity, even among members that have been classified together in the same type. The lack of a common set of surface features has certainly complicated the search for an understanding of the

666 Controlling the Freezing Process with Antifreeze Proteins

details involved in ice-binding and AFP activity. These difficulties notwithstanding, many models of AFP activity have been proposed and investigated in an effort to unravel the mysteries of these interesting proteins.

2.4 Mechanisms of AFP activity While it is widely held that AFPs operate by binding to ice surfaces and inhibiting subsequent ice growth by increasing the local surface curvature of ice crystals, the specific details of this activity remain unanswered. The AFP structures that have been determined have provided considerable assistance in developing models of the molecular details of AFP-ice binding, but they have been unable by themselves to unravel the mechanistic details of antifreeze activity. The major difficulty preventing the development of a detailed explanation of AFP activity lies in our inability to look directly at the molecular interactions occurring between AFPs and ice. For many proteins, it is possible to determine the structure of the complexes with their ligands and so derive a detailed three-dimensional picture of their interactions. Unfortunately, the AFP-ice complex is unsuitable for either X-ray crystallographic or NMR studies (Jia and Davies, 2002). Without the aid of structural details of the AFP-ice complexes, researchers have turned to other methods to investigate AFP activity. The two techniques most prominently reported are mutagenesis, the process of selectively altering the amino acid sequence of a protein to gain insight into its functional elements, and computational modelling. Of primary interest has been determining what part of an AFP’s surface is involved in AFP-ice interactions and what forces are involved in these interactions. In general, binding between two molecules involves shape complementarity, electrostatics, hydrogen bonding, van der Waals interaction, hydrophobicity and other biochemical features. An understanding of the molecular surfaces and the forces involved in binding is instrumental for determining what distinguishes an AFP from a non-AFP, what accounts for the hyperactivity of insect AFPs as compared to fish AFPs and how a synthetic analogue might be created that offers improved antifreeze protection. The most extensively studied AFP has been the fish type I AFP from winter flounder (see Figure 25.5). The regularly-spaced threonine residues along one face of this protein were immediately thought to play an important role in AFP-ice binding, particularly because their spacing, once suitably aligned, matches the oxygen atom spacing of the putative AFP-binding ice plane identified by ice-etching experiments (Yang et al., 1988). Threonine residues are hydrophilic, or water-loving, so the type I AFP face containing these residues appears to be a natural choice for the ice-binding surface (IBS) of this protein, particularly since alanine residues, which make up the majority of the rest of the protein surface, are hydrophobic (water-avoiding). Numerous computer docking simulations between this IBS and the ice plane predicted by iceetching studies to be involved in AFP-binding have given supporting evidence to this hypothesis (Chen and Jia, 1999). Mutagenesis experiments on these threonine residues, however, provided unexpected results (Chao et al., 1997). Replacing the threonines in the type I AFP amino acid sequence with serines, a similar hydrophilic residue,

Antifreeze proteins 667

reduced or even abolished antifreeze activity, while substituting the threonines with valines, a hydrophobic residue, left the antifreeze activity largely unchanged. These findings clouded the role of the threonine residues in ice-binding and left the question of whether hydrophilic or hydrophobic forces play the dominant role in AFP-ice interactions unanswered. It was not until a recent set of mutagenesis experiments investigating another surface of this protein, one that was composed entirely of hydrophobic alanine residues, that general agreement was reached about the IBS for this protein (Baardsnes et al., 1999). Disruptions to any part of this hydrophobic surface caused a dramatic reduction in antifreeze activity and led to the conclusion that hydrophobicity, which relies heavily on surface complementarity, or a ‘snug’ fit between AFP and ice, was critical for antifreeze activity. Figure 25.6 shows the snug fit between AFP molecules and ice that have been derived from several different computer-modelling studies. Similarly, mutagenesis and computational studies have been done with other AFPs. Unlike the repetitive type I AFP from winter flounder, the non-repetitive fish type III AFP from eel pout does not have a clear ice binding motif across its globular surface. Structural analysis and a series of mutagenesis experiments have identified one flat face of this protein as the putative IBS (Jia et al., 1996) and a number of computational studies have added support for this finding (Chen and Jia, 1999). This face, however, containing both hydrophobic and hydrophilic residues, lacks any obvious

c-axis (a)

(b)

c-axis

c-axis (c) Figure 25.6 Molecular modelling. (a–c) Results of computer docking experiments using standard molecular modelling techniques depicting final derived docking orientations between a single bound AFP and a static ice plane. All three show excellent surface complementarity between AFP and ice. (a) Fish type I from shorthorn sculpin. (b) Fish type III AFP from eel pout. (c) Insect AFP from spruce budworm.

668 Controlling the Freezing Process with Antifreeze Proteins

arrangement that might match the regularity of an ice-surface. A docking model using this putative IBS has been proposed for type III AFP (Jia et al., 1996) (Figure 25.6b), but without further supporting evidence it remains only speculative. The two insect AFPs whose structures have been determined to date (the beetle Tenebrio molitor and the spruce budworm moth) have also been subjected to mutagenesis. These AFPs have among the most repetitive structures of any protein structures solved to date (see Figure 25.5). As mentioned previously, although their amino acid sequences are completely different, both fold three-dimensionally to form a face with aligned rows of threonine residues that match the spacing of oxygen atoms on the primary prism plane of ice, the dominant plane (as determined from ice-etching studies) to which these AFPs bind. Mutagenesis on these and other faces of these proteins (Marshall et al., 2002; Graether et al., 2000), coupled with computer modelling (Leinala et al., 2002a) (Figure 25.6c), have provided strong evidence that these faces are the IBSs for these proteins. Other computational efforts have been made better to characterize AFP-ice interactions. Because several AFPs possess unusually flat surfaces, most notably the fish AFGP and types I and III AFPs, one group has analysed ‘flatness’ as a contributing factor towards antifreeze activity (Yang et al., 1998). While ‘flatness’ itself does not directly translate into a general biochemical principle of binding, it does indicate the importance of van der Waals forces in surface complementarity and would perhaps give some indication of the growth patterns of ice surfaces. Correlation among the studied proteins, which included both AFPs and non-AFPs, for flatness and antifreeze activity was found. Our group used a neural network to find any relationship between the antifreeze activity of 13 mutant fish type III AFPs and their various surface properties (Graether et al., 1999). Among these surface properties, hydrophobicity was found to have a significant correlation to antifreeze activity. These studies outline some of the diverse thinking that is currently underway regarding AFPs and their antifreeze abilities. Even with the considerable mutagenesis and computational research that has been reported regarding the mechanisms of AFP activity, some important aspects of this activity remain unclear. Much of the research into antifreeze activity has attempted to determine whether hydrophilic or hydrophobic forces dominate the AFP-ice interaction and numerous attempts have been made to correlate AFP activity with the strength of these forces. However, even granting that AFP-ice interactions are reversible, which is currently in debate, it is clear that the rates of dissociation between AFPs and ice must be very low indeed, given the tremendous growth pressures that exist for ice in supercooled water. Little has been reported as to why these weak interactive forces, be they hydrophilic or hydrophobic, should produce such strong AFP-ice attractions. Moreover, because AFP research has tended to focus on the binding orientations and forces of single AFPs to ice surfaces, other macro-level properties, most notably the cumulative effects of innumerable AFPs on ice growth morphology, have received comparatively little attention. And finally, due mainly to resource limitations, most computational AFP-ice modelling has treated ice as a static entity, instead of as the dynamic crystal that it most assuredly is at the molecular level. In light of these limitations, our recent computational study has taken a new approach to the study of AFPs. Unlike most other computational studies that have used molecular dynamics techniques to model the interactions between single AFPs and

Antifreeze proteins 669

small, static ice blocks, this new study employed a Monte Carlo technique to model the interactions between hundreds of AFPs and dynamic ice crystals containing millions of water molecules (Wathen et al., 2003) (see Figure 25.6d). The study reports the successful simulation of numerous AFP phenomena, including the duplication of iceetching patterns, the inhibition of ice growth within simulation temperatures matching reported antifreeze abilities for several different AFPs and AFP-induced changes to ice morphologies similar to those observed experimentally. From a combination of observations of ice growth inhibition during simulation and deductive reasoning, this study has advanced a new model for antifreeze activity. Because of the tremendous ice growth pressures in supercooled water, any AFP-ice interactions that are not (virtually) irreversible should be incapable of halting ice growth. All AFPs, it is postulated, must interact irreversibly with ice, regardless of the degree of antifreeze protection they can provide. From observations of the inhibition process, this study contends that differences in antifreeze abilities among AFPs are not due to differences in AFP-ice dissociation rates, but are instead due to the different planes of ice to which these AFPs prefer to bind. Because ice growth is anistropic, growth rates are different in different directions and an AFP’s ice-binding plane, together with the AFP’s particular orientation on that plane, will determine, in effect, the maximum ice surface curvatures that can be tolerated, which in turn dictates the amount of antifreeze protection conferred. With the reports of new AFP structures and different ideas about mechanisms of AFP activity, the last few years have seen some dramatic shifts in AFP thinking. Uncovering the details of molecular antifreezes seems tantalizingly close.

2.5 The use of AFPs in food preservation Because of their abilities both to inhibit ice crystal growth and reduce ice re-crystallization, AFPs have long been considered to be natural candidates for use in cold-storage food preservation. For many foods, the freezing process can cause a reduction in either food quality or nutritional value. Cellular-based foods that are eaten after thawing, including, but not limited to, meats, are prone to cell membrane damage from intracellular ice formation. This ultimately results in water loss upon thawing and leads to lower quality food products because of increased drip, a loss of nutrients and a reduced water-holding capacity (Griffith and Ewart, 1995). Foods that are eaten in the frozen state, such as ice cream, can also have their quality degraded over time by the formation of texture-compromising ice crystals. Still other foods, such as fresh produce like strawberries or tomatoes, are prone to severe quality loss from freezing, so much so that freezing is generally avoided. In these cases, efforts are made to maintain these foods at the lowest non-freezing temperature possible so as to minimize spoilage. By inhibiting or altering ice growth, AFPs can play a positive role in preserving food quality and nutritional value in foods that undergo cold storage. The addition of lower concentrations of AFPs to food products can promote ice re-crystallization inhibition, which can increase both the food quality and nutritional value. In frozen foods such as ice cream, ice re-crystallization inhibition can reduce the formation of large ice crystals and so help maintain food texture. Payne et al. (1994) have reported a reduction in

670 Controlling the Freezing Process with Antifreeze Proteins

the size of ice crystals observed in meats following treatment with either fish type I AFPs or AFGPs. Because smaller ice crystals will generally cause less cellular damage than larger ones, AFPs applied to meats and other cellular foods should experience less drip during thawing and thus a better retention of nutrients. The addition of higher concentrations of AFPs may also prove beneficial for foods that should not be frozen at all. Here, the higher AFP concentration will promote ice crystal growth inhibition, thus allowing foods such as strawberries to be stored at low temperatures with a reduced risk of severe quality loss. The use of AFPs to achieve ice growth inhibition, however, is a much more sensitive task than their use in ice re-crystallization inhibition and some evidence exists that the use of particular AFPs under certain conditions might actively damage cellular structures (Hincha et al., 1993; Petzel and DeVries, 1979). Fluids that are supercooled with the help of high concentrations of AFPs tend to experience rapid burst growth once temperatures fall below the level of protection afforded by AFPs and ice growth rates in these fluids can be up to five times faster than in fluids without AFPs (Harrison et al., 1987). This burst growth can cause cellular damage, particularly if the crystal morphology is needle-like. There are two primary considerations that must be addressed when attempting to add antifreeze protection via AFPs to cold-storage food. The first consideration is the selection of the particular AFP to be used. As mentioned, numerous types of AFPs have been found to date in fish, insects, plants and bacteria. In addition, each of these AFP types has numerous isoforms, effectively multiplying the set of AFPs from which to choose. Protein engineering efforts are also being made to improve the antifreeze abilities of these AFPs, adding still more choice. Furthermore, the detection of antifreeze activity in proteins from winter rye not typically thought of as AFPs (Hon et al., 1994) opens the possibility of grafting antifreeze abilities onto other existing proteins. Clearly, there is considerable choice as to the AFP to introduce into a particular food. To complicate this choice even further, it may be more beneficial to select a set of AFPs to provide antifreeze protection instead of just one. If we judge from nature herself, this may in fact be the case, as each organism that derives AFP protection actually produces several types of AFPs concurrently. Although this large collection of AFPs may seem daunting to begin with, in reality it allows one to select the most appropriate choice of AFP(s) for each application. The particular environment in which the AFP is to operate may play a pivotal role in narrowing the selection. For example, the fish type I AFP is an active ␣-helical protein at 0°C, but it remains largely unfolded at higher temperatures, making this AFP a poor choice for foods that must be heated before they are frozen (Griffith and Ewart, 1995). Another consideration when choosing the most suitable AFP for freezeprotection might include qualitative changes to the taste of food. AFPs from winter rye, for example, are similar to thaumatin, an extremely sweet-tasting protein. These AFPs may only be suitable for use in foods that are originally sweet in nature or are sweetened during preparation (Griffith and Ewart, 1995). Still another consideration when choosing an AFP to introduce into a cold-stored food is consumer acceptance. Consumers may be more likely to accept a product that has been infused with an AFP from a more closely-related species than from a more distantly-related one, such as tomatoes that have AFPs from another plant species rather than AFPs from fish.

Conclusions 671

The second consideration that must be addressed when designing a cold-storage system for food products involving AFPs is the manner by which the AFPs are to be introduced into the food. For foods that naturally contain AFPs, this question is obviously much easier to address. In this case, the goal is to promote maximal AFP production in the organism itself. Because AFPs are synthesized in response to cold stress, this might mean, for example, arranging harvest times for vegetable crops after the occurrence of fall frosts. It may even be the case that vegetables such as carrots and cabbage continue to produce AFPs even after they are placed in cold storage, though data for this have not at present been collected. For foods lacking natural AFPs, other means of AFPintroduction must be considered. Because large quantities of many different types of AFPs can be produced through cloning and because AFPs operate extracellulary, it is often possible to introduce AFPs directly into food by mechanical means such as mixing, injecting, soaking or vacuum-infiltration. This enables AFPs to be introduced into a wide variety of foods, including those that are cellular based and those that are not. As mentioned before, meat soaked in a solution containing AFGPs or type I AFP has been shown to contain smaller ice crystals and to have reduced freezing-induced tissue damage. Similarly, AFPs can be mixed into ice cream to minimize the size of ice crystals that can form therein. Besides direct mechanical methods, AFPs can also be introduced into foods by gene transfer. While this method is fraught with pitfalls, once an AFP gene has been successfully transferred to an organism, it and all its progeny would inherit antifreeze protection. To date, several attempts have been made, though success has been limited. Transgenic Atlantic salmon have received the fish type I AFP from winter flounder (Hew et al., 1992), but the protein expression levels have been too low to offer protection. Transgenic tomatoes have also been given the fish type I gene (Hightower et al., 1991), but significant expression of the type I AFP has been limited to the leaves of the plant and not in the fruit. Other organisms have also received the type I AFP gene (Kenward et al., 1993), but have failed to receive antifreeze activity. Clearly, although the rewards are high, this technique is prone to difficulties at many stages. A successful gene transfer must overcome numerous hurdles, including targeting a suitable location in an organism’s DNA, proper and sufficient expression of both mRNA and protein, correct protein folding and stabilization in the organism’s environment, proper susceptibility to protease digestion, correct distribution throughout the organism and extracellular protein targeting. All of these factors depend heavily on the particular AFP chosen for gene transfer and its compatibility to its new environment.

3 Conclusions AFPs, a curious collection of proteins that are produced by many cold-climate organisms, are presently being explored for use in the food processing industry as a means of improving both the quality and nutritional value of food kept in cold storage. Their ability to inhibit ice re-crystallization has been shown to decrease cellular damage in meats and to inhibit the appearance of ice crystals in frozen foods such as ice cream.

672 Controlling the Freezing Process with Antifreeze Proteins

Ice growth inhibition, another ability of these proteins when they are present at elevated concentrations, also holds promise for the food industry, as it may eventually allow food products that are prone to serious damage from freezing to be maintained in supercooled states at temperatures below the freezing point. Of these two abilities, the former is currently considered to be the more promising, partly due to the fact that it requires smaller concentrations of AFPs and partly because of the fact that ice growth inhibition can itself lead to food damage if burst growth is allowed to occur. Because AFPs operate extracellularly, direct mechanical methods are presently the best method of introducing AFPs into food products, though research is progressing in gene transfer methods and several efforts have been made to date to introduce AFPs into organisms at the DNA level. Recent developments in our understanding of how AFPs operate, coupled with ongoing efforts at engineering novel and improved AFPs, suggest that these proteins hold much promise for food preservation.

References Ahlgren JA, Cheng CC, Schrag JD, DeVries AL (1988) Freezing avoidance and the distribution of antifreeze glycopeptides in body fluids and tissues of Antarctic fish. Journal of Experimental Biology, 137, 549–563. Baardsnes J, Kondejewski LH, Hodges RS, Chao H, Kay C, Davies PL (1999) New icebinding face for type I antifreeze protein. FEBS Letters, 463, 87–91. Brown RA, Yeh Y, Nurcham TS, Feeney RE (1985) Direct evidence for antifreeze glycoprotein adsorption onto an ice surface. Biopolymers, 24, 1265–1270. Campbell NA (1987) Biology. Menlo Park: The Benjamin/Cummings Publishing Company Inc, pp. 36–38. Chao H, Houston ME Jr, Hodges RS et al. (1997) A diminished role for hydrogen bonds in antifreeze protein binding to ice. Biochemistry, 36, 14652–14660. Chen G, Jia Z (1999) Ice-binding surface of fish type III antifreeze. Biophysical Journal, 77, 1602–1608. Cheng CC, DeVries AL (1991) The role of antifreeze glycopeptides and peptides in the freezing avoidance of cold-water fish. In Life Under Extreme Conditions (di Prisco G, ed.). Berlin: Springer-Verlag, pp. 1–14. Davies PL, Sykes BD (1997) Antifreeze proteins. Current Opinion in Structural Biology, 7, 828–834. DeVries AL, Vandenheede J, Feeney RE (1971) Primary structure of freezing pointdepressing glycoproteins. Journal of Biological Chemistry, 246, 305–308. Drickamer K, Taylor ME (1993) Biology of animal lectins. Annual Review of Cell Biology, 9, 237–264. Duman JG, DeVries AL (1975) The role of macromolecular antifreezes in cold water fishes. Comparative Biochemistry and Physiology, Part A, Molecular and Integrative Physiology, 52, 193–199. Ewart KV, Rubinsky B, Fletcher GL (1992) Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins. Biochemical and Biophysical Research Communications, 185, 335–340.

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Leinala EK, Davies PL, Doucet D, Tyshenko MG, Walker V, Jia Z (2002b) A ␤-helical antifreeze protein isoform with increased activity: Structural and functional insights. Journal of Biological Chemistry, 277, 33349–33352. Liou YC, Tocilj A, Davies PL, Jia Z (2000) Mimicry of ice structure by surface hydroxyls and water of a ␤-helix antifreeze protein. Nature, 406, 322–324. Madura JD, Karan K, Wierzbicki A (2000) Molecular recognition and binding of thermal hysteresis proteins to ice. Journal of Molecular Recognition, 13, 101–113. Marshall CB, Daley ME, Graham LA, Sykes BD, Davies PL (2002) Identification of the ice-binding face of antifreeze protein from Tenebrio molitor. FEBS Letters, 529, 261–217. Marshall CB, Tomczak MM, Gauthier SY et al. (2004) Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity. Biochemistry, 43, 148–154. Maruyama M, Ashida T, Knight C (1999) Disk crystals of ice grown in air-free water: no effect of dissolved air on the morphology. Journal of Crystal Growth, 205, 391–394. O’Grady SM, Ellory JC, DeVries AL (1983) The role of low molecular weight antifreeze glycopeptides in the bile and intestinal fluid of Antarctic fishes. Journal of Experimental Biology, 98, 429–438. Payne SR, Sandford D, Harris A, Young OA (1994) The effects of antifreeze proteins on chilled and frozen meat. Meat Science, 37, 429–438. Petzel D, DeVries AL (1979) Effect of fish antifreeze agents on cryoprotection of red blood cells in the presence of glycerol and PVP. Cryobiology, 16, 585–586. Ramlov H (2000) Aspects of natural cold tolerance in ectothermic animals. Human Reproduction, 15, 26–46. Raymond JA, DeVries AL (1977) Adsorption inhibition as a mechanism of freezing resistance in polar fish. Proceedings of the National Academy of Science USA, 74, 2589–2593. Stryer L (1995) Biochemistry, 4th edn. New York: W. H. Freeman and Company, pp. 10–11. Turner JD, Schrag JD, DeVries AL (1985) Ocular freezing avoidance in Antarctic fish. Journal of Experimental Biology, 118, 121–131. Wathen B, Kuiper M, Walker V, Jia Z (2003) A new model for simulating 3-D crystal growth and its application to the study of antifreeze proteins. Journal of the American Chemical Society, 125, 729–737. Watson JD, Crick FH (1953) A structure for deoxyribonucleic acid. Nature, 171, 737–738. Wharton DA, Judge KF, Worland MR (2000) Cold acclimation and cryoprotectants in a freeze-tolerant Antarctic nematode, Panagrolaimus davidi. Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 170 (4), 321–327. Yang DSC, Sax M, Chakrabartty A, Hew CL (1988) Crystal structure of an antifreeze polypeptide and its mechanistic implications. Nature, 333, 232–237. Yang DSC, Hon W-C, Bubanko S et al. (1998) Identification of the ice-binding surface on a type III antifreeze protein with a ‘flatness function’ algorithm. Biophysical Journal, 74, 2142–2151.

Minimal Fresh Processing of Vegetables, Fruits and Juices Francisco Artés and Ana Allende Technical University of Cartagena, Department of Food Engineering, Postharvest and Refrigeration Group, Cartagena, Murcia, Spain This chapter tries to summarize the principles and application of hurdle theory together with the development of emerging techniques for the minimal fresh processing or fresh-cut industry to improve the quality, safety and shelf-life of plant-derived commodities in order to satisfy increasing consumer demand. The introduction examines the growing interest of this industry as the consumer demand for healthier and fresher food products is rising every year. The main spoilage changes that affect minimally fresh processed fruit and vegetables, as well as how the traditional processing and preservation techniques solve these problems, are reviewed. Also the need for seeking alternative or secondary technologies which use mild but reliable treatments in order to achieve fresh-like quality and safe products with a high nutritional value is considered. Additionally, the introduction focus on the keys for the production of safe foods, which include screening materials entering the food chain, suppressing microbial growth and reducing or eliminating the microbial load by processing and preventing post-processing contamination. Some successful combinations of subinhibitory processes, based on the application of a combination of various mild treatments, take advantage of the synergisms of the different preservation hurdles known as ‘hurdle technology’. The success of the new technologies also depends on a good understanding of the physiological responses of microorganisms to stresses imposed during food preservation. The second part discusses the factors and processing operations that affect the quality of minimally fresh processed plant foods, which include good agricultural practices in growing crops, good hygienic practices during harvesting and handling, quality of washing water, processing technologies, packaging methods and transportation, processing and storage temperatures. Factors such as the quality of the raw material, worker sanitation in the processing plant, disinfection of the product, antibrowning dips, peeling and cutting steps and the packaging materials and conditions will play a very important role in the final quality of minimally fresh processed commodities. The last part of the chapter addresses the emerging technologies for keeping microbial and sensory quality of minimally fresh processed fruits, vegetables and juices, relating to disinfection of the products and other emerging techniques. Novel modified atmosphere packaging, hydrogen peroxide, ultraviolet-C radiation, ozone, acidic electrolysed water, biocontrol Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

26

678 Minimal Fresh Processing of Vegetables, Fruits and Juices

cultures, organic acids, chlorine dioxide or hot water treatments have been examined. The two most relevant emerging techniques to be applied in minimally fresh processed juices, which are pulsed electric field and high hydrostatic pressure, are commented on and the current applications to particular food products are considered.

1 Introduction Consumers from developing countries are more and more concerned about the nutritional and sensory aspects, as well as the safety, of the food they eat (Da Costa et al., 2000) as a consequence of a higher education level. This motivates an increasing demand for healthy and nutritious products and contributes to a continuous need for new and more differentiated food product assortment (Linneman et al., 1999). For this reason, the development of healthy foods was rated as the most important area of research by a large majority of food companies interviewed, followed closely by developing natural foods (Katz, 2000). Human nutritional research has increasingly shown that a well-balanced diet, rich in fruit and vegetables, promotes good health and may reduce the risk of certain diseases (Meng and Doyle, 2002). Therefore recommendations for a balanced diet must include the consumption of fresh fruit and vegetables, which in fact is a very important part of the diet of people around the world. Minimally fresh processed fruit and vegetables are prepared for consumption by using light combined methods such as washing, cutting, grating, shredding, pulling the leaves off, etc. and packing at chilling temperatures under polymeric films that are able to generate optimum modified atmosphere packaging (MAP) conditions. This kind of plant food, also named fresh-cut or ready-to-eat, is commonly free from additives and only needs minimal or no further processing prior to consumption (Artés, 1992, 2000a; Artés and Martínez, 1996; Odumeru et al., 1997). Traditional preservation methods like freezing, dehydration, curing or salting are never applied and these foods are therefore not stable by design and less heavily processed foods mean also less heavily preserved (Carlin and Nguyen-the, 1997). As consumers increasingly perceive fresh food as healthier than heat-treated food, it motivates a general search for food production methods with reduced technological input. This phenomenon was observed over the last few years since the per capita consumption of fresh fruits and vegetables has increased significantly over the consumption of processed vegetables such as canned vegetables (Orsat et al., 2001). However, a food which meets nutritional requirements is unlikely to be accepted by consumers if they do not like the flavour or other quality attributes (Da Costa et al., 2000). Fruit and vegetables are the major dietary sources of substances with antioxidants and free-radical scavenging properties like anthocyanins and other phenolic compounds, of high importance from the human nutritional point of view. Carotenoids, tocopherols and vitamin C are also appreciated due to their possible role in the prevention of several human diseases. Advances in agronomic, processing, distribution and marketing technologies, as well as the current preservation techniques, have enabled the produce industry to supply nearly all types of high-quality fresh fruit and vegetables to those

Introduction 679

who desire and are willing to purchase them year around. Despite the benefits derived from eating raw fruit and vegetables, safety is still an issue of concern as these foods have long been known to be vehicles for transmitting infectious diseases (Artés and Martínez, 1996; Beuchat, 1996; Allende et al., 2002; Artés, 2004). Whole fruit and vegetable products are highly susceptible to deterioration between harvest and consumption. Since minimal processing damages plant tissues, leading to additional quality losses, the derived fresh-cut commodities are in fact more sensitive to disorders than the original (Huxsoll and Bolin, 1989; Anderson and Lingnert, 1997; Artés, 2000b; Kader, 2002a, b). The main features are the presence of cut surfaces and damaged plant tissues, the minimal processing that cannot guarantee microbial stability of the product, the active metabolism of the plant tissue and the limited shelflife of the product (Orsat et al., 2001). Therefore, deterioration of minimally fresh processed fruits and vegetables is mainly due to further physiological ageing, biochemical changes and microbial spoilage which originate changes in respiration, ethylene emission, transpiration and enzymatic activity of the living tissues after harvesting and processing (Nguyen-the and Carlin, 1994; Ahvenainen, 1996). Many of the compositional changes influence their colour, texture, flavour and nutritive values. The principal spoilage changes affecting minimally fresh processed fruit and vegetables are off-flavours, discoloration, softening (loss of crispness or juiciness) and water loss (Willocx, 1995). As perishable products, minimally fresh processed fruit and vegetables are generally characterized by an irreversible loss of quality. Therefore, sensory quality of these kinds of products can never improve during further storage, instead the quality can only be retained, or its deterioration can be retarded by applying optimal processing and packaging techniques, storage temperature and eventually application of an enzymatic browning inhibitor (Willocx, 1995; Watada and Qi, 1999; Jacxsens, 2002). As mentioned, the traditional processing of this kind of product usually consists of a sequence of operations (trimming, peeling, cutting, washing/disinfection, drying and packaging) and, generally, the extension of the shelf-life depends on a combination of correct chilling treatment throughout the entire chill chain, dips in antibrowning solutions, optimal packaging conditions (usually MAP) and good manufacturing and handling practices in well-designed factories (Artés, 1992; Willocx, 1995; Artés and Artés-Hernández, 2000, 2003). Additionally, some authors have proposed the use of edible coatings in combination with antibrowning compounds to improve the colour preservation of fresh-cut fruit (Wong et al., 1994; Baldwin et al., 1995, 1996). Once these traditional processing and preservation techniques have been able to provide food products with acceptable sensorial and microbial quality, the next step forward is to design mild but reliable treatments in order to achieve fresh-like quality and safe products with a high nutritional value (Soliva-Fortuny and Belloso, 2003). Therefore, the minimal fresh processing industry is currently seeking alternative or secondary technologies to maintain most of the fresh attributes, storage stability and above all safety of fresh processed fruits and vegetables, meanwhile extending their shelf-life, although long shelf-life is not the most important selling argument anymore, with the market trends tending towards more fresh-like products (Jacxsens, 2002). In fact, for producers, handlers, shippers, fresh-cut processors, foodservice,

680 Minimal Fresh Processing of Vegetables, Fruits and Juices

retailers, regulatory agencies and the consumers of perishable edible fresh processed commodities there should be no doubts that prevention and rigorous microbial food safety are critical concerns of the food industry (Sapers, 2001; Suslow, 2002). The contamination of fresh produce within human pathogens appears to be of low probability but of potentially high consequence due to the low number of confirmed cases of illness associated with raw fruit and vegetables, compared to the number of cases due to consumption of food of animal origin (Suslow, 2002). However, recently a wide range of documented cases of contaminated fresh fruit and vegetables, as well as unpasteurized fruit juices, have caused large outbreaks of microbial infections (Anon, 1999, 2000; Meng and Doyle, 2002). Cold adapted psychrotrophic food-spoiling (e.g. Pseudomonas spp. or Micrococcus spp.) and food-poisoning (e.g. Listeria monocytogenes or Yersinia enterocolitica) bacteria remain a concern because they posses cold-adapted proteins and membrane lipids that facilitate growth at chilling temperatures applied for extending shelf-life (Russell, 2002). Production of safe foods includes screening materials entering the food chain, suppressing microbial growth and reducing or eliminating the microbial load by processing and preventing post-processing contamination. Research on alternative or emerging technologies was initially focused on process design, product characteristics and kinetics of microbial inactivation, but it is clear that the success of the new technologies also depends on a good understanding of the physiological responses of microorganisms to stresses imposed during food preservation (Knochel and Gould, 1995; Lado and Yousef, 2002). Additionally, the emergence of microorganisms resistant to conventional food preservation techniques (freezing, thermal treatments, etc.) increases the need for developing new techniques for inhibiting unwanted microbial growth, as microorganisms adapt to survive in the presence of previously effective methods of control (Bower and Daeschel, 1999). As many of these conventional preservation methods are not applicable to minimally processed fruits and vegetables and only a few traditional techniques such as chemical treatments (e.g. antioxidants, chlorination, antimicrobial solutions, acidulants, etc.), MAP storage and mild heat treatments (40–50°C) can be used (Artés and Allende, 2005), many non-conventional methods, such as ultraviolet-C (UV-C) light, ozone, pulsed electric fields, magnetic fields, high-intensity pulsed light, high hydrostatic pressure, antimicrobials of natural origin or new edible coatings are now being investigated. However, in most of the cases, safety of these foods is based on the application of a combination of various treatments, taking advantage of the synergisms of the different preservation hurdles close up, following the principle of the ‘hurdle effect’ or ‘hurdle technology’ as shown in Figure 26.1 (Scott, 1989; Leistner, 1992, 1994, 2000; Carlin and Nguyen-the, 1997; Leistner and Gould, 2002; Ross et al., 2003). According to the hurdle concept, preservation treatments combined at lower individual intensities have additive or even synergistic antimicrobial effects, while their impact on sensory and nutritive properties of the food is minimized (Leistner, 1992). Recently Ross et al. (2003) pointed out that a good understanding of the modes of action of each individual treatment is crucial for selecting effective antimicrobial combinations, although the mechanisms of microbial inactivation by non-thermal

Factors and processing operations that affect quality 681

Food safety Fresh processed commodities

MAP UV-C radiation

Microbial growth

Chlorine, Ozone, Chemicals, Others Preservation techniques

Figure 26.1

Washing Chilling

Raw product in optimal quality

The hurdles concept.

technologies are currently not well understood (Barbosa-Canovas et al., 1998; Leistner, 2000). The base of the hurdle theory is that the microorganisms present (‘at the start’) in a food should not be able to overcome (‘leap over’) the hurdles present during the storage of a product, otherwise the food will spoil or even cause food poisoning (Ross et al., 2003). Additionally, many authors highlight that successful combinations of subinhibitory processes also depend on the technical compatibility of the selected processes and the product characteristics (Barbosa-Canovas et al., 1998; Sizer and Balasubramaniam, 1999; USFDA, 2000; Butz and Tauscher, 2002; Ross et al., 2003).

2 Factors and processing operations that affect quality of minimally fresh processed plant foods Many factors affect the shelf-life and microbial quality of raw prepared fruit and vegetables and they include good agricultural practices in growing crops, good hygienic practices during harvesting and handling, quality of washing water, processing technologies, packaging methods and materials and transportation, processing and storage temperatures (Shewfelt, 1986; Brackett, 1987; Bolin and Huxsoll, 1989; Huxsoll and Bolin, 1989; Ahvenainen et al., 1994; Hurme et al., 1994; Artés and Artés-Hernández, 2000; Artés, 2004). Figure 26.2 summarizes the most common factors that influence the shelf-life of minimally fresh processed fruits, vegetables and juices. The distribution chain of food products is generally composed of many different steps in storage and transportation up until consumption and traceability is today a key concept (Allende et al., 2004a). In this section, special attention is dedicated to those factors and processing steps that affect the final microbial and sensory quality of minimally fresh processed fruits and vegetables and, to a lesser extent, for juices.

682 Minimal Fresh Processing of Vegetables, Fruits and Juices

Mechanical damage Weight loss Decay

Biochemical changes Surface browning Softening

Deterioration of minimally fresh processed fruits and vegetables

Loss of texture

Microbial growth Spoilage Pathogen

Physiological ageing Dehydration Increasing respiration rate Increasing ethylene production Loss of appearance Figure 26.2

Factors that affect minimally fresh processed fruit and vegetables decay and shelf-life.

2.1 Plant material The fresh-cut industry began the minimal processing as a salvage operation to use offgrade and second-harvest products, but it was soon recognized that high quality raw materials were required because of the increased perishability caused by product preparation and the increasing demand for high quality produce. Currently, it is obvious that modern processing factories require raw materials of high quality, homogeneity and stable characteristics fulfilling specific conditions for its specific use (Cantwell and Suslow, 2002; Konstankiewicz et al., 2002). Only a few exceptions to this general rule could be considered, mainly related to external defects in fruit or vegetables. In order to reach the best results in this industrial activity the plant raw materials must be carefully chosen with regard to their ability to support the different processing steps. As some examples, several research studies have been accomplished to select lettuce cultivars with low sensitivity to browning development after minimal processing (Tomás-Barberán et al., 1997; Castañer et al., 1999) or tomato cultivars well adapted to cutting (Aguayo et al., 2001). In addition to this, it is evident that selected fruit and vegetables intended for pre-peeling and cutting must be easily washable and peelable (Ahvenainen, 1996; Watada et al., 1996). To assure the safety and quality of all incoming raw materials, implementation of a quality management standard such as ISO9000:2000 has been recommended as a basis for an agreement between the supplier and the fresh prepared produce manufacturer (Day, 2000), which should include a hazard analysis of critical control points (HACCP) to identify what could go wrong with incoming produce (Leaper, 1997; CHGL, 1999; IFPA, 2001). High quality fruit

Factors and processing operations that affect quality 683

and vegetables are free from damage or symptoms caused by disease and insects. Plant diseases normally cause problems during the period of cultivation, but there may be problems also found throughout the marketing period, especially those caused by microbes, which do not produce severe diseases during the growing season but can develop diseases that will be present in the products during storage and marketing (Tahvonen, 1999). As well as correct quality, proper storage conditions of fruit and vegetables before processing are vital for the production of commodities of good quality (Wiley, 1994). Finally, it is necessary to evaluate rapidly and non-destructively the quality of plant raw materials when received at the factory for such safety aspects as pesticide residues, microbial load, toxic metals, naturally present undesirable compounds and plant growth regulators (Dull, 1986; Yildiz, 1994). The most important characteristic of fresh fruit and vegetable products is that they continue living after harvesting. Respiratory activity is a metabolic process that provides the energy for plant cells to stay alive and develop physiological and biochemical processes. Several factors affect the respiration rate of the product, such as the type and maturity stage of the commodity and the storage conditions after harvesting (Kader, 1989; Gorris and Peppelenbos, 1999; Artés, 2000a; Fonseca et al., 2002). Furthermore, wounding increases the respiration rate of the plant tissue, probably as a consequence of induced ethylene (C2H4) biosynthesis, which stimulates respiration (Brecht, 1995) and, in some plant tissues, may be related to alpha-oxidation of fatty acids to CO2 (Rolle and Chism, 1987). During fresh processing, products are subjected to mechanical damage that provokes a fast physiological and biochemical answer, which is recognized by an increase in their metabolism. The respiratory activity of the plant material stored under MAP reduces the O2 and elevates CO2 sufficiently to create an environment around commodities that would not sustain insect or animal life (Beaudry et al., 1992) that also contributes to MAP storage success. In addition, the respiratory activity of plant tissues under MAP is slowed down by decreasing available O2 as a consequence of the reduction of overall metabolic activity (Smock, 1979; Kader, 1987; Solomos and Kanellis, 1989; Artés, 2000b). C2H4 is a naturally produced, simple two-carbon gaseous plant growth regulator that has numerous effects on the growth, development and storage life of many fruit and vegetables and affects attributes that contribute to appearance (Saltveit, 1999). As expected, the introduction of new cultural practices, cultivars, harvest and handling methods, post-harvest treatments and packaging, influence the effect of C2H4 on fresh processed fruit and vegetables. As example, C2H4 production is promoted by stresses such as chilling (Wang, 1990) or wounding, which increase the production rate of C2H4, sometimes within a few minutes, but usually within 1 h, with peak rates achieved usually within 6–12 h (Abeles et al., 1992). Currently, many actions can be taken to mitigate the effects of exposure of plant tissues to C2H4 such as reducing metabolism by keeping the exposed tissue at its lowest recommended storage temperature and/or O2 concentration, or the source of C2H4 from an enclosed space. For instance, MAP of 3 kPa O2
0 kPa CO2 or 3 kPa O2
3 Pa CO2 reduced C2H4 production in tomato slices (Mencarelli and Saltveit, 1988). Additionally, several chemicals can be used to remove C2H4 from the atmosphere (Loughee, 1987; Saltveit, 1999). Various solid and liquid formulations of KMnO4 could be used to oxidize C2H4. Ozone is also an effective

684 Minimal Fresh Processing of Vegetables, Fruits and Juices

oxidizer. In addition, C2H4 action can be blocked by a variety of compounds including CO2 and a number of unsaturated cyclic olefins, such as 1-methylcyclopropene (1-MCP) (Abeles et al., 1992). The 1-MCP acts as an inhibitor of C2H4 perception and provides commercial potential to control ethylene-dependent processes, however, it was reported that 1-MCP treatment is not as effective for leafy tissues as it is for floral organs. Additionally, due to the short storage period, the use of a C2H4 absorbent within packages did not improve the shelf-life of tomato slices (Gil et al., 2002). Browning of fresh fruit and vegetables is an ever-present problem during post-harvest handling, processing and storage, being one of the major causes of quality loss and spoilage which reduces produce quality and very often being the factor limiting the shelf-life and marketability of minimally fresh processed fruit and vegetables (Vamós-Vigyázó, 1981; Shewfelt, 1987; Bolin and Huxsoll, 1991; Couture et al., 1993; Sapers, 1993; López-Galvez et al., 1996; Pirovani et al., 1997; Artés et al., 1998; Laurila et al., 1998; Peiser et al., 1998). This phenomenon can be due to enzymatic and non-enzymatic reactions. Enzymatic browning or oxidative browning requires different components: enzymes such as polyphenol oxidase (PPO) and peroxidase (POD), a substrate and co-substrates such as O2 and H2O2. The disorder takes place at the cut surface of fruit and vegetables because of decompartmentation that occurs when cells are broken, allowing substrates and oxidizers to come in contact. The brown colour development is related primarily to oxidation of phenolic compounds including monophenols, triphenols, and o- and p-diphenols to o-quinones, a reaction catalysed by PPO and POD (Mayer and Harel, 1979; Artés et al., 1998). These phenolic compounds are produced from the amino acid L-phenylalanine to trans-cinnamic acid catalysed by phenylalanine ammonia liase enzyme (PAL) to p-coumarate. Subsequent reactions produce several new compounds, to form chlorogenic and isochlorogenic acids and with tartaric acid to form caffeoltartaric and dicaffeoltartaric acids (Mateos et al., 1993; Castañer et al., 1996, 1999; Tomás-Barberán et al., 1997). The oxidation products of these reactions, o-quinones, polymerize with each other and react with NH2 or SH groups from amino acids and proteins and with reducing sugars, giving complexes of high molecular weight polymers of unknown structure which leads to the formation of dark brown or black pigments (VámosVigyázó, 1981; Leja et al., 1996). It was reported that wounding also induces synthesis of some enzymes involved in browning reactions or substrate biosynthesis (Brecht, 1995; Peiser et al., 1998). In cut, bruised or senescent plant products this oxidative reaction occurs readily, as fresh processed commodities are highly susceptible to oxidative browning reactions. Given the deleterious effects of PPO activity upon the sensory and nutritional quality of fresh processed produce, it is not surprising that considerable research has been devoted to inhibit the activity of this enzyme.

2.2 Processing line, distribution and storage conditions 2.2.1 Processing line

The main objective of the fresh fruit and vegetable processors throughout all processing operations involved in the production of fresh prepared produce items is the safety

Factors and processing operations that affect quality 685

of their products, quality optimization and loss reduction (Shewfelt, 1999). Common practices consist of the protection of the produce from damage caused by poor handling or machinery, foreign body contamination and/or pest infestation (Day, 2000). In addition, contamination by human handling during manipulation, washing, drying and packaging processing may occur from unhygienic personnel (Hurst, 1995; Francis et al., 1999). Therefore, although worker sanitation is an aspect that is too often neglected in the processing of fresh plant products, good hygiene throughout must be practised and all food handlers must be supervised and trained in food hygiene matters related with their work activities (Brackett, 1992; CHGL, 1999). The sequence of steps needed in a typical industrial factory of minimally fresh processing fruits or vegetables have similarities, although both of them require specific and differentiated steps. Figure 26.3 illustrates the general unit operations and the maximum recommended temperatures for each processing step in the production line of leafy vegetables. However, the most significant steps of the processing chain (washing, cutting, disinfection dips and packaging), which significantly affect the final quality of the products, match up in both production lines. 2.2.1.1 Whole product washing The first step in the minimal fresh processing fruits is generally sanitation of whole fruit to eliminate unwanted dirt, pesticide residues, plant debris, soil, insects and foreign matter and retardation of the enzymatic discoloration reactions (Seymour 1999; Soliva-Fortuny and Martín-Belloso, 2003), as indicated in Figure 26.3. In fact, many researchers have used sodium or calcium hypochlorite and others salts for surface sanitation and sterilization of fruits to prevent microbial inoculation, although after pathogens have infected their host, chlorination is not very effective (Hong and Gross, 1998). For instance, the surface sterilization of whole tomato with sodium hypoclorite, potassium bicarbonate, calcium chloride and calcium lactate extends the shelf-life of fresh-cut tomato (Hong and Gross, 1998; Artés et al., 1999; Gil et al., 2001). Additionally, Aguayo et al. (2003) used an acidified hypochlorite (1.4 mM) water solution at pH 6.4 to disinfect whole melons before their minimal fresh processing. 2.2.1.2 Peeling and cutting Peeling and cutting steps constitute a critical point in the processing line hygiene and the equipment used in this process needs to be cleaned, disinfected and sharpened at regular intervals every working day to avoid build-up of organic residues (Yildiz, 1994; Heard 2000, Soliva-Fortuny and Martín-Belloso, 2003). The physical damage, physiological stress and increase of microbial growth caused by the peeling and slicing steps are well documented (Garg et al., 1990; Barry-Ryan and O’Beirne, 1999; Ahvenainen, 2000). These changes are mainly due to the increase of wound respiration and C2H4 production due to mechanical injuries, which result in the release of intracellular oxidizing enzymes and substrates and leads to various biochemical deteriorations such as browning (Varoquaux and Wiley, 1994; Ahvenainen, 1996) and increased availability of cell juice and nutrients (Jacxsens, 2002). The fluid exudation of bruised and cut surface tissues can lead to high difficulty for keeping quality and shelf-life of some fresh processed plant foods. To prevent this problem some technical

686 Minimal Fresh Processing of Vegetables, Fruits and Juices

Clean area

Dirty area

Maximum recommended temperature Harvesting

25–30°C

Transport

10°C

Pre-cooling and chilling storage

5°C

Manual selection and classification

10°C

Whole product washing

10°C

Cooling

5°C

Trimming operation

10°C

Disinfection washing

5°C

Rinsing

5°C

Dewatering and spin drying

7°C

Weight and optional mixing

7°C

Active or passive modified atmosphere packaging

5°C

Wholesale cold storage and quality control

0–1°C

Cold transport and distribution

5°C

Retail cold storage

5°C

Consumer

5°C

Figure 26.3 General unit operations in a minimally fresh processed vegetable processing plant and the maximum recommended temperatures to each processing step.

implementation has been developed, such as the use of a water-absorbent paper within trays of tomato slices to avoid juice accumulation (Mencarelli and Saltveit, 1988; Artés et al., 1999; Gil et al., 2001). However, Sofos et al. (1998) reported that optimal quality fresh products contain nutrients, numerous phytoalexins and other natural antimicrobial compounds that may enhance or retard the growth of naturally occurring microflora and pathogens.

Factors and processing operations that affect quality 687

Lee and Kader (2000) found that bruising significantly affected the chemical composition of pericarp and locular tissues of tomato fruit and L-ascorbic acid retention in shredded iceberg lettuce was influenced by the slicing method used in the processing chain. The type of cut influenced overall quality and appearance of minimally processed tomato and segments were scored lower than slices due to the separation of the placenta from the pericarp (Aguayo, 2003). Therefore, cutting appears to have a dramatic effect on nutritional value, overall quality and shelf-life of minimally fresh processed fruit and vegetables (Adams et al., 1989; Garg et al., 1990; Watada et al., 1990; Mangles et al., 1993; Barry-Ryan and O’Beirne 1999; Ahvenainen 2000; Artés 2000b). Many different peeling machines are commercially available, but peeling is normally accomplished manually, mechanically, chemically or in high-pressure steam peelers (Wiley, 1994). At the same time, several methods are able for cutting, grating, chopping, shredding, slicing or chipping fresh produce into pieces of various shapes and sizes (Allende et al., 2004a). 2.2.1.3 Washing and disinfection For the fresh-cut industry, washing after peeling and/or cutting is a critical step in the production chain. Washing and disinfection are the only steps in the production chain where a reduction in the microbial load can be obtained, thus minimizing populations of potential pathogens (Nguyen-the and Carlin, 1994; Wiley, 1994; Beuchat et al., 1998; Francis et al., 1999; Day, 2001). However, published efficacy data indicate that theses conventional, time-consuming methods are not capable of reducing microbial population on produce by more than 90–99 per cent, reductions that are insufficient to assure microbiological safety (Beuchat, 1998; Brackett, 1999; Sapers, 2001). Often, some in the produce industry incorrectly assume that chemicals are used to sanitize fruits or vegetables; however, sanitizers are primarily used to maintain bacteriological quality of the water rather than the produce (Brackett, 1999). Sapers (2001) and Suslow (2002) summarize the limiting factors of three alternative sanitizing agents as shown in Table 26.1. Washing can be achieved by simply spraying with potable water, although it generally involves the immersion of the product in chilled (1–10°C) sanitized water in a bath or wash-tanks usually containing between 50 and 150 ppm of chlorine solution and acidified with about 150–200 ppm of citric acid to manage pH values between 6.5 and 7.5 for optimizing the chlorine efficacy (Yildiz, 1994; Francis and O’Beirne, 1997; Simons and Sanguansri, 1997; Artés 2000b; Day, 2000; Artés and Artés-Hernández, 2003; Allende et al., 2004a). Systems that ensure mixing of the wash water and product will improve washing performance, because the turbulence generated by the aeration allows the elimination of almost all traces of soil and foreign matter without damage to the product (Yildiz, 1994; Simons and Sanguansri, 1997). A great number of antimicrobial washing solutions specifically for whole vegetable products have been reported, but probably the most widely used is hypochlorite solution containing 40–150 ppm of available chlorine and quaternary ammonium compounds (Adams et al., 1989; Brackett, 1992; Francis and O’Beirne, 1997; Kim et al., 1999). When chlorine gas (Cl2) or hypochlorite salt (e.g. NaOCl or Ca(OCl)2) is added to water, they will generate Cl2, hypochlorous acid (HOCl), which is the active form, or

688 Minimal Fresh Processing of Vegetables, Fruits and Juices

Table 26.1 Main properties of different disinfection agents currently used in the fresh processed plant foods industry Disinfectant properties

Sodium or calcium hypochlorite

Chlorine dioxide

Ozone

Specificity

Generally effective, including viruses; reference sanitizer. Limited practical effect of parasitic spores

Biofilm penetration

Rapidly attacks bacterial cell walls and is more effective against the thick-walled spores of plant pathogens

Stability

Good as powder, fair as liquid

Good

Highly unstable in water

pH impact

Active at pH 6–7.5

Very low impact, best at 8.5

Unaffected from water pH between 6 and 8.5

Penetration

Poor

Poor

Poor

Concentration

25–200 ppm, 20 000 ppm limited approval for sprout disinfection

3–5 ppm

0.5–2 ppm

Population reduction on produce surfaces

1–2 log units

1–2 log units

1–3 log units

Organic matter

Reacts to form chloramines

Little influence

Reacts but no deleterious by-products

Irritancy

Low

Very irritating vapours

Very irritating to eyes and throat

From Suslow (2000) and Sapers (2001).

hypochlorite ions (OCl) in various proportions, depending on the pH of the solution. However, the antimicrobial activity of hypochlorite solutions is related to the concentrations of undissociated OCl and HOCl (Adams et al., 1989; Jacxsens, 2002). There is no unified criterion about the recommended free chlorine concentration and contact time for the disinfection washing. Therefore, the USFDA recommends 50–200 ppm total chlorine and contact times of 1–2 min for this purpose (USFDA, 1998) and the International Fresh-Cut Produce Association (IFPA, 2001) Model HACCP Plan for shredded lettuce, suggests a maximum chlorination of 100–150 ppm total chlorine at pH 6.0–7.0 and the maintenance of 2–7 ppm free residual chlorine after contact (Flickinger, 1999; Delaquis et al., 2004). It is remarkable that chlorinated washing of produce can effectively remove sand, soil and other debris from fresh fruit and vegetables, but should not be relied upon to remove organisms completely (Beuchat, 1992; Bracket, 1992). In contrast, it was shown that tap water could be just as effective as chlorinated water (Brackett, 1987). Allende et al. (2004a) reported a 2–3 log unit reduction of total microbial growth by washing minimal fresh processed ‘Lollo Rosso’ lettuce with chlorinated (150 ppm of active chlorine) water for 1 min. Additionally, Sinigaglia et al. (1999) showed the effectiveness of chlorine treatment (100 and 150 ppm of free chlorine) against Pseudomonadaceae and Enterobacteriaceae bacteria in cut lettuce salads and shredded carrots. Earlier, Zhang

Factors and processing operations that affect quality 689

and Farber (1996) found that the maximum log reduction of L. monocytogenes on fresh-cut lettuce and cabbage treated with 200 mg/l of chlorine was less than 2 log cfu/g. However, it has been shown that many microorganisms exhibit resistance to chlorine treatments (Dychdala, 1991; Nguyen-the and Carlin, 1994; Simons and Sanguansri, 1997), leaving the food industry to seek alternative agents for disinfection (Bower and Daeschel, 1999). The use of chlorinated water has also raised questions due to some facts, such as even when used at low concentration, it may cause taste and odour defects in treated products, the possibility of health hazards due to the potential toxicity, carcinogenicity and mutagenicity of chlorinated water and chloroorganic compounds formed by reaction with food components and the processing cost and problems associated with disposal of waste chlorinated water (Wei et al., 1985, 1995; Dychdala, 1991; Singh et al., 2002). For all these reasons, alternative chemicals disinfection agents have been tested such as chlorine dioxide, ozone, organic acids, hydrogen peroxide, quaternary ammonium compounds, trisodium phosphate, sucrose esters, iodine compounds, alcohols, anionic and non-ionic surface-active agents, aldehydes, phosphoric and peroxiacetic acids, cisteine, methyl jasmonate and bioflavonoids (Pappalardo et al., 1990; Van de Weyer et al., 1993; Zhuang and Beuchat, 1996; Beuchat et al., 1998; Sapers and Simmons, 1998; Seymour, 1999; Day, 2001). There are several means to control enzymatic browning in fresh processed fruit and vegetables. The most common method used in the industry and the laboratory is the addition of reducing agents to the dipping solution, which prevent browning by reducing the quinones back to their parent o-diphenols, such as sulphites and ascorbic acid, which act as PPO inhibitors and antimicrobial agents. Also, some antioxidants can be used in washing to avoid browning, but only a few chemicals are currently allowed in the European Union (Artés and Allende, 2005). 2.2.1.4 Dewatering As shown in the Figure 26.3, the next critical processing operation is dewatering. Drying or dewatering of wet surfaces must be carried out carefully to avoid unnecessary damage to the plant tissues, reducing the product moisture content and removal of cell leakage that can support microbial growth (Simons and Sanguansri, 1997; SolivaFortuny and Martín Belloso, 2003). Dewatering systems include draining systems, gentle removal with cheesecloth, centrifugal spin driers, vibrating racks, rotating conveyors, hydro-sieves, forced air and spinless drying tunnels (Simons and Sanguansri, 1997; Sapers and Miller, 1998; Seymour, 1999; Gorny et al., 2002). The high centrifugal force not only removes water, but also cracks and crushes the tissues (Ahvenainen, 2000). Forced cold air injected over a perforated conveyor belt, which transports the product, has been recently applied as an alternative to the conventional dewatering systems. This technology is just being used in England, Holland and the USA. However, their main inconvenience is the low efficiency to dry high volumes of product (Artés and Artés-Hernández, 2003). Another new technique that has recently been developed is the use of infrared light to dry the fresh processed commodities. However, this technology still has two main problems for its application in the industry, which are the high initial financial investment and the large area needed in the processing plant to

690 Minimal Fresh Processing of Vegetables, Fruits and Juices

install the device (Artés and Artés-Hernández, 2003). Additionally, between the drying and packaging steps it could be interesting to introduce techniques already applied in the clean room technology by installing a filtered air system that is able to assure the presence of less than 70 particles with a diameter higher than 5 ␮m and less than 10 000 with a diameter higher than 0.5 ␮m (Havet and Hennequin, 1999; Artés and Artés-Hernández, 2003).

2.2.2 Distribution and storage conditions

Virtually all fresh-cut products are refrigerated under MAP to achieve the needed commercial shelf-life. The design and selection of the appropriate polymeric film for trays or bowls as well as for sealing is crucial (Beaudry et al., 1992; Artés, 1993; 2000a; Aguayo et al., 2003; Artés and Artés-Hernández, 2003; Escalona et al., 2004). It is well known that temperature is the most important environmental factor that influences the deterioration rate of harvested commodities. Knowledge about the time-temperature conditions in the cold chain of fresh processed fruit and vegetables is necessary to determine the influence of the actual cold chain on the quality loss and the shelf-life of these products (Willocx, 1995; Jacxsens, 2002). Although throughout the distribution chain commodities must be kept at 1–5°C to ensure quality and shelflife, it is almost impossible to guarantee that this temperature will be maintained during transit, distribution and retail display (Artés and Artés-Hernández, 2000; Orsat et al., 2001). In fact, it has already been demonstrated that these products are often subjected to temperature abuse of about 12°C in the display cabinets for a long time while in the supermarkets (Willocx, 1995). The increased popularity of minimally fresh processed food greatly increases the interest in cold adaptation behaviour of microorganisms and food pathogens, mainly due to the longer time intervals between production and consumption (7–10 days) of food products and the extended use of domestic refrigerators (Abee and Wouters, 1999). Psychrotrophic pathogens such as L. monocytogenes or Y. enterocolitica obtained from fresh processed plant products have received scientific attention in the last few years. Many authors demonstrated the advantages of conventional MAP (3–5 kPa O2, 5–10 kPa CO2 and balanced with N2) to reduce deterioration of fresh-cut vegetables and proliferation of aerobic spoilage microorganisms (Kader, 1986; Gorris and Peppelenbos, 1992; Nguyen-the and Carlin, 1994; Ahvenainen, 1996; Artés and Martínez, 1996; Escalona et al., 2004). The aim of MAP is to create an atmosphere around the packaged produce that retards their respiration and deterioration in such way that the tolerated minimal O2 or maximum CO2 concentrations are not exceeded, in order to avoid a shift towards fermentation or other metabolic or biochemical disorders. Therefore, a well designed MAP is able to generate gas conditions within packages for extending shelf-life, improving safety and maintaining sensory attributes of fresh processed fruit and vegetables by inhibiting metabolic activity and C2H4 biosynthesis and action and minimizing weight loss, decay and risk of chilling injuries (Kader, 1986; Artés, 1993, 2000a; Brecht, 1995; Vermeiren et al., 1999; Jacxsens, 2002). In this way, when temperature abuse (over 5°C) during transport, distribution and, particularly, retail sale

Emerging technologies for keeping microbial and sensory quality 691

could occur, the use of little perforations or microperforations in MAP polymeric films should be suggested. It is known that, on the one hand, atmospheres of high CO2 and low O2 levels could control microbial growth but, on the other hand the risk of recontamination of commodities within packages increases (Escalona et al., 2004; Artés, 2004).

3 Emerging technologies for keeping microbial and sensory quality of minimally fresh processed fruits and vegetables The emphasis in post-harvest fruit protection against quality attributes losses, physiological disorders, diseases and insects has shifted from using agro-chemicals to various alternative techniques, including biological control, cultural adaptations and physical methods such as controlled atmosphere (CA), MAP and irradiation. Given the restrictions of chemical use in plant foods and because many of them cause ecological problems or are potentially harmful to humans and may be withdrawn from use, the advantage of these alternative techniques is that no chemicals are involved (Artés, 1995; Graham and Stevenson, 1997; Reddy et al., 1998; Mathre et al., 1999; Sanz et al., 1999; Daugaard, 2000; Harker et al., 2000; Marquenie et al., 2003). Additionally, preservation techniques are becoming milder in response to demands of consumers for higher quality, more convenient foods that are less heavily processed and preserved and less reliant on chemical preservatives (Abee and Wounters, 1999). The unique way to assure microbial and sensory quality of minimally fresh processed plant products relies on refrigerated storage and distribution, although combination of refrigeration and subinhibitory preservation techniques could prolong their shelf-life. As mentioned above, many non-conventional methods are now being investigated; however, there are some limitations to their application since some methods are not applicable to fresh-cut fruits and vegetables because of damage to plant tissue but only to liquid foods such as fruit juices (Carlin and Nguyen-the, 1997). Therefore, in this section those techniques that can be used to preserve fresh processed plant foods will be revised.

3.1 Disinfection The most critical step in the production chain of minimal fresh processing of fruits and vegetables is washing-disinfection. For this reason, special attention to the alternative sanitizing agents as well as the new technologies for disinfection of these commodities will be given. To develop or improve washing and sanitizing treatments, special attention should be paid to the compatibility of treatments with commercial practices, cost, absence of induced adverse effects on product quality and the need for regulatory approval and consumer acceptance (Sapers, 2001). Some alternatives to

692 Minimal Fresh Processing of Vegetables, Fruits and Juices

sanitizing agents are: O3, ClO2, peracetic acid (about 90–100 ppm), H2O2, organic acids (acetic, lactic, citric, malic, sorbic and propionic acids at 300–500 mg/ml), electrolysed water, radio frequency, hot water treatments and UV-C radiation (Adams et al., 1989; Masson, 1990; Castañer et al., 1996; Tomás-Barberán et al., 1997; Delaquis et al., 1999, 2000, 2004; Sapers, 2001; Suslow, 2002; Jacxsens, 2002; Aguayo, 2003; Allende, 2003).

3.1.1 Hydrogen peroxide

Treatments of hydrogen peroxide (H2O2) seem to be a promising alternative to chlorine for disinfecting minimally fresh processed vegetables (Soliva-Fortuny and Martín-Belloso, 2003). H2O2 is generally recognized as safe (GRAS) for some food applications, but has not yet been approved as an antimicrobial wash. It does not produce residues since it is rapidly decomposed by the enzyme catalase to water and O2 (Sapers, 2001). Various experimental antimicrobial applications of H2O2 for foods have been described, including preservation of vegetable salads, berries and fresh-cut melons (Hagenmaier and Baker, 1997) since it reduces microbial populations and extends the shelf-life without causing loss of quality. Sapers and Simmons (1998) recommended its use for fresh-cut melon as it extended the shelf-life for 4–5 days in comparison to chlorine treatments. However, they demonstrated that H2O2 is injurious to some commodities, causing bleaching of anthocyanins in mechanically damaged berries. H2O2 vapour delayed or reduced the severity of bacterial soft rot in fresh processed cucumber, green bell pepper and zucchini, but no effect on spoilage of fresh-cut broccoli was found (Hagenmaier and Baker, 1997). Additionally, an extended shelf-life was found in fresh processed cucumbers, green bell peppers and zucchini after washing in a 5–10 per cent solution of H2O2 for 2 min (Sapers and Simmons, 1998). It means that the applicability of H2O2 to a broad range of minimally fresh processed vegetables should be determined, especially with commodities that are subject to rapid spoilage.

3.1.2 Acidic electrolysed water

This is a new disinfectant technique for fresh produce that has been shown to be efficient due to its antimicrobial and antiviral activities for fruit and vegetables (Izumi, 1999; Koseki and Itoh, 2000). Electrolysis of water containing a small amount of sodium chloride generates a highly acidic hypochlorous acid solution containing 10–100 ppm of available chlorine. Koseki et al. (2001) found that acidic electrolysed water (pH 2.6, oxidation reduction potential, 1140 mV; 30 ppm of available chlorine) reduced viable aerobes in shredded lettuce by 2 log cfu/g within 10 min, showing a higher disinfectant effect than ozonated water. They reported that the use of this new technique could be applicable for food factory hygiene, meaning that the use of acidic electrolysed water at home or restaurant kitchen just before eating fresh fruits and vegetables could prevent poisoning. According to this, Park et al. (2002) reported population reductions on lettuce leaves exceeding 2.49 log units for E. coli O157:H7 and L. monocytogenes and Horton et al. (1998) reported population reductions of

Emerging technologies for keeping microbial and sensory quality 693

E. coli O157:H7 on apples of 3.7–4.6 log units cfu/g. However, Izumi (1999) only found 1 log unit cfu/g reduction in the microbial population of fresh-cut vegetables. 3.1.3 Chlorine dioxide

Chlorine dioxide (ClO2) is a strong oxidizing agent (about 2.5 times the oxidative capacity of chlorine) having a broad biocide efficacy (Singh et al., 2002), including a good biofilm penetration. To date, the FDA (USFDA, 1998) has allowed the use of aqueous ClO2 in washing of uncut and unpeeled fruit and vegetables. However, ClO2 is unstable and it must be generated on-site and can be explosive when concentrated (Jacxsens, 2002). Zhang and Farber (1996) found that the initial microbial load decreased by 1 log cycle of cfu/g for shredded lettuce inoculated with L. monocytogenes at levels of 5 mg/l ClO2 in aqueous solution. However, Reina et al. (1995) found that bacterial populations present on fresh processed cucumbers were not greatly influenced by ClO2 treatment, even at concentration of 5.1 mg/l. More recently, Singh et al. (2002) found that increasing the concentration of ClO2 in deionized water (5 mg/l for 1 and 5 min) resulted in a decrease in E. coli O157:H7 population on lettuce and baby carrots in comparison to washing with deionized water (control) for the same period. Increasing the washing period from 1 to 15 min with aqueous ClO2 (5 mg/l) showed no significant reduction in the population of E. coli O157:H7 on shredded lettuce. However, after washing baby carrots a reduction in E. coli O157:H7 was found. 3.1.4 Organic acids

Several organic acids have been tested as alternative disinfectants to sanitize fresh-cut vegetable surfaces (Hilgren and Salverda, 2000). They may retard and/or prevent the growth of some microorganisms (Beuchat, 1998). Their antimicrobial activity is not generally due to killing of the cells but they affect the cells’ ability to maintain pH homeostasis, disrupting substrate transport and inhibiting metabolic pathways (Seymour, 1999). Peracetic acid has been recommended for treatment of process water (Hilgren and Salverda, 2000); however, population reductions for aerobic bacteria, coliforms, yeast and moulds on fresh-cut celery, cabbage and potatoes, treated with 80 ppm peracetic acid, were less than 1.5 log units cfu/g (Forney et al., 1991). Wright et al. (2000) obtained a 2 log units cfu/g reduction in apple slices inoculated with E. coli O157:H7 using 80 ppm peracetic acid, with an interval of 30 min between inoculation and treatment. On the other hand, Wisniewsky et al. (2000) found a reduction of less than 1 log unit cfu/g at the same concentration but in an interval of 24 h. Citric acid has been proposed as a very good coadjutant to the washing of fresh-cut fruit and vegetables due to its antibrowning power. It is a phenolase Cu-chelating agent and the inhibition of PPO was attributed to its chelating action (Jiang et al., 1999). Santerre et al. (1988) reported that application of citric acid can prevent browning of sliced apple thus extending shelf-life and it was shown that the combination of citric acid and ascorbic acid exhibited even more beneficial effects (Pizzocaro et al., 1993). Additionally, Jiang et al. (2004) found that the application of citric acid was effective in extending shelf-life and maintaining the quality of fresh-cut Chinese water chestnut slices during storage.

694 Minimal Fresh Processing of Vegetables, Fruits and Juices

3.1.5 Ozone

Ozone (O3) is a strong oxidant and potent disinfecting agent and, when it is applied to food, it leaves no residues since it decomposes quickly. The biocide effect of O3 is caused by a combination of its high oxidation potential, reacting with organic material up to 3000 times faster than chlorine (EPRI, 1997). Even though it is new for the USA, it has been utilized in European countries for a long time (Guzel-Seydima et al., 2004). For instance, it has been commonly used as a sanitizer in water treatment plants since the early 1900s (Gomella, 1972) and also for disinfection of swimming pools, sewage plants, disinfection of bottled water and prevention of fouling of cooling towers in Europe (Gomella, 1972; Rice et al., 1981; Legeron, 1982; Schneider, 1982; Echols and Mayne, 1990; Costerton, 1994; Videla et al., 1995; Strittmatter et al., 1996). In 1997, an expert panel decreed that O3 was a GRAS substance for use as a disinfectant or sanitizer for foods when used in accordance with good manufacturing practices in the USA (Suslow, 2003) and it has now been approved for use as a disinfectant or sanitizer in foods and food processing in the USA (USDA, 1997, 1998). The bactericidal action of O3 has been studied and documented on a wide variety of organisms, including those that are resistant to chlorine, extending the shelf-life of a number of fruit and vegetables (Fetner and Ingols, 1956; Norton et al., 1968; Rice et al., 1982; Foegeding, 1985; Ishizaki et al., 1986; Foegeding and Busta, 1991; Restaino et al., 1995; Beuchat, 1998; Richardson et al., 1998; Aguayo, 2003). In fact, it has been proven that O3 is suitable for washing and sanitizing solid food with intact and smooth surfaces (e.g. fruit and vegetables) and ozone-sanitized fresh produce has recently been introduced in the USA market. The use of O3 to sanitize equipment, packaging materials and the processing environment is currently being investigated (Kim et al., 2003). The modus operandi of O3 implicates the destruction of microorganisms by the progressive oxidation of vital cellular components. The bacterial cell surface has been suggested as the primary target of ozonation (Guzel-Seydima et al., 2004). Khadre and Yousef (2001) compared the effects of O3 and H2O2 against foodborne Bacillus spp. spores and found that O3 was more effective than H2O2. In shredded lettuce treated with O3, Kim et al. (1999) reported that bubbling O3 gas (49 mg/l, 0.5 l/min) in a lettuce-water mixture decreased the natural microbial load by 1.5–1.9 log unit cfu/g in 5 min. As a consequence, a number of patents have been issued for using O3 to treat fruit and vegetables. However, the results obtained by Singh et al. (2002) have shown that treatment with ozonated water (5.2 mg/l) did not result in any significant reduction in E. coli O157:H7 populations during 1–15 min of washing in shredded lettuce, although they found a reduction in microbial counts on baby carrots after 10 min exposure to 5.2 mg/l ozonated water. The reduced efficacy of ozonated water during lettuce washing might be due to more O3 demand of organic material in the medium as it was also found in melon fresh-cut pieces (Aguayo, 2003). It was shown that the use of O3 in the storage of vegetable products could have detrimental effects, as happened in some berries with very thin skin which can be easily penetrated by O3, oxidizing the fruit (Norton et al., 1968; Rice et al., 1982). The antimicrobial efficacy can be enhanced considerably when ozonation is combined with other chemical (e.g. H2O2) or physical (e.g. UV-C radiation) treatments. Mechanical action is also needed as a means to dislodge microorganisms from the surface of the food and expose them to the action of the sanitizer (Kim et al., 2003).

Emerging technologies for keeping microbial and sensory quality 695

3.1.6 Hot water treatments

Heat preservation is one of the oldest forms of preservation known to man and has the potential to provide barriers to reduce microorganisms and inhibit enzyme activity, but this treatment is incompatible with fresh processed plant food since heat is associated with destruction of flavour, texture, colour and nutritional quality (Orsat et al., 2001). However, hot water treatments used to reduce or eliminate pathogens offer an alternative means to control the quality deterioration of fresh fruit and vegetables, as well as a means of enzyme inactivation (Bolin and Huxsoll, 1991). These mild heat treatments consist of subjecting the products to temperatures of 50–90°C for periods of time not exceeding 1–5 min. Loaiza-Velarde et al. (1997) reported that dipping lettuce in water at 45–55°C would extend the shelf-life and visual quality of minimally fresh processed lettuce by inhibiting the activity of PAL, which is the enzyme that initiates biosynthesis of phenolic compounds that leads to visible discoloration along the cut edge of the lettuce leaf (López-Gálvez et al., 1996). Additionally, Li et al. (2001) suggest that heat (50°C) treatment combined with 20 mg/l free chlorine for 90 s may have delayed browning and reduced initial populations of some groups of microorganisms naturally occurring on iceberg lettuce, but enhanced microbial growth during subsequent storage due to tissue damage. Delaquis et al. (1999, 2000) found a reduction of 2 log cfu/g in initial microbial load in lettuce washed with chlorinated water (100 ␮l/l) at 47°C for 3 min, compared to washing at 4°C. However, in 2004, Delaquis et al. found that comparison between lettuce washed at 4°C and 50°C revealed that disinfection of the lettuce was improved by heat, although the difference in total microbial populations was only 1 log cfu/g. The application of mild heat treatments is commonly by using hot air, hot water or steam. Among them, hot water is the easiest conditioning treatment since it offers a great flexibility and easiest control (Barkai-Golan and Philips, 1991). However, Orsat et al. (2001) have demonstrated that it is possible to treat carrot sticks thermally with radio-frequency energy in less than 2 min at an internal temperature of 60°C, to reduce the microbial load before packaging while minimizing the detrimental effects on the sensory quality of the fresh-like product. The main difference in using this treatment is that in radio-frequency heating, the energy is absorbed directly within the material, the heating is rapid and uniform throughout the material and the technology is relatively simple to adapt to an existing processing line.

3.1.7 UV-C radiation

One strategy to minimize the risks implicated with the consumption of fresh fruit and vegetables involves either reducing or eliminating external surface contamination (Yaun et al., 2004). UV-C radiation acts both directly by damaging the microorganisms on the exposed crop surface and indirectly by stimulating defence mechanisms in the treated product (Stevens et al., 1998; Nigro et al., 1998). Treatment with UV energy offers several advantages to food processors as it does not leave a residue, it is lethal to most types of microorganisms, with no legal restrictions and it requires no extensive safety equipment for use (Yousef and Marth, 1988; Wong et al., 1998; Bintsis et al., 2000). Additionally, as Figure 26.4 shows, the UV-C application device is simple and cheap.

696 Minimal Fresh Processing of Vegetables, Fruits and Juices

Net UV-C lamps

Metal structure Figure 26.4

UV-C equipment used to apply different UV-C doses to plant products.

The code of Federal Regulations (Title 21, Part 179) of the USA permits the use of UV radiation (wavelengths of 220–300 nm with 90 per cent emission at 253.7 nm) on food products to control surface microorganisms (Rhim et al., 1999). However, there are relatively few applications of UV disinfection technology in the food processing industries. The main reasons are that UV irradiation of foods has often been associated with undesirable changes to both the appearance and nutritional value of foods and the restricted range of commercially available equipment for disinfecting solids (Gardner and Shama, 2000; Shama, 2000). Other critical factors include the transmissivity of the product, the geometry configuration of the reactor, the power, wavelength and physical arrangement of the UV source(s), the product flow profile and the radiation path length (Sastry et al., 2001). Despite this, UV-C radiation is widely used in the industry for fruit juices and disinfection of water supplies and food contact surfaces. Its uses have demonstrated a beneficial effect when low doses were applied to different food products (Stermer et al., 1987; Liu et al., 1993; Stevens et al., 1999; Duffy et al., 2000; Mercier et al., 2000; Erkan et al., 2001). Recently, UV-C radiation has been considered an alternative treatment for preserving vegetable products (Maharaj et al., 1999) and fresh processed lettuce (Allende and Artés, 2003a, b; Allende et al., 2003c). Exposure to low UV-C radiation doses has been reported to reduce post-harvest decay of onions (Lu et al., 1987), sweet potatoes (Stevens et al., 1999), carrots (Mercier and Arul, 1993), tomatoes (Liu et al., 1993; Maharaj, 1995), strawberry (Marquenie et al., 2003), apples (Wilson et al., 1997), peaches (Stevens et al., 1998), lemon fruits (Ben-Yehoshua et al., 1992), table grape (Nigro et al., 1998) and zucchini squash (Erkan et al., 2001). It has been found that the use of low UV-C doses (between 0.81 and 8.14 kJ/m2) causes respiratory stress in minimally fresh processed ‘Lollo Rosso’ and ‘Red Oak Leaf’ lettuces tissues and was effective in reducing microbial growth (psychrotrophic bacteria, coliforms, yeast and moulds) without affecting the sensory quality of the product, as can be observed in Figure 26.5 (Allende and Artés, 2003a, b). Marquenie et al. (2003) confirmed that the intensity of these treatments should be minimized to prevent quality loss as an undesired side effect and this can be achieved by combining different techniques. They proposed the combined use of pulsed white light and UV-C or mild heat treatment to inactivate conidia of Botrytis cinerea and Monilia fructigena. When combining UV-C with light pulses, there was an increase in

Emerging technologies for keeping microbial and sensory quality 697

10

0

8 6 4

10

3

2

Control 0.40 kJ/m2 0.81 kJ/m2 2.44 kJ/m2 4.07 kJ/m2 8.14 kJ/m2

0

7

5

Figure 26.5 Overall visual quality (OVQ) of minimally fresh processed ‘Lollo Rosso’ lettuces non-radiated and radiated with different UV-C doses and stored under passive MAP at 5°C for 10 days.

inactivation for both B. cinerea and M. fructigena and synergism was observed. Despite all the research done, there are a relatively few applications of UV disinfection in the food processing industries so more work should be carried out.

3.2 Other emerging techniques 3.2.1 Biocontrol

Although the use of synthetic fungicides is the primary means for controlling postharvest diseases, several reasons, such as the need to reduce the use of chemicals after harvest, have encouraged the rapid development of alternative approaches such as the search for microbial antagonists to control post-harvest decay (Eckert et al., 1994; Ippolito and Nigro, 2000). Holzapfel et al. (1995) suggested that the use of protective cultures should only be considered as a supplement to good manufacturing practices, not as a substitute for the proper handling and packaging of minimally fresh processed food products. The use of biocontrol cultures may therefore be considered to enhance existing hurdle technology to prevent the growth of pathogens (Breidt and Fleming, 1997). Lactic acid bacteria (LAB) have been used for centuries for the fermentative preservation of many foods and some attempts have been made for the preservation of fresh-cut fruit and vegetables (Vescovo et al., 1996; Carlin and Nguyen-the, 1997). The only factor that should be taken into account is that the quality of the product must be checked after application of LAB, because they are potentially able to ferment or acidify vegetable substrates (Carlin and Nguyen-the, 1997). There is a wide range of inhibitory metabolites of LAB such as organic acids (lactic and acetic acids), H2O2 enzymes (lactoperoxidase system with H2O2 and lysozyme), low-molecular-weight metabolites (reuterin, diacetyl and fatty acids) and bacteriocins (nisin and other) (Breidt and Fleming, 1997). Additionally, the production of organic acids and bacteriocins, which inhibit growth of pathogens in a food, increases as conditions become

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more favourable to bacterial growth. Therefore, the addition of antimicrobial agentproducing strains or the use of culture permeate, combined with refrigerated storage and good hygienic handling practices could be helpful to control spoilage and pathogenic bacteria in fresh-cut vegetables (Torriani et al., 1997).

3.2.2 Novel MAP

It is well-known that MAP has been successfully used to maintain the quality of minimally fresh processed fruits and vegetables (Kader 1986; Gorris and Peppelenbos, 1992; Nguyen-the and Carlin, 1994; Ahvenainen, 1996; Artés and Martínez, 1996; Artés, 2000a, b, 2004). However, novel MAP technologies that allow an extension of the shelf-life are still much demanded by producers and distributors (Nguyen-the and Carlin, 1994; Ahvenainen, 1996). It was observed that exposure to high O2 alone did not strongly inhibit microbial growth and was highly variable (Jacxsens, 2002). On the other hand, many authors have found that superatmospheric O2 (higher than 70 kPa O2), when combined with increased CO2 concentrations, inhibits enzymatic discolorations and microbial growth in fresh-cut vegetables and prevents anaerobic fermentation reactions (Heimdal et al., 1995; Amanatidou et al., 1999, 2000; Day, 2001; Allende et al., 2001, 2002, 2003a, b, 2004b). Therefore, it could be considered as a good alternative to conventional MAP with moderate-to-low O2 and high CO2 levels (Day, 2001). Many authors have described the antimicrobial activity of CO2 and its use in MAP. CO2 is the most important component of gas mixtures applied to minimally fresh processed plant foods, because of its antimicrobial activity due to the increase of the lag phase and the generation time of spoilage microorganisms as well as by forming carbonic acid and thus possibly lowering the pH of the food to bacteriostatic levels (Gorris and Peppelenbos, 1992; Ahvenainen, 1996; Devlieghere et al., 1997; Artés, 2004). Superatmospheric O2 levels affect metabolism and different properties of vegetable commodities such as respiration rate, colour and texture, reduction of microbial growth and decay (Gregory and Fridovich, 1973; Amanatidou et al., 1999; Kader and Ben-Yehoshua, 2000; Wszelaki and Mitcham, 2000; Jacxsens et al., 2001; Allende et al., 2002). Furthermore, the effect of superatmospheric O2 on microbial growth can be very contradictory (Amanatidou et al., 1999). The O2 molecule is known to have a low reactivity, therefore, its toxicity stems mostly from its excited state (singlet O2) or its semi-reduced radical forms that can cause deleterious or lethal oxidative damage to cells (Gille and Sigler, 1995). The reactive oxygen species  1 (ROS), notably O 2 and hydroxyl (OH ) radicals, H2O2 and singlet oxygen ( O2), generated during the aerobic cellular metabolism induce DNA and nucleoprotein damage as well as lipid and protein damage in microorganisms (Moradas-Ferreira et al., 1996). Therefore, the antimicrobial effect is mainly attributed to the formation of superoxide radicals (O 2 ) (Gregory and Fridovich, 1973; Amanatidou et al., 1999). It was recently found that the effect of superatmospheric O2 on the growth of aerobic microflora was variable. Because of the different behaviour that microorganisms have under this atmosphere, it is necessary to study the effect of superatmospheric O2 on each microorganism and vegetable product before use. Thus LAB and members of the Enterobacteriaceae were inhibited, while growth of yeast and A. caviae seems to be

Emerging technologies of minimally fresh processed fruit juices 699

stimulated and growth of psychrotrophic bacteria and L. monocytogenes was not affected. The overall visual appearance (mainly colour) of the mixed vegetable salads and fresh processed spinach baby leaves was better maintained and the shelf-life prolonged when packaged under O2 levels higher than 50 kPa (Allende et al., 2002, 2004b). It is clear from all the published studies that more information is needed to guarantee the success of the superatmospheric O2 atmospheres to prolong the shelf-life of different fresh processed commodities. The development of new packaging materials will allow definitive avoidance of anaerobic conditions and a reduction in respiration rate, ethylene emissions, browning as well as weight loss in order to keep the fresh properties of minimally fresh processed fruits and vegetables longer, attenuating undesirable changes in sensory quality and controlling microbial growth (Artés, 2004). It is known as ‘active’ and ‘smart’ packaging, which responds actively to changes in the food package. As an example, smart packaging can now include materials designed to absorb or emit chemicals during storage, thereby maintaining a preferred environment within the package which maximizes product quality and shelf-life (Ohlsson, 2000). Therefore, the use of non-conventional MAP combined with antimicrobial, moisture absorbers and edible films or those films fitted with porous substrates covered with side-chain crystallizable polymers or with an O2 emitter and/or CO2 or C2H4 scavenging devices will also have many potential applications.

3.2.3 Genetic engineering technology

The possible use of genetic engineering to develop higher production and more resistant plant foods is relatively well known. Currently, this technology is being used to introduce desirable attributes such as improved colour, aroma, flavour and taste of different fruit and vegetable products. In fact, the first transgenic product introduced as a food commodity was a tomato with reduced polygalacturonase activity. Although the huge advance of these technique was in the last decade, there is still a lack of published information about the development of genetically modified fruit and vegetables which overcome some relevant problems of the post-harvest science such as chilling injury resistance, longer storage duration and pathogen resistance. Therefore much more effort should be done in this area and recent advances in functional genomics should bring candidate genes to manipulate (Artés, 2004).

4 Emerging technologies of minimally fresh processed fruit juices The market of minimally processed refrigerated fruit juices, like ready-to-eat plant foods, has experienced substantial growth over the past few years. Traditionally, fruit juices were subjected to heat treatments between 60 and 100°C for a few seconds. However, by using this technology, undesirable reactions may take place producing unwanted changes in the product or by-product formation, which decrease the overall

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quality of the juice. Therefore, the development of emerging technologies, which use a lower temperature to the traditional heat treatment and guarantee a final food product which preserves the fresh properties of the fruit juices as much as possible, is needed. Their success relies on a mild preservation treatment (generally, heat) combined with chilling to keep flavour and nutritional properties. Some researchers contrast minimal processing techniques with thermal processing, however, developments in thermal technologies have been considered ‘minimal’ where they have minimized quality losses in food compared to conventional thermal techniques (Ohlsson, 1996). The emergence of novel spoilage microorganisms in juices also poses a new challenge for the correct preservation of these food products. Fruit juices have been considered for many years susceptible to spoilage only by yeast, moulds and LAB. Their acid pH, lower than 4.0 in most cases, was considered sufficient to prevent growth of almost all spore-forming microorganisms. This fact has allowed the fruit beverage industry to apply successfully a hot-fill-hold process to pasteurize these products. However, in the last few years an increasing number of incidents of spoilage of acid foods, such as fruit juices, has been reported. Most of these spoilage incidents have been related to spore-forming thermo-acidophilic microorganisms. Spoilage caused by this kind of microorganisms is difficult to detect. The juice appears normal or has light sediment and no gas is produced. Often, the only evidence of the alteration is a ‘medicinal’ or ‘phenolic’ off-flavour (Walls and Chuyate, 1988). For instance, microorganisms of the genus Alicyclobacillus have a pH range for growth of 2.0–7.0 and are able to grow in a temperature range of 20–71°C. In particular, A. acidocaldarius will survive easily any heat treatment currently applied in the food industry to process fruit juices. Moreover, the extremely high heat resistance of this microorganism makes impossible the design of any heat treatment to inactivate the spores, as it would also damage dramatically the quality of the food. Fortunately, the high minimum growth temperature of this microorganism makes it improbable to cause spoilage in pasteurized acid foods like fruit juices. However, after storage of such foods for long periods at high temperatures, which can be reached in hot weather climates, growth of surviving microorganisms in the fruit juices should not be discarded (Palop et al., 2000). Only in the last ten years has there been a real recognition of mild preservation treatments as non-thermal methods to preserve food products (Barbosa-Cánovas et al., 1998) and there is a growing interest for non-heat-treatment of juices. The juices can be processed by using pulsed electric fields, high hydrostatic pressure, high intensity pulsed light, irradiation, new chemical and biochemical additives and, of course, the hurdles technology. The use of membrane disrupting novel preservation techniques, such us ultrasound, high pressure or pulsed electric field is based in their potentially synergistic effects with chill storage or mild heat treatment (Russell, 2002). However, there is a safety concern about the use of these new technologies since it is necessary to inactivate and control spoilage and pathogenic microorganisms that may be present in these products. It is well known that these preservation methods are physical and chemical hurdles that can be adjusted to guarantee the safety of the food products. The physical parameters include temperature of the process and storage, water activity, pH, redox potential of the product, etc. and the chemical and

Emerging technologies of minimally fresh processed fruit juices 701

biochemical parameters include antimicrobial, antioxidant, bactericide, etc. These parameters can be controlled at levels that can inhibit or inactivate the microbial load to produce safe products (Barbosa-Cánovas et al., 1998). In this respect, the application of non-thermal technologies could be a suitable alternative allowing an efficient preservation without causing heat damage to the product. This section only briefly mentions some of the current uses of these emerging technologies in fresh processed fruit juices since all of them are extensively discussed in different chapters of the present book.

4.1 Pulsed electric fields Pulsed electric fields (PEF) have been shown to be able to reduce the microbial population of refrigerated fruit juices, such as apple (Evrendilek et al., 1999) or orange and carrot juice (Rodrigo et al., 2001). At the same time, this technology induces sublethal damage in bacteria, which causes a significant delay in their ability to grow and spoil the product. However, PEF can be only applied to liquid products. It was demonstrated that 35 pulses in a electric field of about 35 kV/cm applied to fresh orange juice reduced the microbial growth in 5 log unit cfu/g without modifing the nutrient composition or the sensory properties of the product. Additionally, the shelf-life of the orange juice processed with PEF was extended to 14 days, meanwhile the non-treated juice was not acceptable after 4 days of storage. However, to prevent spoilage of orange-carrot juice, it would be necessary to combine an efficient PEF treatment with chilling temperatures during the distribution and storage periods and to guarantee low initial concentrations of contaminating bacteria in fresh-squeezed juice (Selma et al., 2004).

4.2 High hydrostatic pressure The application of high hydrostatic pressure to processing food products consists of a pressure treatment in the range of 4000–9000 atm (Barbosa-Cánovas et al., 1998). The high hydrostatic pressure is used to inactivate microbial growth as well as certain enzymes to prolong the shelf-life of the food products, although the microbial inactivation will depend on the pH, food composition, osmotic pressure and the temperature of the environment. It is known that Gram-negative bacteria are inhibited at lower pressure than Gram-positive bacteria. The inhibition of microbial spores can be managed by combining the high pressure treatment with chilling temperatures. The production of non-pasteurized citric juices by using high hydrostatic pressure makes it possible to obtain fruit juices with a similar flavour to the fresh juice without vitamin C losses and a shelf-life of about 17 months (Farr, 1990). One of the most important benefits of this emerging technique is that the plastic package of the product is filled with the fruit juice and then subjected to a pressure of about 4000–6000 atm for 1–30 min. Therefore it is easy to avoid contamination of the product after packaging (Barbosa-Cánovas et al., 1998).

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5 Conclusions Recommendations for a balanced diet include the consumption of fresh fruit and vegetables as a very important part of the diet of people around the world. Consumers increasingly perceive fresh food as healthier than heat-treated food, which motivates a general search for new minimally fresh processed fruit and vegetables. However, despite the benefits derived from eating fresh foods, safety is still an issue of concern due to a wide range of documented cases of contaminated fresh fruits and vegetables, as well as unpasterized fruits jucies, which have caused large outbreaks of microbial infections. Minimally fresh processed fruit and vegetables are very perishable products, highly susceptible to deterioration and the minimal processing reduced shelf-life leading to additional quality losses. Therefore, the derived processed produces are in fact more sensitive to disorders than the original ones. Many factors affect the shelf-life and microbial quality of raw prepared fruit and vegetables and they include good agricultural practices, good hygienic practices during harvesting and handling, quality of washing water, processing technologies, packaging methods and materials and processing, storage, transportation, distribution and retail sale temperatures. Common practices consist of the protection of the produce from damage caused by poor handling or machinery, foreign body contamination and/or pest infestation. The traditional processing of this kind of product usually consists of a sequence of simple operations (trimming, peeling, cutting, washing/disinfection, drying and packaging) and generally, the extension of the shelf-life depends on a combination of correct chilling treatment throughout the entire chill chain, dips in antibrowning solution, optimal MAP conditions and good manufacturing and handling practices. However, the minimal fresh processing industry is currently seeking alternative or secondary technologies to maintain most of the fresh attributes, storage stability and, above all, the safety of fresh processed fruit and vegetables while extending their shelf-life. Many of the conventional preservation methods that assure the safety of the plant food products such as high temperatures are not applicable to minimally processed fruit and vegetables and only a few traditional techniques such as chemical treatments, chilling and MAP can be used. For these reasons, the development of emerging processing techniques and the application of the hurdle concept represent a good solution for this type of food industry. According to the hurdle theory, preservation treatments combined at lower individual intensities have additive or even synergistic antimicrobial effects, while their impact on the sensory and nutritive properties of the food is minimized. Therefore, preservation techniques are becoming milder in response to demands of consumers for higher quality, more convenient foods that are less heavily processed and preserved and less reliant on chemical preservatives. Many non-conventional methods are now being investigated and some of the most important technologies that can be applied to preserve fresh processed plant foods are hydrogen peroxide, acidic electrolysed water, chlorine dioxide, ozone, mild heat water treatments, UV-C radiation, biocontrol, novel MAP, genetic engineering technologies, pulsed electric fields and high hydrostatic pressure. All these techniques help the minimal fresh processing industry to develop safer and healthier plant products to fulfill the current consumer demand.

References 703

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Minimal Processing of Ready Meals S J James and C James University of Bristol, Food Refrigeration and Process Engineering Research Centre, Langford, North Somerset, UK

There is a very large and growing, domestic and catering market for chilled and frozen ready meals. A chilled or frozen ready meal will ultimately be heated in a conventional domestic or microwave oven. The consumer expects a meal with an eating quality at least as good as if they had cooked and prepared it themselves. The most important quality consideration is that all the components that form the complete ready meal have been processed in a manner that destroys non-sporing pathogenic microorganisms. Most systems for the production of consumer packs of chilled or frozen ready meals cool the cooked meal before it is sealed. To avoid contamination of the cooked product, some systems hot-fill the packs and seal them before cooling and there are a few systems where the raw ingredients are assembled, sealed in the pack and the pack cooked and cooled. Many heating and cooling systems are used in the production process. However, despite the rapid development and ever increasing market for chilled, frozen and ambient stable ready meals most are processed in very traditional manners. With current market drivers, versatile equipment that allows small batches of product to be produced are more cost-effective than continuous but less versatile processes.

1 Introduction There is no strict definition of ‘ready meals’; the term is generally applied to a pack containing a full meal, or main course, with a meat, fish or a totally vegetable base that requires little further preparation and cooking. Ready meals are supplied to the consumer in five main forms: dried, canned, ambient stable, chilled and frozen. Ranges of dried meals, typically with an Indian or Chinese image, such as curries, chow mein, etc. appeared in the 1950s and still have a place in the market. Again a range of canned meals such as chilli-con-carne, spaghetti bolognaise, chicken supreme, etc. have an established market and some are available in duel cans with cooked rice in the second to make a complete meal. There are clear distribution advantages with ambient stable ready meals and a number of complete Italian dishes (pasta plus a sauce) are available. However, the domestic ready meal market is dominated by the chilled and frozen sector and it is their production that is primarily covered in this chapter. The chilled ready meal market in the UK in 2003 was stated to be worth £1433 m and growing at a rate of Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

27

718 Minimal Processing of Ready Meals

13 per cent per year (Anon, 2005). Traditionally chilled and frozen ready meals are packaged after production and the individual packs sold from refrigerated display cabinets. In the 1990s multipacks with a complete Indian, Chinese, Italian, etc. meal appeared and there is a growing trend for retailers to provide a serve-over service. Large trays of different dishes are kept in chilled display cabinets and the consumer makes a selection, which is transferred to individual packs that are sealed in the store before sale. There are also specific production processes such as cook-chill, cook-freeze and sous-vide, for ready meals that are supplied to the catering sector. These processes are also covered. A chilled or frozen ready meal will ultimately be heated in a conventional domestic or microwave oven. The consumer expects a meal with an eating quality at least as good as if they had cooked and prepared it themselves. Eating quality is defined by appearance, aroma, flavour and texture. James et al. (1987) described the engineering problems in ready meal production. In a processing operation the meat, fish etc. either have to be pre-cooked to varying extents before assembly and the cooking completed in the pack or cooked individually. Cooling the cooked product cannot be instantaneous, so some further cooking occurs during the cooling process. The need to avoid surface freezing, which can impair the quality of chilled meals, limits the temperature of the cooling medium that can be used and therefore the maximum rate of cooling that can be achieved. When using microwaves localized high and low temperatures can occur during re-heating (Swain and James, 2005) and the production system must allow for this. The most important quality consideration is that all the components that form the complete ready meal have been processed in a manner that destroys non-sporing pathogenic microorganisms. In cooking, a minimum temperature of 70°C has to be achieved to satisfy food safety requirements. However, many companies heat to over 80°C for a few minutes to satisfy organoleptic requirements. The shelf-life of a chilled ready meal is typically 4–10 days and is limited by the growth of food spoilage bacteria. The processing system is therefore required to produce a hermetically sealed chilled ready meal. Once the meal has left the factory the extent of bacterial growth is primarily a function of the product time/temperature history. The storage life of frozen ready meals is governed by the rate of rancidity development and aroma changes in the product. These changes are primarily a function of the raw materials used in the recipe, the packaging system and the storage temperature.

2 Design of total system Most systems for the production of consumer packs of chilled or frozen ready meals cool the cooked meal before it is sealed. A cold-fill system will contain most, if not all, the following operations but not necessarily carried out in the same order: 1 2 3 4

Delivery and storage of raw ingredients Preparation of ingredients Mixing of prepared ingredients Bulk cooking of ingredients

Design of total system 719

5 6 7 8 9 10 11 12 13

Bulk cooling Mixing of cooked components and addition of gravy/sauce Deposition of cooked components/mixtures into consumer packs Addition of hot or cooled sauce/gravy to pack Pasteurization of packs for chilled distribution Sealing of packs Inserting packs in outers Cooling/freezing of packs and Assembly of pallets and storage before distribution.

To avoid contamination of the cooked product, some systems hot-fill the packs and seal them before cooling and there are a few systems where the raw ingredients are assembled, sealed in the pack and the pack cooked and cooled. The constituents of these multicomponent products require different cooking methods, times and temperatures, therefore most production systems are a mixture of bulk and individual item or pack processing operations, which can be grouped under several types.

2.1 Solid/liquid mixtures Two main types of system are used: open top vessels and closed pressurized vessels. The open top vessel is used in many small and medium scale operations. The advantage of such systems being low cost, ease of cleaning and versatility. As they are open topped they allow components to be conveniently added at different times through the cooking process. Disadvantages of using this system are that considerable temperature stratification occurs, with differences up to 50°C (Burfoot et al., 1987). The vessels are also energy inefficient with up to 15 per cent of heat lost to the environment via evaporation from the surface. Cooling is a major problem and often the vessel is emptied into a large bin that is allowed to cool in ambient temperatures or in a refrigerated room. Temperatures as high as 65°C have been recorded in the centre of bins after 16 h of ‘cooling’ and during this period the product continues to cook with a consequent deterioration in texture and flavour (James, 1990). Spore-forming bacteria will survive these cooking operations and proliferate at temperatures between 10 and 50°C. Closed pressurized vessels are water and sometimes vacuum cooled. Heating is usually via steam jackets. Direct steam injection into the product is more efficient and reduces processing time. The design and operation of pressurized systems is dependent on:

• The optimum time/temperature relationship required during cooking and cooling for the range of products processed.

• Overall heat transfer coefficients and their variation with temperature, agitator speed and design, dimensions of vessel and product characteristics. • Optimum agitator design for each product. Soups, sauces and gravies containing pieces of meat can be heated continuously in pipe or spiral heat exchangers, controlled by scraped surface heat exchangers, ohmic heaters

720 Minimal Processing of Ready Meals

or microwaves. The high rates of heating obtained with all three methods may not meet the requirements of the product. For example, although low quality pieces of meat can be heated rapidly, a long holding time is required to gelatinize the collagen responsible for toughness. Since other meal components could suffer quality loss if heated for such periods, it may be necessary to pre-cook the meat before it is introduced into the continuous system. An ohmic heater can raise the average temperature of the product to 140°C in minutes, but if over-cooking is to be avoided in sensitive products a similarly rapid rate of temperature reduction is required. The simplest method of cooling the foodstuff is passing a cooled liquid over the surface of the pipe. The cooling of solid particles, which may be as large as 25 mm cubes, will always be conduction controlled (Burfoot and Self, 1988). As the outer layer of liquid phase cools, its viscosity will increase to a point where it is no longer economical to pump and will limit the rate of cooling that can be achieved. Other methods of cooling are the direct addition of liquid carbon dioxide into the pipeline, separating the solid and liquid phase and cooling them independently before re-combining and also vacuum cooling.

2.2 Solid foods Solid components, i.e. portions, cubes or pieces of chicken, red meat, fish or vegetables are processed on large trays or individual packs. Cookers are designed to use either forced air or steam to transfer heat into the product. Most forced air cookers are simple rectangular chambers into which racks of products are heated by circulated air. Direct steam addition or water sprays control relative humidity. In small products, heat transfer into the surface is the rate-controlling factor and this is a direct function of the air velocity over the product. In many cooking operations low temperatures and high humidities are used to minimize weight loss from large products. Continuous cookers such as the ECHO-system from Ireland are being introduced (Albers, 1997). This utilizes high speed collimated air streams to penetrate the resistant boundary layer of air that surrounds the food and is a barrier to efficient heat transfer. It is claimed that roasting, baking, steaming, broiling in one facility is possible due to independent control over temperature, air and humidity both above and below the product. To avoid excessive handling, racks of cooked product are cooled in conventional chill rooms or specially designed blast cooling units. In some of the cooking cabinets cold water sprays are installed and the initial cooling of the cooked product is a mixture of spray and evaporative cooling. In air cooling the temperature of the air is usually above
2°C to avoid surface freezing of the food. Pressure cookers achieve higher surface heat transfer coefficients and, consequently, shorter cooking times. The majority of existing systems operate at 100 kPa or less, which considerably limits the vapour temperature that can be achieved.

2.3 Consumer packs Consumer packs are either cooled to 0–2°C for chilled, or
18°C for frozen. Freezing can be achieved in 1–2 h using a blast freezer, a manual or automatic plate system or

Cook-chill 721

a spiral freezer, or less than 1 h in a liquid nitrogen tunnel. Despite the relatively poor conductivity of the food and its packaging, the large temperature difference between the food and the freezing media ensures rapid heat removal. In all three systems there will only be a brief period between 1 and 50°C (5–30 min) where microorganisms can multiply and the extent of growth will be very small. There will be no increase during subsequent storage.

3 Cook-chill Cook-chill is a catering system based on the full cooking of food followed by fast chilling and storage in controlled low temperature conditions above freezing point (0–⫹3°C) and subsequent thorough reheating close to the consumer before consumption. Cook-chill products are stored above their freezing point, so it is essential to handle these products in accordance with the guidelines (Anon, 1989) to minimize the growth of any microorganisms that may be present. A product can be stored for up to 5 days, including the day of cooking, but no longer as food quality diminishes. The system of cook-chill is commonly used by hospitals, schools and airline catering companies and is satisfactory, provided that certain basic principles are adopted and followed at all times (Table 27.1). Any abuse of temperature control in the preparation, storage, distribution and reheating may result in public health hazards. Since pathogenic organisms can grow freely at temperatures above 10°C, this temperature should be regarded as the critical safety limit in the storage and distribution of precooked chilled foods. Pre-planning is an essential factor in achieving successful cook-chill operations. It is strongly recommended that the following in particular should be considered in detail when planning a system: 1 Suitability of existing buildings for conversion 2 Food to be produced 3 Special equipment required including bulk cooking, refrigeration and reheating equipment Table 27.1 Requirements in cook-chill guidelines Stage in process

Requirements

Initial cooking Time for chilling to begin after initial cooking cycle Chilling time

Minimum ⫹70°C for not less than 2 minutes 30 minutes 1.5 hours to ⫹3°C 2.5 hours or sooner for larger meats Meat joints are recommended not to exceed 3 kg or 100 mm thick 0°C (32°F) to ⫹3°C (37°F) 0.2°C tolerance 5 days ⫹5–⫹10°C. Consume within 24 hours Above ⫹10°C destroy Minimum ⫹70°C for not less than 2 minutes

Storage temperature Shelf-life at storage temperature Critical temperature during storage Re-heating temperature

722 Minimal Processing of Ready Meals

4 Design of central production unit and satellite unit; food distribution and 5 Hazard analysis; quality assurance and staff training. In order to achieve the recommended chilling process a chiller must be used which has a performance specification showing it is capable of reducing the temperature of a 50 mm layer of food from ⫹70 to ⫹3°C or below in a period not exceeding 90 minutes when fully loaded. With certain foods it may not be possible to achieve this temperature reduction on a 50 mm layer of food, in which case the depth of food should be reduced to allow the required performance to be achieved. Three typical methods of chilling are: 1 The use of clean high velocity recirculating air at low temperatures in mechanical apparatus 2 The use of cryogenic apparatus involving the use of non-oxidizing gas at low temperatures and 3 The immersion of packed products in a safe and suitable refrigerated liquid. Evans et al. (1996) provide design graphs for predicting the cooling times of cooked products in air chilling systems and Ketteringham and James (1999) in immersion systems.

4 Cook-freeze Cook-freeze is a process by which hot or cooked food is immediately frozen for eventual distribution in the frozen state. These techniques are particularly applicable to the production of prepared and pre-cooked meals, as well as individual items such as meat pies and pasties. This is a well-established process having been first used in the USA in 1945, primarily for the feeding of airline passengers. Advantages of the cook-freeze process are obtained through the centralized production of meals, which allows flowline-processing operations to be used. Centralized meal production also allows strict monitoring of hygiene and quality standards. Another advantage of the system is that when it is applied to prepared meals, extensive variation of the menu can be arranged, this allows for different tastes and needs to be catered for ensuring a versatile product range. Cook-freeze is defined as a catering system based on full cooking followed by fast freezing, storage at controlled low temperature conditions well below freezing point (
18°C or below) and subsequent thorough reheating close to the consumer before prompt consumption. The requirements for cook-freeze processing are given in Table 27.2. The frozen product can be stored for at least 8 weeks without significant loss of quality, after this time products that contain meats with a high fat content may start to develop rancidity. Products with little or no fat content may be stored for longer periods without quality loss. A well-organized system of product identification marking along with production date, expiration date and batch identification should be operated.

Sous-vide 723

Table 27.2 Requirements of cook-freeze guidelines Stage in process

Requirements

Initial cooking Time for freezing to begin after initial cooking cycle Freezing time Storage temperature Shelf-life at storage temperature

Minimum ⫹70°C for not less than 2 minutes 30 minutes 1.5 hours to
5°C at centre
18°C In general up to 8 weeks without significant changes Partly/completely thawed food not to be refrozen. Food thawed at unknown temperatures not to be consumed Minimum ⫹70°C for not less than 2 minutes

Critical temperature during storage

Re-heating temperature

5 Sous-vide Sous-vide may be considered to be a specific type of cook-chill. An exact definition is difficult to find and the term can be used to describe quite different processes. The main aspect in common with all processes described as ‘sous-vide’ is vacuum packing and par-cooking. Two main processes are often described as sous-vide: 1 Products are par-cooked, vacuum packed and then subjected to a heavy in-pack pasteurization and 2 Products are vacuum packed when raw and fully or partially cooked in the pack. The products are then refrigerated until use. The products are then either warmed or fully reheated to complete the cooking. The process is claimed to provide higher quality than conventional cook-chill by preventing evaporative loss of flavour volatiles. There may also be a reduction in loss of nutrients through oxidation and leaching. Oxidative rancidity and aerobic spoilage are reduced and there is a reduced risk from aerobic pathogens such as Listeria monocytogenes and Salmonella typhirium, however, Clostridium botulinum is a potential hazard. For this reason there is great concern regarding sous-vide parcooking processes and many recommendations are that heavy pasteurization is carried out instead. UK recommendations for sous-vide production are given in Table 27.3. The sous-vide method involves: 1 The pre-packaging, in specialized plastic bags or pouches, of prepared raw or par-cooked foods 2 Sealing the prepared foods in the bags (or pouches) under vacuum to remove the air surrounding the food 3 Cooking the vacuum-sealed food for immediate consumption or to controlled pasteurization temperatures 4 Rapidly chilling the pasteurized food product

724 Minimal Processing of Ready Meals

Table 27.3 Recommendations for sous-vide production Stage in process

Requirements

Initial cooking Portioning Chilling time Storage temperature Shelf-life at storage temperature

Minimum ⫹70°C, time not specified ⬍10°C within 30 minutes 1.5 hours to ⫹3°C
18°C In general up to 8 weeks without significant changes ⫹5–⫹10°C. Consume within 12 hours Above ⫹10°C destroy For short periods insulated containers suffice. For longer periods, refrigerated transport is required Minimum ⫹70°C

Critical temperature during storage Distribution Re-heating temperature

5 Storing the cooked, chilled product under controlled conditions until required recommended 5 days maximum under the present Department of Health CookChill Guide lines) and 6 Reheating (regenerating) the food product and serving. It is claimed that the method of cooking stabilizes and maximizes the quality of stored foods, retaining (within time and temperature limitations) the special aroma, flavour, texture and nutritional contents of the food. Due to the impermeable nature of the pouch, nothing escapes from within; natural flavours are not lost or diminished to the same extent as in conventional cooking so flavour enhancers and additives (including salt) may be reduced or eliminated altogether. Armstrong and McIlveen (2000) reported that sous-vide processed bolognaise meat sauce and chicken tikka masala largely retained their level of sensory quality and acceptance throughout the 40 days storage at 1.5°C. However, Church and Parsons (2000) in their studies on sous-vide processing of chicken breast and of sliced potatoes in cream reported that, ‘in some products, the heat treatments necessary to ensure microbiological safety cause an appreciable loss of sensory quality’. Creed (1995) stated that, ‘Few data are available which are consistent and provide quantitative scientific evidence for this method’s undoubted gastronomic appeal. Similarly, fewer data are available to support the supposed superiority in retention of vitamins.’ The system can be used to rationalize food production in a central kitchen, using proven recipes, unskilled operatives to prepare raw food items and insert them into pouches, vacuumize and cool under the supervision of a small number of professionally qualified and skilled chefs and technicians. Dishes can be prepared in advance of need, dated to ensure stock rotation, kept at low chilled temperatures and reheated (regenerated) at the point of sale by trained staff. Only small regeneration kitchens (satellite kitchens) are necessary to reheat the food, with minimal equipment and less skilled manpower than a conventional restaurant. Once a standard recipe has been developed with ingredients, cooking temperatures and times established and satisfactory microbiological levels achieved, the product can be

Novel and alternative processing options 725

reproduced to the same standard in quantity without measurable variation. Random microbial testing should be carried out at regular intervals to ensure quality standards are maintained. Capital expenditure can be recovered through the rationalization of manpower, output of production, portion yields, cost control and reduction of waste/spoilage.

6 Novel and alternative processing options Despite the rapid growth in the ready meal market and the ever-growing range of products available, most of current production uses the traditional methods already described. However, there has been interest in using alternative pasteurization and sterilization techniques, such as microwave and ohmic heating, hydrostatic pressure, high voltage pulses and surface decontamination treatments. These can either be used on the raw materials to allow minimal processing during the cooking or applied to the complete meal. Aseptic processing techniques are used to produce a range of ready meals, which can be either chilled or sold as an ambient stable product. In addition, irradiation of raw materials or total meals is a well-researched technology that is increasingly but slowly gaining acceptance.

6.1 Microwave heating Most convenience meals are reheated rather than fully cooked in the home or at a catering establishment. The majority of these products are designed for rapid reheating in a domestic or commercial microwave oven as well as in conventional ovens (Swain and James, 2005). Investigations carried out since the late 1980s (Burfoot et al., 1990; James, 1993) have revealed considerable variability in the ability of different models and types of domestic microwave oven to reheat chilled convenience meals. They have also revealed a large degree of non-repeatability during heating. Some of the reasons for the variability and non-repeatability have been identified, while others are less defined. The development of a protocol to produce microwave reheating instructions for ready meals needs to take into account all the factors that are currently undefined as well as those that can be quantified (James et al., 2002; Swain et al., 2004). Although much attention has been focused on microwave reheating of ready meals, the industrial use of microwaves in their production has received less. Current chilled meal production produces packs whose chilled shelf-life is generally limited to 5–7 days as a result of contamination of the components during assembly and/or from bacteria, which may be airborne or present on the tray. Meals are therefore made in small production runs, which incur high costs from substantial labour and cleaning requirements and require well-controlled refrigerated distribution systems, which are expensive to set up and maintain. If the chilled shelf-life could be increased, longer production runs could be used.

726 Minimal Processing of Ready Meals

Table 27.4 Advantages and disadvantages of different pasteurization systems Method

Advantages

Disadvantages

Forced air convection

Inexpensive and technology available

Product may ‘burn’, discolour or overcook. Long heating time 120 min at 115°C

Pressurized steam

Technology available

Batch system unless expensive equipment is used. Condensate on product in unsealed packs. Intermediate heating time of ⬃22 min at 115°C can affect delicate foods

Microwave

Fast ⬇ 160 s at 6 kW

High capital cost, ‘burn’, discolouring and uneven heating

It is believed that heating sealed products containing meat to a temperature between 80 and 85°C, holding at that temperature for approximately 3 minutes and then rapidly cooling will achieve a safe shelf-life of 3 weeks. Initial investigations (Burfoot et al., 1988) studied the feasibility of using conventional conduction based heating systems, either forced air or steam under pressure, or an experimental microwave pilot plant. The relative advantages and disadvantages of the three systems are given in Table 27.4. The very long processing times, typically 2 hours for forced air, would necessitate very large plants and produce substantial over-cooking in most products. In small throughput operations with reasonably robust products the 22-minute pasteurization time achieved with steam would produce a feasible operation. However, large batch or automatic pressure systems are expensive and the quality of delicate foods could suffer. Only the microwave system seemed to offer the potential to produce a very rapid increase in product temperature and achieve pasteurization conditions without quality problems. Only two microwave frequency bands, 2450 (50) MHz and 896 (l0) MHz, are currently available for commercial use in Europe. An investigation was therefore carried out into the feasibility of achieving pasteurization temperatures in chilled meals using a continuous 6 kW multi-mode oven operating at 2450 MHz or a continuous singlemode oven operating at 7 kW and 896 MHz. Tests were also carried out in a domestic 0.6 kW multi-mode oven operating at 2450 MHz which might offer a low-cost alternative in a robotic serviced small throughput operation. Table 27.5 contains data on some of the treatment combinations used and the resulting highest, lowest and average temperatures measured in the packs. Mean temperatures above 80°C were achieved when operating the domestic microwave oven at 0.6 kW for 240 or 300 s. However, the minimum temperatures of 42 and 48°C are only marginally above that required for the maximum growth of Salmonella and Clostridia perfringens. The large standard deviations of temperature illustrate the difficulty of measuring temperatures reproducibly in this type of multicomponent meal in which the relative position of the strands of pasta varies from pack to pack. All experiments on the 2450 MHz multi-mode tunnel produced mean temperatures higher than 77.5°C, but the minimum temperature was as low as 50°C, which is

Novel and alternative processing options 727

Table 27.5 The mean, lowest and highest temperatures measured in packs of spaghetti bolognaise after heating in three microwave plants (standard deviation in brackets) Microwave conditions Power (kW) 2450 MHz domestic oven 0.36 0.6 0.6 2450 MHz continuous tunnel 6.0 6.0 6.0 6.0 896 MHz continuous tunnel 7.0 7.0

Time (s)

Film cover

Temperature (°C) Mean

Lowest

Highest

300 240 300

Pierced Pierced Pierced

77.4 (18.4) 87.0 (11.1) 90.4 (8.3)

32.4 41.7 47.6

96.0 97.2 97.3

125 125 158 158

Pierced None Pierced None

77.7 (9.5) 77.5 (10.7) 86.5 (8.2) 82.1 (9.1)

55.0 50.1 62.3 58.8

91.6 94.3 98.0 94.7

144 144

Pierced None

90.2 (3.8) 90.8 (5.5)

80.3 65.9

95.9 97.2

clearly insufficient for pasteurization. The trailing edge of the top surface of the products heated for 125 s showed very slight drying, whereas products heated for 158 s had a very dry, almost crusty, region on the upper leading and trailing edges. Mean temperatures above 80°C were obtained in both pierced and uncovered packs in the 896 MHz tunnel and the lowest temperatures measured during the six tests with pierced packs were all between 80.3 and 85.5°C. Minimum temperatures below 80°C (75.2 and 65.9°C) were measured in two of the tests with uncovered packs. Temperatures in pierced packs were slightly more uniform than those measured in uncovered packs. After heating in the 2450 MHz tunnel, the minimum temperatures were also higher in the covered packs. The presence of the covering film probably reduced heat losses and maintained a warm, moist environment above and between the strands of pasta thereby reducing temperature variations within the product. In Europe, at least five manufacturers have developed microwave pasteurization systems: Alfastar (Sweden), Berstorff (Germany), Omac (Italy), APV-Baker (UK) and Microwave Systems (UK). The Alfastar system differs from the others in that the product is immersed in water during preheating, microwave (2450 MHz) and cooling treatments. Alfastar AB claimed to halve processing times compared to conventional systems and increase product shelf-life to 25–90 days. A plant running at 400 kg/h was supplying test products for the Swedish market in the late 1980s (Burfoot et al., 1988). With the Berstorff system, product was hot filled into trays, sealed and then preheated in air followed by microwave heating and further holding in air. The temperature at the surface of foods can increase rapidly to the boiling point during microwave heating and the vapour generated leads to a pressure rise and consequent distortion or bursting of the packaging. Berstorff overcome this problem by hot filling the product at 55°C to reduce the amount of microwave energy needed to pasteurize the food. At least two microwave pasteurization systems from Berstorff operated at full production

728 Minimal Processing of Ready Meals

rates (1000 kg/h) in Europe. One particular system used almost 100 magnetrons to operate at an overall power of 120 kW. Omac described a pressurized microwave system, which could be used for pasteurization or steri1ization of prepared meals. Omac claimed such equipment could be designed to operate up to 250 kPa pressure, utilize 250 kW of power and process 2000 kg/h of product. One commercial unit sterilized 750 kg/h of Italian style prepared meals producing products with an ambient stable life of 9 months (Mullen, 1996). The APV system differed from the first three in that it (i) operates at 896 MHz as opposed to 2450 MHz; (ii) uses high power generators; and (iii) incorporates metallic structures within the cavity to improve the distribution of the electromagnetic field and consequently the product temperature profile. The use of the lower frequency was claimed almost to double the energy efficiency of the microwave process leading to an efficiency of 85 per cent. In the Microwave Systems plant, the meals are pressurized and passed through a pressure vessel where microwave energy is applied first from a 915 MHz generator and then from 2450 MHz units. The use of a pressurized system resists the bursting of packs, although they are allowed to flex slightly during heating and cooling. Also by using two frequencies, they claimed to achieve more uniform heating. Few details are available about the process but it was operated in Europe producing 390 kg/h of 175 g packs. Despite these developments and the production of successful microwave pasteurization and sterilization systems for ready meals their penetration into the industry has been marginal.

6.2 Ohmic heating The ‘ohmic heater’ was originally developed by EA Technology at Capenhurst, UK and the theory of its operation is described by Sastry (1994) and Fryer (1996). In 1984, APV Baker secured a licence for the heating system and then developed a commercial process. Commercial scale systems were made with power outputs of 75 and 300 kW, corresponding to product capacities about 750 and 3000 kg/h. In the system, food passes from a product pump into a vertical or near-vertical pipe containing a series of electrodes. Sufficient pressure is maintained in the column to ensure that the material does not boil; this can be up to 400 kPa for sterilization at 140°C. Each electrode housing is machined from PTFE and contains a cylindrical cantilever electrode (supported only at one end) across the tube. Alternating current from a 3-phase supply flows between the electrodes and thus through the food. The connecting tubes are made from stainless steel lined with insulating plastic and are made of different lengths so that the section between each pair of electrodes is always of similar impedance. The tubes increase in length from inlet to outlet, reflecting the increase in electrical conductivity with temperature. From the heater, food passes to a holding tube, in which it is held for long enough to ensure sterility, prior to cooling and aseptic packaging. Cooling may be provided solely by water flowing on the outside of the cooling tube, or precooled liquid can be pumped into the tube after the holding section to allow an increase in the heating rate.

Novel and alternative processing options 729

The process enables the sterilization of foods of high solids fraction, up to about 60 per cent, heating rates in the region of l°C/s. Large particles, up to 25 mm in diameter, can be processed using the technique. It is now in commercial use in the UK, the USA, Europe and Japan and has been further developed for a number of specific applications for the processing of solid-liquid mixtures:

• Aseptic processing of high added value ready prepared meals for storage and distribution at ambient temperature • Pasteurization of particulate food products for hot-filling • Preheating of food products prior to in-can sterilization and • Hygienic production of high added value ready prepared meals for storage and distribution at chilled temperatures.

6.3 Hydrostatic processing Hydrostatic pressure technology is a novel non-thermal processing technology where foods at room temperature are subjected to high hydrostatic pressure, typically 100–600 MPa. In a similar way to high temperature inactivation of microorganisms, the high pressure will inactivate vegetative microorganisms, spores and enzymes thus extending the shelf-life of the treated food. The European, Japanese and US food industries invested a great deal of research and development effort into this technology in the 1990s. An increasing number of products produced using this technology are now entering the food market. These include jams, fruit dressings, yoghurt, fruit juices, dairy products and non-frozen tropical fruits. It has an obvious role in treating both the raw material used in ready meals and the total product. However, the authors have not located any information on its commercial use in ready meal production.

6.4 Surface decontamination techniques The deep musculature of animals and fish is essentially sterile at the time of death and the same can be said for fruit and vegetables. The main bacterial load is present on the surface of the raw material and this surface load is further increased during any handling or processing operation. There is considerable interest in methods of substantially reducing if not completely eliminating pathogens and spoilage organisms on raw materials used in ready meals. If this was achieved then far less severe processing methods would be required and a product with improved eating quality could be safely produced. Many chemical and physical methods of surface decontamination have been researched and James and James (1997) have reviewed them in detail. In general physical methods, especially those reliant on rapid surface heating are preferred over the application of chemicals. However, none are currently know to be used in the pre-treatment of raw materials for ready meals.

730 Minimal Processing of Ready Meals

6.5 Aseptic processing Characteristically, in an aseptic process, the food is subjected to rapid heating then held for a sufficient time to achieve commercial sterility and then cooled before filling a previously decontaminated container in an aseptic filling zone. The resulting products have a long storage life at ambient temperature. Some raw materials for ready meals, such as tomato paste, are routinely produced in large (200 kg) aseptic packs. There is also a niche domestic market for ambient stable ready meals for use on camping and other outside activities. In addition, a market exists for a small number of, usually pasta based, ambient stable ready meal packs. However, in general chilled and frozen meals have a higher quality image.

6.6 Irradiation Over six decades of research and development food irradiation has been shown to be an effective and safe method of food preservation. Consumers appear to find the quality of irradiated chilled meals to be acceptable. Stevenson et al. (1995) carried out trials where 170 consumers assessed the sensory quality of a chilled irradiated (2 kGy) and non-irradiated ready meal, consisting of beef and gravy, Yorkshire pudding, carrot, broccoli and roast potato 4 days after treatment. The irradiated meal was moderately to very acceptable and was not significantly different to the non-irradiated meal. The beef and gravy component of the meal was most liked by consumers. Loaharanu (1995) lists a whole range of food, including spices, spinach, poultry meat, yams, Camembert cheese, potatoes, fermented pork sausage and fruits that have been successfully irradiated and marketed in different parts of the world. Increasingly, some of the ingredients for ready meals, especially spices, have been irradiated before use. Irradiated ready meals are regularly supplied to astronauts, armed forces and patients with poor immune systems. However, concerns about consumer resistance have kept them away from the chilled and frozen ready meal market. Studies have shown that irradiation can substantially increase the safety of chilled meals. Foley et al. (2001) contaminated a steak, gravy and mashed potato product with L. monocytogenes, then irradiated and stored it at 4°C for 3 weeks. Listeria was not recovered from any sample at any time point, even after selective enrichment, for product treated at 5.7 kGy. Furthermore, sensory tests revealed that the irradiated meals were no less acceptable than non-irradiated meals. Experiments have been carried out to combine medium dose gamma irradiation with sous-vide cooking (Farkas et al., 2002). This increased considerably the microbiological safety and the keeping quality of the meals studied. However, approximately 40 per cent loss of thiamine content occurred as an effect of combination treatments and adverse sensorial effects could limit the feasible radiation doses. Grant and Patterson (1995) reported that irradiation increased the heat sensitivity of L. monocytogenes. They suggested that, ‘As cook-chill products are intended to be reheated prior to consumption the results of the present study suggest that any L. monocytogenes present in a cook-chill product would be more easily killed during reheating if it were to be treated with a low dose of gamma radiation during manufacture’.

References 731

7 Conclusions Despite the rapid development and ever increasing market for chilled, frozen and ambient stable ready meals, most are processed in very traditional manners. Many large producers still operate with equipment that is basically scaled up versions of that found in traditional kitchens. Raw materials are still cooked and cooled in batch systems and the meal assembled by a combination of hand and automatic dosing systems. With current market drivers these processes are unlikely to change because they offer a high degree of versatility. The supermarkets and consumers demand an ever increasing and changing range of ready meals. Some meals are only in fashion for less than 6 months before they are replaced. In this situation, versatile equipment that allows small batches of product to be produced are more cost-effective than continuous but less versatile processes. There have been developments in continuous tunnel cookers that can be programmed automatically to optimize cooking conditions for different products. As the cost of human operators rises and their availability decreases then we may see the increasing automation of ready meal production.

References Albers D (1997) Preparing of cooked foods on a large scale. Fleischwirtschaft, 77 (7), 626–627. Anon (1989) Chilled and frozen. Guidelines on Cook-Chill and Cook-Freeze Catering Systems. London: Department of Health and Social Security, HMSO. Anon (2005) Welcome to Geest. http://www.geest.co.uk/ Armstrong GA, McIlveen H (2000) Effects of prolonged storage on the sensory quality and consumer acceptance of sous vide meat-based recipe dishes. Food Quality and Preference, 11 (5), 377–385. Burfoot D, Self KP (1988) Prediction of heating times for cubes of beef during water cooking. International Journal of Food Science Technology, 23, 247–257. Burfoot D, Griffin WJ, James SJ (1988) Microwave pasteurization of ready meals. Journal of Food Engineering, 8, 145–156. Burfoot D, Hayden R, Badran R (1987) Simulation of a pressure cook/water and vacuum cooling processing system. In Process Engineering in the Food Industry – Developments and Opportunities (Field RW, Howe JA, eds). London: Elsevier Applied Science, pp. 27–42. Burfoot D, James SJ, Foster AM, Self KP, Wilkins TJ, Phillips I (1990) Uniformity of reheating in domestic microwave ovens. In Process Engineering in the Food Industry: 2 Convenience Foods and Quality Assurance (Field RW, Howell JA, eds). London: Elsevier Science, pp. 33–44. Church IJ, Parsons AL (2000) The sensory quality of chicken and potato products prepared using cook-chill and sous-vide methods. International Journal of Food Science and Technology, 35 (2), 155–162. Creed PG (1995) The sensory and nutritional quality of sous vide foods. Food Control, 6 (1), 45–52.

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Evans J, Russell S, James SJ (1996) Chilling of recipe dish meals to meet cook-chill guidelines. International Journal of Refrigeration, 19, 79–86. Farkas J, Polyak-Feher K, Andrassy E, Meszaros L (2002) Improvement of microbiological safety of sous-vide meals by gamma radiation. Radiation Physics and Chemistry, 63 (3–6) 345–348. Foley DM, Reher E, Caporaso F, Trimboli S, Musherraf Z, Prakash A (2001) Elimination of Listeria monocytogenes and changes in physical and sensory qualities of prepared meal following gamma irradiation. Food Microbiology, 18 (2), 193–204. Fryer P (1996) Electrical resistance heating of food. In New Methods of Food Preservation (Gould GW, ed.). London: Blackie Academic and Professional, pp. 205–235. Grant IR, Patterson MF (1995) Combined effect of gamma-radiation and heating on the destruction of Listeria monocytogenes and Salmonella typhimurium in cook-chill roast beef and gravy. International Journal of Food Microbiology, 27 (2–3), 117–128. James C, James SJ (1997) Meat decontamination – the state of the art. MAFF Advanced Fellowship in Food Process Engineering, University of Bristol, EU concerted action programme CT94 1881. James C, Swain MVL, James SJ, Swain MS (2002) Development of a methodology for assessing the heating performance of a domestic microwave oven. International Journal of Food Science Technology, 37, 879–892. James SJ (1990) Cooling of products. In Proceedings of International Institute of Refrigeration Commissions B2, C2, D1, D2/3 – Dresden, Germany, Paper 30. James SJ (1993) Factors affecting the microwave heating of chilled foods. Food Science and Technology Today, 7 (1), 28–36. James SJ, Burfoot D, Bailey C (1987) The engineering aspects of ready meal production. In Process Engineering in the Food Industry – Developments and Opportunities (Field RW, Howell JA, eds). London: Elsevier Applied Science, pp. 43–58. Ketteringham L, James SJ (1999) Immersion chilling of trays of cooked products. Journal of Food Engineering, 40, 256–267. Loaharanu P (1995) Food irradiation: current status and future. In New Methods of Food Preservation (Gould GW, ed.). London: Blackie Academic and Professional, pp. 90–111. Mullen J (1996) Microwave processing. In New Methods of Food Preservation (Gould GW, ed.). London: Blackie Academic and Professional, pp. 112–134. Sastry SK (1994) Ohmic heating. In Minimal Processing of Foods and Process Optimisation – An Interface (Singh RP, Oliveira FAR, eds). London: CRC Press, pp. 17–34. Stevenson MH, Stewart EM, Mcateer NJ (1995) A consumer trial to assess the acceptability of an irradiated chilled ready meal. Radiation Physics and Chemistry, 46 (4–6), 785–788. Swain MJ, James SJ (2005) Factors that affect heating performance and the development of products for heating/cooking in domestic and commercial microwave ovens. In Microwave Processing of Foods (Regier M, ed.). Cambridge: Woodhead Publishing Ltd, pp. 221–242. Swain MVL, Russell SL, Clarke R, Swain MJ (2004) Development of food simulants for microwave oven testing. International Journal Food Science Technology, 39 (6), 623–630.

Modified Atmosphere Packaging for Minimally Processed Foods Robert W Lencki University of Guelph, Department of Food Science, Guelph, Canada

Since its beginnings in the 1960s, the design of modified atmosphere packaging (MAP) has progressed from a trial-and-error process to one that is firmly based on sound phenomenological models developed with a good understanding of the underlying mechanisms. This chapter examines the equations used to quantify the physical and metabolic properties of the foods commonly packaged under MA conditions. Next, models used to characterize the transport properties of polymeric and porous films are presented. The chapter concludes with a review of how the various models are combined to predict the behavior of both non-respiring and respiring food materials.

1 Introduction It has been known for over a century that the shelf-life of some foods can be extended by storage in gas environments different from that which is normally observed in the earth’s atmosphere (i.e. 78.08 vol% N2, 20.95 vol% O2). By 1938, a significant percentage of the meat exported from Australia and New Zealand to England was being shipped with added dry ice, which improved its quality compared to crushed ice shipments (Brody, 1995; Dixon and Kell, 1989). The storage of apples and pears in warehouses under reduced O2 and elevated CO2 conditions was also first exploited in the 1930s where it was shown to double produce shelf-life (Brody, 1995). Controlled atmosphere (CA) storage is the term commonly applied to these mobile or large-scale fixed enclosures in which gas concentrations are maintained via various mechanical systems. Starting in the 1960s, packaging materials also began to be used to alter the gas makeup surrounding smaller-scale consumer and restaurant-sized food servings (Brody, 1995). Known as modified atmosphere packaging (MAP), this technology has grown Emerging technologies for food processing ISBN: 0-12-676757-2

Copyright © 2005 Elsevier Ltd All rights of reproduction in any form reserved

28

734 Modified Atmosphere Packaging for Minimally Processed Foods

substantially since then and is now used for a wide variety of food products. In the last few decades, a number of books (Brody, 1989; Ooraikul and Stiles, 1991; Parry, 1993; Blakistone, 1998b) and review articles (Church and Parsons, 1995; Devlieghere et al., 2002; Mullan and McDowell, 2003) have appeared that summarize the progress made in the field of MAP. Numerous other articles have been published that focus on the MAP of a particular commodity group such as meat and poultry (Calvert, 1996; Blakistone, 1998a; Narasimha and Sachindra, 2002), fish (Stammen et al., 1990; Ashie et al., 1996; Davis, 1998; Sivertsvik et al., 2002), dairy and dried foods (Subramaniam, 1988), pasta and bakery products (Farber et al., 1993; Seiler, 1998; Guynot et al., 2003), or fruit and vegetables (Kader et al., 1989; Day, 1994; Lee et al., 1995, 1996; Calvert, 1996). Other reviews have examined various subjects relevant to MAP such as microbial safety (Hintlian and Hotchkiss, 1986; Farber, 1991; Reddy et al., 1992; Phillips, 1996) or packaging materials (Greengrass, 1998) and machinery (Hastings, 1998). The purpose of this review will be specifically to summarize work on the modelling and design of MAP systems. This topic has been previously reviewed for specific commodities like fruit and vegetables (Yam and Lee, 1995; Gorney, 2003). This work will take a broader view and examine in more detail the parameters that influence MAP performance along with the equations that quantify the various relevant phenomena. In the past, the MAP literature has been handicapped by a lack of rigour when presenting system parameters such as film permeability and produce respiration rate, making the comparison of results between publications difficult (Banks et al., 1995). When possible, MAP parameters will be presented using the currently recommended SI units (Banks et al., 1995). In addition, all model equations will be developed in a consistent, rigorous manner.

2 Properties of packaged food 2.1 Optimal gas atmospheres The gas atmosphere surrounding a particular food product can be altered to retard chemical and metabolic processes that are detrimental to product quality or to inhibit the growth of undesirable microbial populations. The optimal gas atmosphere inside the package depends on the composition of the contained food and the microbial contaminants that could potentially be present. Table 28.1 lists the gas compositions commonly used for a variety of MA packaged non-respiring foods. Oxidation is a reaction that generally has a negative impact on food quality. In particular, lipid oxidation leads to rancidity, so foods with elevated fat levels (e.g. nuts, snack foods, cheese and oily fish) are usually packaged with as much oxygen removed as possible. However, there are some exceptions. Retail red meat, which contains substantial amounts of fat, is usually packaged in elevated levels of O2 in order to maintain myoglobin’s consumer-appealing bright red colour (Livingston and Brown, 1981). Fresh meat has been packaged under low O2 conditions, but this technology has been restricted to bulk industrial packaging because of the unsightly blue appearance of the

Properties of packaged food 735

Table 28.1 Modified gas atmospheres commonly used for some non-respiring food products (Dodds, 1995) Commodity

N2 (vol%)

CO2 (vol%)

Oily fish White fish Crustaceans Red meat Poultry Bakery and pasta Cheese Coffee Potato chips

40–60

40–60 60 80–100 15–30 20–30 50–80 0–70

70–80 20–50 30–100 100 100

O2 (vol%)

40 70–85

Table 28.2 Optimal gas atmosphere for some fresh produce commonly packaged under MAP conditions (Gorney, 2003) Commodity

O2 (vol%)

CO2 (vol%)

Broccoli florets Shredded cabbage Carrot sticks Chopped romaine lettuce Diced onion Potato Apple Kiwifruit Strawberry Watermelon cubes

2–3 5–7.5 2–5 0.5–3 2–5 1–3 1 2–4 1–2 3–5

6–7 15 15–20 5–10 10–15 6–9 4–12 5–10 5–10 10

meat under these conditions (Taylor, 1985). Of course, O2 levels do not strongly affect the colour of cooked meat, so these products can be packaged under low O2 conditions (Gill, 1995). Many marine fish contain the osmoregulator trimethylamineoxide (TMAO). Spoilage organisms can use this compound as a terminal electron acceptor, reducing TMAO to the unpleasant-smelling compound trimethylamine. Marine fish should be packaged in O2 concentrations
30 per cent in order to inhibit this reaction (Boskou and Debevere, 1998). Post-harvest fresh fruit and vegetables continue to respire and thus packaged produce requires a constant supply of oxygen. Otherwise, anaerobic respiration will lead to the creation of off-flavour producing compounds such as ethanol and acetaldehyde. However, if MAP O2 concentrations are decreased below subatmospheric levels, but are kept above levels that would induce anaerobic respiration, the rate of aerobic respiration can be significantly reduced, leading to increased shelf-life (Lee et al., 1995). Table 28.2 provides suggested optimal O2 concentrations for various MAP produce. Some work has suggested that very high O2 levels can have a significant antimicrobial effect with packaged produce (Day, 1996), but more work needs to be done to determine if this packaging strategy truly has significant positive effects (Kader and Ben-Yehoshua, 2000).

736 Modified Atmosphere Packaging for Minimally Processed Foods

Because of its antimicrobial properties, CO2 is added to a wide variety of systems. Several mechanisms have been proposed to explain this inhibitory effect (Farber, 1991). Depending on the buffering capacity of the food, CO2 dissolution can reduce the pH of the aqueous phase, making it more difficult for some microbial species to grow (Daniels et al., 1985). CO2 can also penetrate into microbial cells, disrupting cell membrane function (Farber, 1991). Bicarbonate ion produced from CO2 hydration and ionization is also known to be inhibitory to some important cellular metabolic enzymes (Mathiew et al., 1986). However, it would appear that CO2 has, in most cases, only a moderately inhibitory effect on the aerobic respiration of fruit and vegetables (see section 2.3). Furthermore, care must be taken with some produce like lettuce, because elevated CO2 concentrations cause metabolic problems that lead to the formation of ‘brown stain’ (Brecht et al., 1973). Table 28.2 also provides suggested optimal CO2 concentrations for some MAP fruit and vegetable systems. For non-respiring foods, 100 per cent CO2 is rarely used and is often blended with cheaper N2 (see Table 28.1). Because of its high solubility in both the aqueous and fat phases of food, large amounts of CO2 can dissolve in the food, reducing the pressure in the void volume, which leads to the collapse of the package and subsequent product compression (Zhao et al., 1995). This phenomenon can be inhibited by using CO2/N2 mixtures. In low water activity foods that are much less susceptible to microbial spoilage, pure nitrogen is typically used. With snack food such as potato chips, the nitrogen-filled MAP can also cushion the fragile product. For less fragile foods like cooked meats, simple vacuum packaging can produce an optimal atmosphere (Zhao et al., 1995).

2.2 Gas solubility in foods In order to obtain the desired final optimal conditions, it is necessary to quantify how added gasses dissolve in the various food phases. For non-respiring foods, once the package has been flushed with a gas mixture and sealed, a readjustment of the various equilibria can occur, resulting in gas dissolution in the food product or transfer of previously dissolved gases from the food to the package void volume. Gas solubility is typically characterized using Henry’s law. For example, with CO2: l pCO2  K CO CCO 2

2

(1)

where pCO2 is the carbon dioxide partial pressure in the package void volume (Pa), l CCO is the concentration of carbon dioxide in the liquid phase (kmol/m3) and KCO 2 is 2 the Henry’s law constant (Pa m3/kmol). The KN2, KO2 and KCO2 values and at 0°C in pure water are 9.66  107, 4.65  107 and 1.33  106, respectively (Tchobanoglous and Burton, 1991). Evidently, CO2 gas is more than an order-of-magnitude more soluble in water than O2 or N2. Another important phenomenon that must be taken into consideration is that, once the CO2 dissolves in the aqueous phase, a significant amount of other hydrated carbonate species can also form inside the food’s aqueous phase. The various carbonate

Properties of packaged food 737

species present in solution at a particular pH can be calculated using the following equilibria: K

1



CO2 (l)  H2O ↽


⇀
H2CO3

(2)

K

2



H2CO3 ↽


⇀
HCO3  H

(3)

K3





HCO3 ↽


⇀
CO32  H

(4)

Literature values for K1, K2 and K3 for pure water at 0°C are 1.2  103, 2.5  104 and 4.9  1011, respectively (Roughton, 1941). At the slightly acidic pH found in many food products, the dissolved gas (CO2(l)) would first hydrate, creating carbonic acid (Equation (2)). The hydrated species would then ionize via Equation (3), releasing bicarbonate and an H ion that could potentially lower pH. The degree to which the internal pH drops would depend on the food’s buffering capacity. As a result, the total amount of CO2 present in the liquid phase is made up of the following compounds: lt l l l CCO  CCO  CHl CO  CHCO   CCO 2

2

2

2

3

3

(5)

Consequently, depending on the pH, the total concentration of all dissolved CO2 species can be orders-of-magnitude higher than O2(l) or N2(l). In fact, it has been shown that, in MAP of cut rutabaga, the amount of O2 or N2 dissolved in the produce is negligible compared to that in the package void volume (Lencki et al., 2004). On the other hand, the amount of dissolved CO2 species in the produce can be almost 38 per cent of the total CO2 found inside the package (Burton, 1974). Gases also have significant solubility in the lipid phase of foods. For example, in vegetable oil at 0°C, Henry’s law constants for N2, O2 and CO2 are 9.4  108, 1.9  107 and 2.3  106 Pa m3/kmol, respectively (Formo et al., 1979). To put these values into perspective, 1 l of vegetable oil, when exposed to the pure gas at 0°C, can absorb approximately 6 ml of N2, 12 ml of O2, or almost 1 l of CO2 at standard temperature and pressure (STP). In various meat fats, CO2 solubility can range from 0.6 to 1.3 l at STP/kg depending on the temperature and tissue source (Gill, 1988). It would appear that the amount of dissolved CO2 is a complex function of the physical properties of both the aqueous and lipid phases. Because of its high solubility in many foods, it is often necessary to add additional gas to compensate for this dissolution effect, in order to obtain a desired final gas concentration and package pressure (Zhao et al., 1995). On the other hand, when products with relatively high lipid concentrations such as meat (Enfors and Molin, 1984) or coffee (Jenkins and Harrington, 1991) are packaged under vacuum, some of the initially dissolved CO2 can re-enter the gaseous phase. This can create a protective modified atmosphere for the meat, but it can also lead to undesirable expansion of coffee packages. This latter phenomenon can be counteracted by the addition of CO2 scavenging materials (see section 3.3).

738 Modified Atmosphere Packaging for Minimally Processed Foods

2.3 Tissue respiration 2.3.1 Respiration rate measurement

The design of MAP systems for respiring fruit and vegetables requires a means of quantifying produce respiration behaviour. Plant respiration principally occurs via the following reaction: C6H12O6  6O2 : 6H2O  6CO2

(6)

Thus, respiration rate (r) in kmol/kg/s when glucose is the substrate can be defined as: l Vl dCC6H12O6 r  W dt

(7)

where Vl and W are the liquid volume (m3) of the produce and its weight (kg), respectively. Since changes in glucose concentration (kmol/m3) cannot easily be determined without destroying plant material, more indirect methods, such as the rate of O2 uptake or CO2 production rate are typically used. These two rates can be determined using either open flow-through or closed systems. The development of an expression for respiration rate for a particular measurement system begins with the law of conservation of mass:  Material  Material     output     input     through    through the     boundary   system   bo   oundary

  Material     generation      within the     system     boundary  

  Material     consumption      within the     system     boundary  

  Material     accumulation      within the     system     bo   oundary

         

(8)

In an open system, a gas of known composition is continuously flushed through a container containing the produce. With this steady-state system, the right-hand side of Equation (8) equals zero. As a result, simple expressions can be obtained for the respiration rate under a particular gas atmosphere:

r

( pO2inFin  pO2out Fout ) ( pCO2out Fout  pCO2inFin ) RTW



RTW

(9)

where F is the volumetric flow rate (m3/s). In a closed system, the produce is simply placed inside a sealed chamber and the pO2 and pCO2 are monitored with time. In this unsteady-state system, the first two terms in Equation (8) are zero. The third term in an oxygen balance would also be zero, whereas the consumption term would simply be equal to the product of the produce’s respiration rate (r) and weight (W). Oxygen would be depleted – which is considered a negative accumulation – in the surrounding atmosphere and also within the

Properties of packaged food 739

liquid phase of the produce. Thus, putting the appropriate expressions into Equation (8) and rearranging gives: rO   2

t Vt dCO2 1  6W dt 6W

 dC l Vg dpO2  O2 V   l  dt RT dt   

(10)

where Vt and Vg are the total container and gas void volumes, respectively, of the system (i.e. Vt  Vl  Vg), COt 2 is the total kmol of oxygen per m3 of respiration chamber, COl 2 is the total kmol of oxygen dissolved per m3 of produce aqueous phase, and pO2 is the oxygen partial pressure inside the container void volume. Because of the low solubility of O2, the amount of this gas present in the produce would be negligible compared to that in the surrounding void volume (Lencki et al., 2004). As a result, Equation (9) becomes: rO   2

Vg

dpO2 6WRT dt

(11)

Therefore, the respiration rate can be calculated directly if it is known how the pO2 in the surrounding atmosphere changes with time. A similar development can be performed for CO2 to give: rCO

2

t Vt dCCO2 1   6W 6W dt

 dC lt Vg dpCO2  CO2 V   l  dt RT dt   

(12)

t lt where CCO is the total kmol of carbon dioxide per m3 of respiration chamber, CCO is 2 2 3 the total kmol of carbon dioxide dissolved per m of the produce aqueous phase and pCO2 is the carbon dioxide partial pressure in the surrounding void volume. In this case, the CO2 found in the liquid and gaseous phases are both significant so the simplification that was applied to O2 is inappropriate (Lencki et al., 2004). However, lt CCO can be related to pCO2 by incorporating Equations (1) to (4) into Equation (12) 2 to give:

rCO

2

1  6W

  Vg Vl   RT  K CO2  

    dpCO2 K K K K K 1 2 3 1 2 1 K    1 2   CHl l (CH )  dtt 

(13)

The most striking difference between the respiration rate equations determined for steady-state flow-through systems (Equation (9)) and unsteady-state closed systems (Equations (11) and (13)) is that the latter is a function of both produce loading (i.e. Vl/Vg) and internal pH, whereas the former is independent of these system parameters. This can lead to significant differences in the respiration rates determined by these two methods, particularly if CO2 is the gas monitored (Lencki, 2004). Another important phenomenon that is often observed with plant tissue is that the respiration rate determined by O2 uptake (Equation (11)) can be significantly different

740 Modified Atmosphere Packaging for Minimally Processed Foods

from that determined by CO2 production (Equation (13)). In other words, even though the stoechiometry of Equation (6) dictates that the respiration coefficient (RQ) value, which is the ratio rCO 2/rO 2, should be equal to one, often it is not. One explanation for this effect is that the plant has several potential substrates, not just glucose as illustrated in Equation (6). The relative amounts of O2 metabolized and CO2 produced are dependent on the degree of oxidation of the substrate and the pathways by which it is metabolized. For example, the theoretical RQ when hexoses are aerobically metabolized is 1.0, but this value changes to 0.8 or 1.33 if fat or organic acids, respectively, are the principal substrate (Wills et al., 1981). Higher RQ values can also be obtained if oxygen drops below a critical value, leading to the onset of fermentation. In closed respiration systems, large swings in RQ can occur over a period of only a few hours (Zhu et al., 2001). However, such rapid RQ changes do not generally occur in steadystate respiration systems (Lencki, 2004). It is likely that the large RQ variations observed in closed respiration chambers are a result of changes in pH and dissolved CO2 species concentrations within the produce (Lencki, 2004). 2.3.2 Respiration models

Numerous expressions have been used to characterize the respiration rate of various produce. Equations based on enzyme kinetics generally take on the form: rO or rCO  2

2

Vmax pO2 K M  pO2

(14)

where Vmax (kmol/kg/s) and KM (Pa) are fitted parameters. In this same vein, it has been shown that, among the various enzyme inhibition models, the uncompetitive inhibition form appears to best describe the effect of CO2 on produce respiration (Peppelenbos and van’t Leven, 1996): rO or rCO  2

2

Vmax pO2  pCO2   pO K M  1   K  2

(15)

I

where KI (Pa) is the fitted inhibition constant. Polynomial respiration models have also been used and typically take on the form (Zhu et al., 2001): rO or rCO  a0  a1pO2  a2 pCO2  a3 pO22 2

2

 a 4 pCO22  a5 pO2 pCO2

(16)

where ai (i  0 to 5) are the fitted constants. A linear model would simply be Equation (16) with the constants a3, a4 and a5 equal to zero. Exponential models like the following have also been used (Beaudry et al., 1992): a rO or rCO  a0 1 exp(a1pO2 )  2 2

2

(17)

Properties of packaged food 741

1 14 13 12 11 10 9 8 7

Dimensionless oxygen respiration rate

0.9 0.8

6 5 43 2

0.7

1 0.6 0.5 0.4

0.3 0.2

0.1 0

2

4

6

8

10

12

14

16

18

20

Oxygen partial pressure (kPa) Figure 28.1 Dimensionless oxygen respiration rate as a function of pO2 at 4 to 7°C. 1: strawberry (Talasila et al., 1992); 2: cut rutabaga (Zhu et al., 2001); 3: raspberry (Joles et al., 1994); 4: green pepper (McLachlan and Stark, 1985); 5: apple (Makino et al., 1996); 6: cut cucumber (McLachlan and Stark, 1985); 7: cut garlic (McLachlan and Stark, 1985); 8: blueberry (Song et al., 2001); 9: cut carrot (McLachlan and Stark, 1985); 10: coleslaw (McLachlan and Stark, 1985); 11: cut apple (Lakakul et al., 1999); 12: cut broccoli (Hagger et al., 1992); 13: blueberries (Beaudry, 1993); 14: cut broccoli (Talasila et al., 1994).

The relative effects of pO2 and pCO2 can be compared for a wide range of produce by normalizing the various models. Figure 28.1 shows a plot of dimensionless respiration rate or r OD2 (defined as rO2 at a particular pO2 divided by rO2 at pO2  21 kPa) as a function of pO2 for various published literature models at temperatures typically used in MAP (i.e. 4–7°C). It is evident from Figure 28.1 that the hyperbolic enzyme kinetic model is the one most commonly used (Fonseca et al., 2002). Unfortunately, rarely is a proper statistical analysis performed to justify this model choice. In fact, statistical analyses have shown that linear or quadratic models usually provide a better fit to respiration data than enzyme kinetic models (Segall and Scanlon, 1996; Zhu et al., 2001). The influence of pO2 is evidently very produce-dependent, having a dramatic effect on strawberries but only a modest effect on, for example, cut broccoli (Figure 28.1). Figure 28.2 compares four models that characterize the effect of temperature on r nO2. Evidently, low pO2 has a much more significant effect on respiration rate at room than at refrigeration temperatures. It is unlikely that temperature has such a large effect on the KM of the various produce oxidases (see Equation (14)). A more likely

742 Modified Atmosphere Packaging for Minimally Processed Foods

1 12 11 10 7 10

0.9

11

Dimensionless oxygen respiration rate

0.8

7 0.7 12 0.6 0.5 0.4

0.3 0.2

0.1 0

2

4

6

8

10

12

14

16

18

20

Oxygen partial pressure (kPa) Figure 28.2 The effect of temperature on the dimensionless oxygen respiration rate: ——  4 to 7°C; ----  22 to 25°C (see Figure 28.1 for model definitions).

explanation is that, at higher temperature, respiration rates are much higher and O2 solubility is lower, which would lead to conditions that create a substantial O2 concentration profile within the produce. This diffusion-limited condition would lead to an apparent increase in KM and a much more pronounced O2 effect (Tucker and Laties, 1985). The exact effect of pCO2 on produce metabolism is open to debate, with several researchers finding that it has a negligible influence on respiration rate (Beaudry, 1993). However, care must be taken when trying to separate the effects of pO2 and pCO2 on produce, particularly in closed respiration experiments, because these two concentrations are often strongly correlated and this can have a significant impact on regression analysis (Segall and Scanlon, 1996; Zhu et al., 2001). For the work that observed an inhibitory effect, the degree to which pCO2 affects rO2 can be compared for various models by calculating the ratio: rOD2 (at pCO2  10 kPa)/rOD2 (at pCO2  0 kPa). This ratio is plotted as a function of pO2 for various produce in Figure 28.3. In most cases, pCO2 has less of an effect at lower pO2. However, the trend with regard to temperature is less clear. The lack of consensus regarding the effect of pCO2 on respiration is perhaps not surprising since it would appear that inhibitory effects are transient and are a strong function of produce internal pH and the degree to which the produce has time to adjust to higher CO2 concentrations (Lencki, 2004).

Properties of packaging materials 743

Rnor(pCO2  10 kPa)/Rnor(pCO2  0 kPa)

1 0.9

19 18

0.8

2 10

0.7

0.6

17 16 15

0.5

1 7

0.4

4 12

0.3

9

0.2

0.1 0

2

4

6 8 10 12 14 Oxygen partial pressure (kPa)

16

18

20

Figure 28.3 A plot of r DO2 at 10 kPa pCO2 divided by r DO2 at 0 kPa pCO2: ——  4 to 7°C; ----  22 to 25°C. See Figure 28.1 for some model definitions. 15: mung bean, 16: cut broccoli (Lee et al., 1991), 17: cut chicory, 18: cut broccoli, 19: asparagus (Peppelenbos and van’t Leven, 1996).

3 Properties of packaging materials 3.1 Film permeability The amount of a particular gas or vapour diffusing across a packaging film can be characterized using Fick’s first law of diffusion: Ji  Di

∂C i ∂x

(18)

where Ji is the diffusive flux of species i through the film (kmol/m2/s), Di is the diffusion coefficient (m2/s), Ci is the gas concentration (kmol/m3) in the air or polymer film and x is a variable representing the coordinate along a direction perpendicular to the film (m). As discussed by Banks et al. (1995), partial pressure (i.e. pi) is a more convenient unit for gas concentrations because it incorporates variations in gas status due to changes in total internal pressure. For non-porous polymeric films, it is usually assumed that all the flux resistance is present in the film, so the gas flux (JFi ) can be determined as follows: JiF  Pi (piout  piin ) 

Pi out (p  piin ) X i

(19)

744 Modified Atmosphere Packaging for Minimally Processed Foods

Table 28.3 Permeabilities of films commonly used in barrier MAP at 25°C (Brown, 1992) Polymer

PO2 (kmol/s/m/Pa)

PCO2 (kmol/s/m/Pa)

Ethyl vinyl alcohol Polyvinylidene chloride Amorphous nylon Polyethylene terephthalate Oriented polypropylene High density polyethylene Polystyrene

0.6–1.4  1022 2.0  1022 2.4  1021 9.4  1021 2.9  1019 3.6  1019 7.2  1019

0.8–3.9  1022 4.9  1022 7.9  1021 3.9  1020 1.1  1018 1.1  1018 1.8  1018

where P⬘i is the permeance (kmol/s/m2/Pa), Pi is the permeability (kmol/s/m/Pa) for gas i, and X is the film thickness. The permeance is the product of Di and Si, the latter being the solubility of the gas in the film. Some food products, such as coffee and nuts, are often packaged in glass or metal containers. For snack food such as potato chips, metallized laminates are typically used (Del Nobile, 2001). If sealed properly, these glass or metallic packages have gas permeabilities approaching zero, so they are ideal for extended shelf-life foods. However, with higher water activity products, such as meat or bread, that have relatively short shelf-lives, consumers need to see the product to confirm its quality. Consequently, clear polymeric films are more appropriate. Extensive lists of the gas and water vapour permeabilities of plastic polymers have been published in several sources (Brown, 1992; Exama et al., 1993; Al-Ati and Hotchkiss, 2002). Ideally, non-respiring food materials packaged under MAP conditions should use films or laminates with very low gas permeabilities. Films containing polymers such as polyvinylidene chloride or ethylene vinyl alcohol are often used for this purpose (Table 28.3 lists some of the more commonly used polymers). However, because of its high cost and sensitivity to high humidity, ethyl vinyl alcohol is typically sandwiched between other polymers (Brown, 1992). For laminated or coextruded films, the overall permeability can be calculated as follows: n

∑Xj PiT 

j1 n

Xj j j1 Pi

(20)

∑

where Xj and Pj are the thicknesses and permeabilities of the n polymer layers. In some cases, such as with bread, the higher cost of laminated or coextruded films cannot be justified. Cheaper polymers such as low density polyethylene, which have relatively much higher gas permeabilities, can provide enough of a barrier to give the desired few extra days of shelf-life (Seiler, 1998). Using barrier polymers for respiring produce would lead to anaerobic conditions and rapid quality loss. Thus, for MAP of fruit and vegetables, high gas permeability films are required (Table 28.4). In order to obtain the desired pCO2 and pO2 steady-state

Properties of packaging materials 745

Table 28.4 Permeabilities and ␤ value at 4°C for films used in steady-state MAP Polymer

PO2 (kmol/s/m/Pa)

PCO2 (kmol/s/m/Pa)

␤ value

Silicon rubbera Natural rubbera Ethyl cellulosea Polyvinyl chloridea Low density polyethylenea Plasticized LDPEb

3.4  1016 2.9  1017 4.7  1017 7.0  1018 3.7  1018 7.0  1016

2.2  1015 2.1  1016 1.1  1016 4.3  1017 2.5  1017 9.1  1016

6.5 6.1 2.4 6.1 6.7 1.3

a

Exama et al., 1993, b Zhu et al., 2002.

conditions inside the package, it is also necessary to have the proper PCO2 to PO2 ratio or  value (Gorney, 2003). For some produce, such as green pepper and Brussels sprouts,  ideally should be around 6.0, whereas strawberry requires a ratio of 1.1 (Exama et al., 1993). With most pure polymers, CO2 is much more permeable than O2, with  values ranging from 2.4 to as high as 10.2 (Exama et al., 1993). However, certain polymer additives have been found to have a large effect on PO2 (Gorney, 2003). Therefore, by controlling additive addition, polymers with high gas permeabilities and  values close to unity are available and these films have been shown to be very effective in MAP produce systems (Zhu et al., 2002). Another strategy for creating high gas flux systems has been through the use of films containing macroscopic (Lee and Renault, 1998) or microscopic pores (Fonseca et al., 2000). The characterization of the flux through porous or microporous films is more complex due to the lack of a phase change as the gas crosses the film. For any practical commercial package containing pores, effusion or Knudsen diffusive transport mechanisms do not play a significant role (Zanderighi, 2001). Several researchers have characterized the pore gas fluxes using Stephan-Maxwell equations (Renault et al., 1994; Lee and Renault, 1998), but this approach is perhaps unnecessarily complex considering that the diffusivities of the various gases crossing the film are very similar (Zanderighi, 2001). A simpler approach using an approximation of Fick’s first law gives the following expression for diffusion through a pore (Paul and Clarke, 2002): n

JiPD  y i ∑ J PD j  j1

Dim ∂pi RT ∂x

(21)

where yi is the mole fraction and Dim is the diffusion coefficient (m2/s) for component i in a mixture containing n gases. Equation (21) can be integrated to give an expression for the total diffusive flux (kmol/s) through a pore (NiPD): n  d2  Dim   NiPD  y i ∑ N PD (ppiout  piin ) j   4  RTl j1 eff

(22)

where leff is the effective length of the pore (m) and d is its diameter (m). Paul and Clarke (2002) have shown that for straight pores, leff  l  7/6d where l is the actual pore length.

746 Modified Atmosphere Packaging for Minimally Processed Foods

If the produce inside a porous MAP has an RQ value 1 – which is often the case (Lencki, 2004) – or the pores are combined with a high-permeability film, then the total internal pressure can drop below that of the surrounding atmosphere, creating a pressure driving force and convective flow through the pores (Paul and Clarke, 2002). In fact, very small differences in pressure can create a convective flow that overwhelms any diffusive flux (Del-Valle et al., 2003). Poiseuille’s law has been used (Del-Valle et al., 2003) to characterize the total convective flux (kmol/s) through a pore (NiPC ): NiPC  y i

d4 pTout (p out  pTin ) 128lRT T

(23)

where  is the gas viscosity (Pa s), yi is the mole fraction of gas i in the outside atmosin phere, and pout T and p T are the total pressures (Pa) outside and inside the package, respectively. The diffusive and convective terms are additive and can be summed to provide a general expression for pore gas transport in MAP. Because the gas molecules diffusing through the pores have very similar diffusivities, the  value of pore systems is essentially unity. As a result, if a relatively low pO2 value is required inside the package (e.g. 2 kPa), the corresponding internal pCO2 would be excessively high (i.e. 19 kPa), which could damage CO2 sensitive produce like lettuce. One strategy for circumventing this problem is to combine polymeric films and pores to create barriers with lower  values to produce optimal internal pO2 and pCO2 simultaneously (Paul and Clarke, 2002). Another problem with both polymeric and microporous films is that their permeabilities are not a strong function of temperature (Exama et al., 1993). Depending on the polymer, permeabilities will usually increase by approximately a factor between one and two for every 10°C increase in temperature (Exama et al., 1993). On the other hand, produce respiration is much more sensitive to temperature, with rates increasing by a factor of two to three for every 10°C. Evidently, an optimal design at low temperatures could cause anaerobic conditions under high temperature stress. Thus, it is essential that MAP packages are maintained at refrigeration temperatures through the food distribution system to avoid anaerobic conditions at elevated temperatures.

3.2 Package configuration MAP systems have taken on a wide range of shapes and sizes, but the most common forms are plastic bags and thermoformed trays (Greengrass, 1998). The package surface-to-volume (S/V) ratio is an important design parameter since it determines the amount of active surface for gas transport. For barrier systems where gas permeation is minimized, a more cubic shape with a lower S/V would be preferable whereas, with MAP containing produce, a flatter shape with a higher S/V is desirable. However, one is often limited by the shape of the product being packaged (e.g. sliced meat). Scalingup of MAP, particularly with packaged produce, can be a challenge because, for a fixed geometry, the S/V decreases with increasing size. Therefore, in order to obtain the same system performance in larger packages, the film permeability must be increased, the produce loading decreased, or the geometry altered.

Modified atmosphere packaging design 747

Package void volume also is a key design parameter, as it has been shown significantly to affect both unsteady-state behaviour and the final gas partial pressures in produce MAP (Lencki et al., 2004). Typical void volumes observed with numerous produce have been tabulated along with produce densities (Exama et al., 1993), which can be used to convert produce weight (the unit used to characterize respiration) to volume (the unit used to determine package capacity).

3.3 Gas scavenger and generator systems Another strategy for altering the gas environment is to add gas scavengers or generators inside a MAP (Brody et al., 2001). This technology is popular in Japan but has yet to be used extensively in other parts of the world. O2 absorbing materials such as iron powder are typically contained in gas-permeable sachets in order to minimize food contamination. With these sachets, O2 can be reduced to as low as 100 ppm, levels that cannot be obtained by vacuum or gas flush systems (Smith et al., 1995). CO2 from packaged coffee can also be absorbed using Ca(OH)2. Simultaneous O2 removal and CO2 generation can be accomplished with a combination of iron carbonate and ascorbic acid and these systems have been shown to be effective with products such as peanuts. Other technologies for the adsorption of ethylene and the generation of ethanol vapour are also available. Scavenging kinetics can be controlled by modifying particle size, controlling moisture and applying various encapsulation technologies (Smith et al., 1995).

4 Modified atmosphere packaging design 4.1 Barrier systems Even if a food material like coffee is vacuum packaged or gas flushed before being sealed in a very high gas barrier package such as a metal can, changes in gas environment can occur due to a repartitioning of gases between the food product and surrounding void volume. A molecular balance on gas constituent i during this repartitioning process takes on the form:  kmol of gas i    initially dissolved   in food 

  kmol of gas i        initially present     in void volume  

  kmol of gas i        finally dissolved     in food  

  kmol of gas i           finally present       in void volume    

(24)

This balance becomes: piinitial p p /v p MAP p MAP MAP V Vl  i Vginitial  i Vl  i Ki RT Ki RT g

(25)

where piinitial is the partial pressure of gas i to which the food is initially at equilibrium before packaging, pip/v is the partial pressure of gas i in the initial purge or vacuum

748 Modified Atmosphere Packaging for Minimally Processed Foods

gas, piMAP is the final equilibrium partial pressure of gas i inside the MAP, Ki is the Henry’s law constant as defined in Equation (1) and Vl and Vg are the liquid and gas volumes, respectively. Rearranging Equation (25) gives an expression for the final gas partial pressure:  Vginitial  V   piMAP   piinitial l  pip /v  RT  Ki

MAP  V  l Vg   K  RT   i 

(26)

A summation of the partial pressure of all the gasses present provides a measure of the total final pressure inside the package. If the barrier film surrounding the food has significant gas permeability, then some degree of gas transfer will occur during the shelf-life of the product (Robertson, 1993). If there is a maximum gas concentration in the food product above which ), then the shelf-life (t) can be calcuserious degradation occurs (for example, COcritical 2 lated by integrating Equation (18) over the whole package surface (A in m2). Assuming that pO2out

pO2MAP, then: t

X Vl APO pO2out 2

   critical pO2MAP  C    O2 K O   2

(27)

4.2 Steady-state systems In MAP of produce, the internal gas atmosphere is modified by controlling the final steady-state established between the produce respiration and permeation across the gas transfer medium. The uptake of O2 of a fruit or vegetable can be calculated by multiplying the O2 consumption rate at the in-package gas composition by the package weight (W): (28)

QO  WrO 2

2

where QO2 and W have the units of kmol/s and kg, respectively. A similar expression can be written for the CO2 production rate: QCO  WrCO 2

2

(29)

where QCO2 is the amount of CO2 produced per unit time (kmol/s). For a MAP constructed of a polymer film, Equation (19) can be used to characterize the diffusion of gases in and out of the package. A mass balance similar to Equation (8) can then be conducted to provide expressions for the kinetic behaviour of the various gases. For example, with oxygen: A JOF  QO  2

2

Vg dpO2 RT dt

(30)

Modified atmosphere packaging design 749

Substituting in Equation (19) and rearranging gives:  dpO2in RT  PO2 out  pO in  Q  pO  A ( ) 2 2 O2  dt Vg  X  

(31)

Similar expressions can be derived for CO 2in and N in 2: dpCO2in RT  dt Vg

 PCO   2 A ( pCO out  pCO in )  Q  CO2  2 2  X  

 dpN2in RT  PN2 out  pN in  pN  A ( ) 2  2 dt Vg  X  

(32)

(33)

An expression for water transport can also be included:  dpH2O in RT  PH2O out  pH O in   A pH O ( ) 2 2  dt Vg  X  

(34)

Although pH2O is relatively small at refrigeration temperatures (0.77 kPa at 3°C) and is usually ignored, it can become significant at elevated temperatures (e.g. pH2O  3.2 kPa at 25°C). For most MAP systems, pO 2out, pCO2out, pN2out and pH2O out are those found in the surrounding atmosphere. The boundary conditions used to integrate Equations (31) to (34) depend on the particular MAP package configuration. For rigid packages, Vg is constant so the total internal pressure will vary with time. An overall balance can also be conducted to yield an expression for total internal pressure pin T: dpTin dpO2in dpCO2in dpN2in dpH2O in     dt dt dt dt dt

(35)

For flexible packaging, at least initially, pTin will be constant (i.e. dpTin/dt  0) and Vg can vary with time. However, as is usually the case, Vg decreases with time and eventually a minimum volume will be reached below which further shrinkage is impared by the contained produce and Vg  Vgmin. At this point, Equation (35) will once again be valid. Equations (31) to (35) can be numerically integrated to provide curves that predict the various gas partial pressures as a function of time inside an MAP containing produce (Zhu et al., 2002). The unsteady-state portion of these evolution curves can last for hours or days, depending on the respiration rate of the contained produce and the package loading (i.e. W/(Vl  Vg)). This transition period can be reduced by gas flushing (Jacxsens et al., 1999). Eventually, a steady-state will be reached where dpini/dt and dpinT /dt  0 and the various gases will reach their steady-state values (i.e. p*i ). The final concentrations

750 Modified Atmosphere Packaging for Minimally Processed Foods

can be calculated by equating Equations (31), (32) and (33) to zero and rearranging: pO2*  pO2out 

QO X 2

PO A

(36)

2

pCO2* 

QCO X 2

PCO A

 pCO2out

(37)

2

The optimal pO*2 and pCO*2 values can be obtained from Table 28.2 or the literature (Gorney, 2003). By inserting the appropriate packaging parameters, the required PO2, PCO2, and film  values can be calculated. A similar development can be conducted for MAP systems containing pores or microperforations (Paul and Clarke, 2002). In this case, the total amount of material crossing the polymeric film (jTi in kmol s1) would be: jiT  AJiF  m(NiPD  NiPC )

(38)

where m is the total number of pores in the package. The unsteady-state solutions for the resulting set of differential equations is much more complex than for simple polymer systems. Nevertheless, steady-state solutions have been presented for these combined polymer/micropore systems (Paul and Clarke, 2002).

5 Conclusions The use of MAP has greatly expanded over the past few decades and is now used around the world to extend the shelf-life of a wide range of food products. Models now exist to predict the gas transport behaviour of a wide variety of MAP systems. Even equations that characterize the complex behaviour of steady-state microporous membrane systems have been recently developed. These models have the potential greatly to reduce the amount of experimentation required to optimize MAP products. Even though much progress has been made, there still needs to be more research conducted on how various gases affect the MA packaged food and the associated microbial populations. For example, the exact mechanism by which CO2 affects the respiration rate of various produce is still not well understood. More work in this regard will expand the applicability and further improve the economic viability of MAP systems.

Nomenclature ai A CHl  COl 2, CCl 6H12O6

produce respiration model constants package surface area available for gas transport (m2) hydronium ion liquid phase concentration (kmol/m3) concentration of oxygen or glucose in the liquid phase (kmol/m3)

Nomenclature 751

l l l 2 , CCO CCO , CHl 2CO3, CHCO , 2 3 3

CltCO2 t COt 2, CCO 2

COcritical 2 d Di, Dim Fin, Fout jTi Ji JFi, JPi K1, K2, K3 KM, KI KO2, KCO2, KN2 Ki l, leff NiPD, NiPC pO2, pCO2, pN2, pH2O pO2in, pO2out, pCO2in, pCO2out

pO2MAP pi, piin, piout pTin piinitial, pip/v, pMAP i Pi, Pi PTi QO2, QCO2 r

concentration of dissolved carbon dioxide gas, carbonic acid, bicarbonate and carbonate ions in the liquid phase (kmol/m3) total concentration of all CO2 species in the liquid phase (kmol/m3) total concentration of oxygen or carbon dioxide species in the liquid and gas phases inside an MAP critical oxygen concentration above which serious degradation occurs (kmol/m3) pore diameter (m) diffusivity of gas i in a binary or complex mixture (m2/s) gas volumetric flow rate into and out of an open respiration chamber (m3/s) total flow of gas i through a combined pore/membrane system (kmol/s) diffusive flux for gas i (kmol/m2/s) diffusive flux of gas i across a polymeric film and pore (kmol/m2/s) equilibrium constants for carbonate species (kmol) fitted parameters for the enzyme respiration model (kPa) Henry’s law constant for oxygen, carbon dioxide and nitrogen (kPa m3/kmol) Henry’s law constant for a particular gas i (kPa m3/kmol) pore length and effective pore length (m) total diffusive and convective flux through a pore (kmol/s) partial pressures of oxygen, carbon dioxide, nitrogen and water (Pa) gas partial pressure flowing into and out of an open respiration chamber or the gas partial pressures inside and outside of a MA package (Pa) final oxygen partial pressure inside a MAP (Pa) partial pressure of gas i inside and outside of an MAP (Pa) total pressure inside an MAP (Pa) initial, after purging or vacuum application and final partial pressure of gas i inside an MAP (Pa) permeance (kmol/s/m2/Pa) and permeability (kmol/s/m/Pa) of a polymeric film total permeability of a laminated or coextruded polymer (kmol/s/m/Pa) total oxygen consumption or carbon dioxide production rate inside an MAP (kmol/s) glucose respiration rate (kmol/kg/s)

752 Modified Atmosphere Packaging for Minimally Processed Foods

rO2, rCO2 rOD2 R T t Vl, Vg, Vt Vmax VMAP W x X yi 

produce respiration rate based on an oxygen uptake or carbon dioxide production basis (kmol/kg/s) dimensionless respiration rate gas constant (8314 Pa/m3/K kmol) temperature (K) time or shelf-life (s) produce liquid volume, package gas void volume and total package volume (m3) fitted parameter for the enzyme respiration model (kmol/kg/s) final volume of a MA package (m3) produce weight (kg) coordinate along a direction perpendicular to the film (m) film or layer thickness (m) mole fraction (dimensionless) viscosity (Pa s)

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Index

Acetic acid, 389–90 Acidic electrolysed water, 692 Adiabatic heating, 7, 15 Adipic acid, 395 Agitated thin-film evaporators, 253 Air drying: microwave vacuum drying and, 521–2 osmotic dehydration and, 235–6 Airborne powders, precipitation, 336–7 Alfalfa seeds, radio frequency processing, 460, 462 Alkaline phosphatase (ALP): pulsed electric field processing effects, 167–8 pulsed light effects, 298 Antifreeze proteins (AFPs), 653, 655–72 discovery of, 658–62 mechanisms of activity, 666–9 structures and evolution, 662–6 use in food preservation, 669–71 Antimicrobials, 114–16, 387–8, 407–9 enzymes, 401–4 glucose oxidase, 402–3 lactoperoxidase, 403 lysozyme, 401–2 plant-derived antimicrobials, 396–9 with high pressure processing, 55–6 with pulsed electric field processing, 114–15, 119–26, 148, 192 See also specific chemicals Apple juice: pulsed electric field processing, 121–2 shelf-life, 206 radio frequency electric fields processing, 315–19 See also Fruit juices

Bacillus, see Bacteria Bacteria, 10–11 high pressure effects on, 11–12, 56–8 biochemical reactions, 58 cell wall and membrane, 57–8 genetic mechanisms, 58 morphology, 56–7 importance of food-borne disease, 354–5 irradiation effects on, 363, 364, 369 ohmic heating effects, 492–4 pulsed electric field effects on, 74, 143–4, 194–203 radio frequency electric fields effects on, 315–17 pilot scale inactivation, 317–19 sublethal injuries: in high pressure processing, 57 in pulsed electric field processing, 74 ultrasonic inactivation, 330–2 see also Microorganisms Bacterial spores, 12 high pressure effects on, 12, 58–9 radio frequency electric fields effects on, 315–17 Bakery products: microwave baking, 427–9 NMR and MRI applications, 565–7 vacuum cooling, 586–7 Batch systems, 6 ohmic heating, 476–7 pulsed electric field processing, 106–7, 159 ultrasonic, 328–9 Beer, pulsed electric field processing, 126 Benzoic acid, 392–3

758 Index

Beverages: direct osmosis, 271 osmotic membrane distillation, 269–71 see also Fruit juices; Liquid foods; specific beverages Biocontrol, 697–8 Blanching: microwave, 435–6 ohmic heating, 494–5 Bread: microwave baking, 428 NMR and MRI baking, 565–7 Broad spectrum pulsed light (BSPL), 292, 293 see also Pulsed light technology (PLT) Carvacrol, 115 Centrifugal force, with osmotic dehydration, 234–5 Chemical shift, 557–8 Chitin/chitosan, 399–401 Chlorine dioxide disinfection, 693 Cider, pulsed electric field processing, 121–2 Citric acid, 393–4 Cold storage, antifreeze protein applications, 669–71 Colours, see Natural food colours Controlled atmosphere (CA) storage, 733 see also Modified atmosphere packaging (MAP) Cooking: NMR, 567–9 ohmic heating, 494 see also Specific cooking processes Cooling, See Freezing; Vacuum applications Cranberry juice, pulsed electric field processing, 126, 206 see also Fruit juices Dairy products, ultrasound effects on, 342–3 see also Milk Dehydration, see Drying Dielectric heating, 446–51 dielectric properties of foods, 420–2, 511–12 see also Microwave processing; Radio frequency (RF) processing

Diffusion ordered spectroscopy (DOSY), 560–1 Dips, see Salads Direct osmosis (DO), 263–7 future developments, 271–2 mathematical models, 264–5 membranes, 266, 268–9 process design and economics, 267 process parameter effects, 266–7 DNA, pulsed light effects on, 284–5, 286 Dressings, see Salads Drying, 535–6 air drying, osmotic dehydration and, 235–6 fresh fruits and vegetables, 689–90 hybrid drying systems, 535–47 fluidized bed drying (FBD), 541–3 heat pump drying (HPD), 538–41 microwave drying, 429–32, 544–6 freeze-drying, 524–5 with superheated steam drying, 547 see also Microwave vacuum drying ohmic pre-treatment, 496 pressure regulating drying, 547–8 product quality degradation, 537 radio frequency drying (RFD), 461–3, 543–4 agricultural products, 462 foods, 462–3 wood, 461–2 rotating jet spouted bed (RJSB), 548–9 ultrasound, 338–40 Egg: pulsed electric field processing, 120 ultrasound effects on, 344 ‘Elcrack’ process, 71 Electrical conductivity, 474–6, 488–9 Electrohydraulic treatment, 70 Electrolysed oxidizing (EO) water, 407 Electromechanical instability theory, 72, 103–4 Electroplasmolysis, 71 Electroporation, 69–71, 141–2, 143 mechanisms of action, 72–4, 103–5, 141–2 microbial inactivation, 186–7 see also Pulsed electric field (PEF) processing

Index 759

‘Electropure’ process, 70 ‘Elsetril’ process, 71, 100 Emulsification, ultrasound application, 333–4 Enzyme activity: antimicrobial enzymes, 401–3 glucose oxidase, 402–3 lactoperoxidase, 403 lysozyme, 401–2 high pressure freezing effects, 639 high pressure processing effects, 37–9 combination treatments, 174–5 fruits and vegetables, 37–8 meats, 39 microwave vacuum processing effects, 526–7 pulsed electric field processing effects, 144–5, 156–76 alkaline phosphatase (ALP), 167–8 influencing factors, 158–61 lipase, 169–70, 172, 173 lipoxygenase (LOX), 167, 172, 175 mechanisms of enzyme inactivation, 157 modelling, 170–4 pectin methyl esterase (PME), 163–5, 171, 172, 173, 175 peroxidase (POD), 166–7 polygalacturonase (PG), 165, 171–2 polyphenoloxidase (PPO), 165–6, 171 protease, 168–9, 173–4 storage of PEF processed foods, 175 pulsed light effects, 298 ultrasonic inactivation, 332 Escherichia coli, see Bacteria Essential oils, antimicrobial properties, 397–9 Ethylene production, 683–4 Evaporative concentration, 252–3 ohmic heating, 495 Extraction: ohmic pre-treatment, 495 ultrasound application, 340–2 Falling film evaporator, 253 Filtration: microfiltration, 254–5 ultrasound application, 338

Fish: irradiation, 369 combination treatments, 372–7 pulsed electric field processing effects, 146–8 vacuum cooling, 587 Flow systems, ultrasonic, 329 Fluidized bed drying (FBD), 541–3 two-stage system, 542 Foam formation and destruction, 334–6 Food colours, see Natural food colours Food safety: high pressure (HP) processing and, 9–16 importance of food-borne disease, 353–6 pulsed electric field (PEF) processing and, 143–4, 183–208 chemical safety, 206–7 see also specific processes Fourier transformation, 559 Free induction decay (FID), 559 Freeze concentration, 253–4 Freeze-drying, 538 microwave, 524–5 Freezing, 627–8 antifreeze protein applications, 669–71 ice and freeze survival, 655–8 freeze-avoidance, 656–8 freeze-tolerance, 656, 657 MRI studies, 569–70 osmotic dehydration applications, 236–7 see also High pressure freezing; Ultrasonic assistance of food freezing Fruit juices, 251–2 direct osmosis, 263 high hydrostatic pressure, 701 minimal processing, 699–702 osmotic membrane distillation, 256, 269–71 pulsed electric field processing, 121–6, 701 effects on microorganisms, 185–6 radio frequency electric fields (RFEF) processing, 307 ultrasound effects on, 343–4 see also Liquid foods; specific juices

760 Index

Fruits, 678–9 deterioration after harvest, 679 high pressure effects on enzyme activity, 37–8 irradiation, 368–9 combination treatments, 370–2 minimal fresh processing, 677–99, 702 biocontrol, 697–8 dewatering, 689–90 disinfection, 687–9, 691–7 factors affecting quality, 681–91 genetic engineering technology, 699 novel modified atmosphere packaging (MAP), 698–9 processing line, 684–90 osmotic dehydration, 223 in jam manufacture, 240–41 vacuum cooling, 584–6 see also Fruit juices Frying, osmotic dehydration applications, 237–8 Fumaric acid, 394–5 Fungi, 12–13 high pressure effects on, 12–13, 56–8 biochemical reactions, 58 cell wall and membrane, 57–8 genetic mechanisms, 58 morphology, 56–7 pulsed electric field processing effects on, 203–5 see also Microorganisms; Yeasts GdL (glucono-delta-lactone), 395 Genetic engineering, 699 Glucose oxidase, 402–3 Grape juice, pulsed electric field processing, 124–5 see also Fruit juices Heat pump drying (HPD), 538–41 infrared-augmented, 541 radio frequency (RF)-assisted, 540 Heating, see Microwave processing; Ohmic heating; Radio frequency (RF) processing; Temperature Herbs, antimicrobial properties, 397–9 High intensity electric field pulses, see Pulsed electric field (PEF) processing

High intensity pulsed light technology, see Pulsed light technology (PLT) High pressure freezing, 20–22, 627–47 additives and, 637–9 enzymatic inactivation, 639 future perspectives, 645–6 microbial inactivation, 639 modelling, 24–5, 639–45 convective phenomena, 641–2 temperature variation after adiabatic pressure change, 641 thermophysical properties under pressure, 640–41 processes, 629–37 high-pressure assisted freezing (HPAF), 630–3, 642 high-pressure induced freezing (HPIF), 637 high-pressure shift freezing (HPSF), 633–7, 642–5 see also Freezing High pressure (HP) processing, 4–27, 33–43, 47–60 current commercial status, 9 factors affecting effectiveness of treatment, 48–56 combined treatments, 54–6 conditions of treatments, 52–4 food products, 49–52 types of organisms, 48–9 food quality and, 16–19 food colour, 17, 39 food sensory quality, 18 food texture, 17–18, 39–42 food yield, 18–19 nutrient content, 42 food safety and stability, 9–16 effects on enzyme activity, 37–9 microbiological effects, 10–14, 35–7, 48–9, 56–60 factors influencing microbial sensitivity, 14–15 modelling HP processes, 23–4 outlook, 25–7 packaging requirements, 8 process description, 5–7 process principles, 7–8 ready meals, 33–43, 729 regulations, 15–16

Index 761

salads, 33–4, 37, 41–3 with irradiation, 370 with osmotic dehydration, 230 with pulsed electric field processing, 118–19, 148, 192–3 see also High pressure freezing High pressure non-frozen storage, 22–3 High pressure pasteurization, 35 High pressure sterilization, 35 High pressure thawing, 22 High temperature short time (HTST) processes, 434, 457, 469 Homogenization, ultrasound application, 333–4 Hot water treatments, 695 Hydrogen peroxide disinfection, 692 Ice, 655 see also Freezing Ice cream, ultrasonic assistance of freezing, 620–2 Infrared heating: heat pump drying and, 541 with microwave heating, 424–5 Insects: irradiation effects on, 363, 364–6 radio frequency processing and, 458, 460–61 Ionizing radiation, see Irradiation Irradiation, 353–78 biological effects of, 363–8 microorganisms, 363–4 parasites and insects, 364–6 ripening delay, 367 sprouting inhibition, 367–8 viruses, 367 combined treatments, 369–77 definition, 356–7 doses of irradiation, 357–8 fruit and vegetables, 368–9, 370–2 meat and fish, 369, 372–7 ready meals, 730 wholesomeness of irradiated foods, 358–63 government regulations, 358–61 public acceptance, 361–3 with high pressure processing, 55 with osmotic dehydration, 233 Isostatic Rule, 7–8

Jam manufacture, osmotic dehydration applications, 240–1 Lactic acid, 391–2 Lactobacillus, see Bacteria Lactoferrin, 405–6 Lactoperoxidase, 403 Le Chatelier’s Principle, 7 Lettuce, vacuum cooling, 584–5 Lipase, pulsed electric field processing effects, 169–70, 172, 173 Lipoxygenase (LOX), pulsed electric field processing effects, 167, 172, 175 Liquid foods, 99, 251–2 direct osmosis, 263 existing concentration methods, 252–5 evaporative concentration, 252–3 freeze concentration, 253–4 membrane processes, 254–5 osmotic membrane distillation, 256 pulsed electric field processing, 119–26 vacuum cooling, 591 See also specific foods Liquid whistle, 329 Listeria, see Bacteria Long chain fatty acids, 395–6 Lysozyme, 401–2 pulsed electric field processing and, 114–15, 119–25, 192 Magnetic resonance imaging (MRI), 553–4 baking, 565–7 freezing studies, 569–70 instruments, 562–3 pulse sequences, 562 temperature mapping, 434 theory of, 561–2 Magnetrons, 509 Malic acid, 394 Mango juice, pulsed electric field processing, 125 see also Fruit juices Manothermosonication (MTS), 331, 332 see also Ultrasound Marx generator, 76 Mass transfer: microwave processing, 423–5 ohmic pre-treatment, 495–6

762 Index

Mass transfer (contd) osmotic dehydration, 222, 223–6 effect of process parameters, 226–7 methods to increase mass transfer rate, 229–35 osmotic membrane distillation (OMD), 257–9 Meats: colour change, 39 high pressure effects: on enzyme activity, 39 on texture, 40–1 irradiation, 369 combination treatments, 372–7 NMR use in cooking, 567–9 pulsed electric field processing effects, 146–8 radio frequency processing, 459 vacuum cooling, 588–90 modelling, 591–2 see also Ready meals Microfiltration, 254–5 Microorganisms, 9–10 high pressure freezing effects, 639 high pressure processing effects, 10–15, 35–7, 48–9, 56–60 factors influencing microbial sensitivity, 14–15 irradiation effects, 363–4 microwave vacuum processing effects, 526–7 ohmic heating effects, 492–4 pulsed electric field processing effects, 74, 185–6, 194–205 combination treatment, 191–3 influencing factors, 187–91 mechanism of inactivation, 186–7 modelling, 193–4 pathogenic microorganisms, 194–201 spoilage microorganisms, 201–5 pulsed light effects, 284–6, 287–91, 293–5 radio frequency electric fields effects, 314–19 radio frequency processing effects, 458–60 sublethal injuries: in high pressure processing, 10, 48, 57 in pulsed electric field processing, 74

ultrasonic inactivation, 330–2 see also Bacteria; Fungi; Prions; Viruses Microwave processing, 419–37 baking, 427–9 blanching, 435–6 dielectric properties of foods, 420–2, 511–12 difference between radio frequency and microwaves, 447–8 drying, 429–32, 544–6 freeze-drying, 524–5 with superheated steam drying, 547 see also Microwave vacuum drying future developments, 436–7 heat generation, 423–4 mass transfer, 423–5 pasteurization and sterilization, 433–5 ready meals, 725–8 roasting, 435 thawing and tempering, 432–3 thermal properties of food, 512 Microwave vacuum drying, 507–28, 545–6 combination processes, 521–2 commercial potential, 527 dehydration costs, 519–21 drying rate, 513–15 enzyme inactivation, 526–7 equipment, 522–3 commercial driers, 522–3 research driers, 523 microbial inactivation, 526–7 modelling, 523–4 quality attributes of products, 515–19 rehydration potential, 515–16 retention of chemical components, 517–19 texture modification, 516–17 tempering and thawing, 526 see also Drying; Microwave processing Microwaves, 509–11 Milk: pulsed electric field processing, 119–20 effect on microorganisms, 185 enzyme inactivation, 159, 167, 168–9, 173–4 shelf-life, 206 ultrasound effects on, 342–3

Index 763

Minimal processing: fruit juices, 699–702 high hydrostatic pressure, 701 pulsed electric fields, 701 fruits and vegetables, 677–99, 702 biocontrol, 697–8 dewatering, 689–90 disinfection, 687–9, 691–7 factors affecting quality, 681–91 genetic engineering technology, 699 novel modified atmosphere packaging (MAP), 698–9 processing line, 684–90 ready meals, 717–31 cook-chill system, 721–2 cook-freeze system, 722–3 design of total system, 718–21 processing options, 725–30 sous-vide system, 723–5 Modified atmosphere packaging (MAP), 370, 371, 733–50 fresh fruit and vegetables, 683, 690–1 novel technologies, 698–9 packaged food properties, 734–42 gas solubility in foods, 736–7 optimal gas atmospheres, 734–6 tissue respiration, 738–42 packaging design, 747–50 barrier systems, 747–8 steady-state systems, 748–50 packaging material properties, 743–7 film permeability, 743–6 gas scavenger and generator systems, 747 packaging configuration, 746–7 Moulds, 12, 13 see also Fungi Mushrooms, vacuum cooling, 585–6 see also Vegetables Natural food colours, 252 direct osmosis, 263 existing concentration methods, 252–5 evaporative concentration, 252–3 membrane processes, 254–5 osmotic membrane distillation, 256, 269–71

Nisin, 404–5 high pressure processing and, 55–6 pulsed electric field processing and, 114–15, 119–25, 148, 192 Non-thermal processing, 100 Nuclear magnetic resonance (NMR), 553–71 future directions, 570 instruments, 562–3 multivariate data analysis, 563–4 theory of, 554–61 chemical shift, 557–8 detection and Fourier transformation, 559 nuclear spins and energy levels, 554–7 pulse sequences, 559–61 relaxation, 557 thermal processes and, 564 baking, 565–7 cooking, 567–9 rheo-NMR, 564 Ohmic heating, 469–99 electrical conductivity, 474–6 generic configurations, 476–9 batch configuration, 476–7 collinear ohmic heating, 478 technical considerations, 479 transverse ohmic heating, 477–8 non-Newtonian liquids, 479–82 on-line treatment validation, 496–7 pre-treatment, 489–92, 495–6 dehydration, 496 diffusion and extraction, 496 principles, 470–3 electrical heat generation, 472–3 product suitability, 489–92 ready meals, 728–9 solid/liquid mixtures, 483–9 effects of parameters, 486–9 electrical heat generation, 484 heat transfer, 485–6 mass conservation, 484–5 thermal treatments, 492–5 blanching, 494–5 cooking, 494 evaporation, 495 stabilization, 492–4 thawing, 494

764 Index

Open pan evaporators, 252 Orange juice: pulsed electric field processing, 122–4, 164–5, 175 shelf-life, 205–6 ultrasound effects on, 344 see also Fruit juices Organic acids, 388–96, 693 Osmotic dehydration, 221–43 applications, 235–41 air drying, 235–6 freezing, 236–7 frying, 237–8 jam manufacture, 240–1 rehydration, 238–40 limitations, 241 mass transfer, 222, 223–6 effect of process parameters, 226–7 mechanism, 223–6 methods to increase mass transfer rate, 229–35 centrifugal force, 234–5 gamma-irradiation, 233 high electric field pulse pre-treatment, 230–3 high hydrostatic pressure, 230 ultrasound, 233 vacuum, 234 microwave vacuum drying and, 522 moisture and solid diffusion coefficients, 227–9 osmotic solution management, 241–2 Osmotic membrane distillation (OMD), 256–63 applications, 269–71 future developments, 271–2 mathematical models, 257–60 heat transfer, 259–60 mass transfer, 257–9 membranes, 260–1, 268–9 process design and economics, 262–3 process parameter effects, 261–2 Ovalbumin, pulsed electric field processing effects, 144 Ozone, 406 minimal fresh processing, 694 pulsed electric field processing and, 116

Packaging, in high pressure (HP) processing, 8 see also Modified atmosphere packaging (MAP) Parabens, 393 Parasites, 355 high pressure effects on, 59 irradiation effects on, 363, 364–5, 369 Partial lease squares (PLS) regression, 564 Pasteurization, 470, 493 microwave, 433–5 ohmic heating, 492, 493 radio frequency processing, 458 Pectin methyl esterase (PME): high pressure processing effects, 37, 38 pulsed electric field processing effects, 163–5, 171, 172, 173, 175 Pediocin, high pressure processing and, 55–6 Peroxidase (POD), pulsed electric field processing effects, 166–7 pH: high pressure processing and, 14 pulsed electric field processing and, 112–13 Phenolic acids, 395–6 Plant-derived antimicrobials, 396–9 Plate evaporators, 253 Polygalacturonase (PG): high pressure processing effects, 37, 38 pulsed electric field processing effects, 165, 171–2 Polyphenoloxidase (PPO): high pressure processing effects, 38 pulsed electric field processing effects, 165–6, 171 pulsed light effects, 298 Poultry, see Meat Power ultrasound, 324–5 see also Ultrasound Preferential sorption capillary flow (PSCF) model, 265 Pressure effects, see High pressure freezing; High pressure (HP) processing Pressure regulating drying, 547–8 Pressure shift freezing (PSF), 20–2 modelling, 24–5 Principal component analysis (PCA), 563–4

Index 765

Prions, 13–14 Propionic acid, 390–1 Protease, pulsed electric field processing effects, 168–9, 173–4 Puffing, 515–16 Pulsed electric field (PEF) processing, 69–90, 100–29, 155–6, 184 applications, 83–9 disintegration of biological material, 84–6 preservation of liquid media, 86–9 stress induction, 83–4 combination treatment, 113, 114–15, 119–26, 148–9 antimicrobials, 114–15, 119–26, 148, 192 enzyme inactivation, 174–5 high pressure processing, 118–19, 148, 192–3 microbial inactivation and, 191–3 pre-treatment for osmotic dehydration, 230–3 temperature, 81, 100–1, 113, 119–26, 159–60, 191 enzyme inactivation, 156–76 combination treatments, 174–5 enzyme activity during storage, 175–6 influencing factors, 158–61 mechanisms, 157 modelling, 170–4 solid foods, 144–5 specific enzymes, 161–70 food safety aspects, 143–4, 183–208 chemical safety, 206–7 combination treatment, 191–3 effects on microorganisms, 185–6, 194–205 factors affecting microbial inactivation, 187–91 mechanism of microbial inactivation, 186–7 modelling microbial inactivation, 193–4 shelf-life of foods, 205–6 historical background, 70–71, 100–101 liquid foods, 119–26, 701 mechanisms of action, 72–4, 103–5, 141–2, 143

problems and challenges, 89–90 process models, 126–8 processing parameters, 79–83 cell characteristics, 82–3, 116–18 electric field strength, 79–80, 158–60, 187 enzyme inactivation and, 158–60 microbial inactivation and, 187–9 temperature, 81, 113, 128, 159–60, 188–9 treatment medium, 81–2, 112–16 treatment time, specific energy and pulse geometry, 80–81, 110–12, 158–60, 187–8 solid foods, 141–3, 144–8 effects on proteins and enzyme activity, 144–5 effects on texture and microstructure, 146–8 target differences, 82–3, 116–18 treatment medium factors, 81–2, 112–16 air bubbles and particles, 82, 191 antimicrobials, 114–16 composition, 114, 161, 190–1 conductivity, 82, 114, 160–1, 189–90 ionic effect, 116 microbial inactivation and, 189–91 pH, 112–13, 161, 190 treatment system, 74–9, 101–3, 105–9 batch treatment system, 106–7, 159 continuous treatment system, 107–9, 159 generation of pulsed electric fields, 75–9, 101–2 treatment chamber design, 77–9, 102–3 variables, 109–12 see also Radio frequency electric fields (RFEF) processing Pulsed light technology (PLT), 279–303 effects on food, 298–9 enzymes, 298 nutritional properties, 298 sensory properties, 298–9 effects on microorganisms, 284–6, 287–91, 293–5 history, 279–80 principles, 280–4

766 Index

Pulsed light technology (PLT) (contd) process optimization, 286–97 spectral distribution and treatment intensity, 292–5 target parameters, 296–7 time parameters, 295–6 systems, 299–302 examples of experimental plants, 300–302 Radiation, see Irradiation Radio frequency electric fields (RFEF) processing, 307–20 bacterial inactivation, 315–19 pilot scale, 317–19 electrical costs, 319 equipment, 309–13 history, 307–8 modelling, 313–14 yeast inactivation, 314–15 Radio frequency (RF) processing, 445–63 adoption of, 453–7 applicator design, 455–7 standardized technology, 454–5 difference between radio frequency and microwaves, 447–8 drying applications, 461–3, 543–4 agricultural products, 462 foods, 462–3 heat pump drying and, 540 vacuum drying and, 544 wood, 461–2 heating applications, 457–61 product disinfestation/disinfection, 460–1 seed treatments, 460 thermal treatment of food products, 458–60 heating mechanism, 448–51 material properties, 451–3 see also Radio frequency electric fields (RFEF) processing Ready meals, 34–5, 717–31 cook-chill system, 721–2 cook-freeze system, 722–3 design of total system, 718–21 consumer packs, 720–1 solid foods, 720 solid/liquid mixtures, 719–20

high pressure processing (HPP), 33–4, 42–3, 729 effects on enzyme activity, 37–9 effects on texture, 39–41 microbial inactivation, 35–7 importance of, 34–5 processing options, 725–30 aseptic processing, 730 hydrostatic processing, 729 irradiation, 730 microwave heating, 725–8 ohmic heating, 728–9 surface decontamination techniques, 729 sous-vide system, 723–5 vacuum cooling, 590–1 see also Meats; Vegetables Rehydration, 238–40 microwave vacuum-dried products, 515–16 Respiration, See Tissue respiration Reuterin, 406–7 Reverse osmosis, 255 Rheo-NMR, 564–5 Rice wine, pulsed electric field processing, 126 Rising film evaporator, 253 Roasting, microwave, 435 Rotating jet spouted bed (RJSB), 548–9 Salads, 34 high pressure processing (HPP), 33–4, 42–3 effect on nutrient content, 42 effects on food texture, 41–2 microbial inactivation, 37 importance of, 34 see also Vegetables Salmonella, see Bacteria Sauces, vacuum cooling, 587–8 Scraped surface heat exchanger (SSHE), 620–1 Seeds, radio frequency processing, 460, 462 Shelf life: fresh fruit and vegetables, 681 irradiation effects, 367–9 pulsed electric field (PEF) processing effects, 205–6 Slip velocity effect, 486

Index 767

Sorbic acid, 392 Soups, vacuum cooling, 587–8 Sous-vide process, 723–5 Spices, antimicrobial properties, 397–9 Sterilization, 470, 493 microwave, 433–6 ohmic heating, 492, 493 radio frequency processing, 458 Supercooling, 20–22 Supercritical fluid extraction (SFE), 341–2 Superheated steam (SHS) drying, 547 Tartaric acid, 395 Temperature: direct osmosis and, 267 heat generation in microwave processing, 423–4 high pressure processing and, 15, 52, 641 irradiation and, 365–6, 368–9, 370, 371 ohmic heating and, 474–6, 481 osmotic membrane distillation and, 262 pulsed electric field processing and, 81, 100–101, 113 enzyme inactivation relationship, 159–60 liquid foods, 119–20, 121–5 microbial inactivation relationship, 191 pulsed light and, 285–91 radio frequency processing, 458–9 Tempering, 432, 526 Thawing: high pressure application, 22 microwave application, 432–3 microwave vacuum processing, 526 ohmic heating, 494 Thermal hysteresis (TH) gap, 658 Thermal properties of food, 512 Thyratron switches, 75, 102 Time temperature indicator (TTI), 434 Tissue respiration, 738–42 models, 740–2 respiration rate measurement, 738–40 Tomato juice: pulsed electric field processing, 124, 175 shelf-life, 206 ultrasound effects on, 343–4 Transmembrane potential, 72, 79–80, 103–5, 143 Treatment temperature, see Temperature

Ultrafiltration, 255 Ultrasonic assistance of food freezing, 603–24 acoustic effects on freezing process, 612–13 applications, 620–3 freezing and storage of fresh foodstuffs, 622–3 ice cream, 620–2 moulded frozen products, 522 factors affecting efficiency, 618–20 acoustic duration, 619–20 acoustic power level, 618–19 functions in food freezing, 613–17 acceleration of freezing process, 614 control of crystal size distribution, 615 improvement of frozen food microstructure, 616–17 initiation of ice nucleation, 613–14 preventing incrustation on cold surface, 617 see also Ultrasound Ultrasound, 323–45 acoustic effects, 609–12 on gas, 611 on liquid, 609–11 on solid, 611–12 as a processing aid, 333–42 drying, 338–40 extraction, 340–2 filtration, 338 foam formation and destruction, 334–6 mixing and homogenization, 333–4 precipitation of airborne powders, 336–7 effects on food properties, 342–4 dairy products, 342–3 egg products, 344 juices, 343–4 enzyme inactivation, 332 freezing, see Ultrasonic assistance of food freezing microbial inactivation, 330–2 power ultrasound generation, 604–9 air-borne power ultrasonic system, 608–9 power generator, 604–5 ultrasonic bath, 607 ultrasonic probe system, 607 ultrasound transducers, 605–7

768 Index

Ultrasound (contd) power ultrasound principles, 325–7 in gases, 326–7 in liquid systems, 325–6 processing equipment, 327–9 batch systems, 328–9 flow systems, 329 laboratory scale, 327 large scale, 327–9 with osmotic dehydration, 233 Ultraviolet (UV) light, 284–6, 293, 695–7 effects on DNA, 284–5, 286 see also Pulsed light technology (PLT) Vacuum applications: cooling, 579–97 advantages, 592–4 bakery products, 586–7 cooked meat joints, 588–90 disadvantages, 593, 594–5 equipment, 581–4 factors affecting cooling process, 595–7 fishery products, 587 fruit and vegetables, 584–6 mathematical modelling, 591–2 principles, 580–1 process, 581 ready meals, 590–1 sauces, soups, particulate foods, 587–8 radio frequency drying and, 544 with osmotic dehydration, 234 see also Microwave vacuum drying Vegetables, 678–9 deterioration after harvest, 679 high pressure effects: on enzyme activity, 37–8

on nutrient content, 42 on texture, 39–40 irradiation, 368–9 combination treatments, 370–2 minimal fresh processing, 677–99, 702 biocontrol, 697–8 dewatering, 689–90 disinfection, 687–9, 691–7 factors affecting quality, 681–91 genetic engineering technology, 699 novel modified atmosphere packaging (MAP), 698–9 processing line, 684–90 ohmic heating, 490–2 osmotic dehydration, 223, 238–9 vacuum cooling, 584–6 see also Ready meals; Salads Viruses, 13, 355 high pressure effects on, 13, 59–60 irradiation effects on, 367 see also Microorganisms Visible pulsed light (VPL), 292 see also Pulsed light technology (PLT) Water, importance for life, 653–5 Water activity (Aw), high pressure processing and, 14, 51 Wood drying, 461–2 Working pressure, 6–7 Yakju (rice wine), pulsed electric field processing, 126 Yeasts, 12–13 radio frequency electric fields effects on, 314–15 see also Fungi

Food Science and Technology International Series

Maynard A. Amerine, Rose Marie Pangborn, and Edward B. Roessler, Principles of Sensory Evaluation of Food. 1965. Martin Glicksman, Gum Technology in the Food Industry. 1970. Maynard A. Joslyn, Methods in Food Analysis, second edition. 1970. C. R. Stumbo, Thermobacteriology in Food Processing, second edition. 1973. Aaron M. Altschul (ed.), New Protein Foods: Volume 1, Technology, Part A—1974. Volume 2, Technology, Part B—1976. Volume 3, Animal Protein Supplies, Part A— 1978. Volume 4, Animal Protein Supplies, Part B—1981. Volume 5, Seed Storage Proteins—1985. S. A. Goldblith, L. Rey, and W. W. Rothmayr, Freeze Drying and Advanced Food Technology. 1975. R. B. Duckworth (ed.), Water Relations of Food. 1975. John A. Troller and J. H. B. Christian, Water Activity and Food. 1978. A. E. Bender, Food Processing and Nutrition. 1978. D. R. Osborne and P. Voogt, The Analysis of Nutrients in Foods. 1978. Marcel Loncin and R. L. Merson, Food Engineering: Principles and Selected Applications. 1979. J. G. Vaughan (ed.), Food Microscopy. 1979. J. R. A. Pollock (ed.), Brewing Science, Volume 1—1979. Volume 2—1980. Volume 3—1987. J. Christopher Bauernfeind (ed.), Carotenoids as Colorants and Vitamin A Precursors: Technological and Nutritional Applications. 1981. Pericles Markakis (ed.), Anthocyanins as Food Colors. 1982. George F. Stewart and Maynard A. Amerine (eds.), Introduction to Food Science and Technology, second edition. 1982. Malcolm C. Bourne, Food Texture and Viscosity: Concept and Measurement. 1982. Hector A. Iglesias and Jorge Chirife, Handbook of Food Isotherms: Water Sorption Parameters for Food and Food Components. 1982. Colin Dennis (ed.), Post-Harvest Pathology of Fruits and Vegetables. 1983. P. J. Barnes (ed.), Lipids in Cereal Technology. 1983. David Pimentel and Carl W. Hall (eds.), Food and Energy Resources. 1984.

770 Food Science and Technology: International Series

Joe M. Regenstein and Carrie E. Regenstein, Food Protein Chemistry: An Introduction for Food Scientists. 1984. Maximo C. Gacula, Jr., and Jagbir Singh, Statistical Methods in Food and Consumer Research. 1984. Fergus M. Clydesdale and Kathryn L. Wiemer (eds.), Iron Fortification of Foods. 1985. Robert V. Decareau, Microwaves in the Food Processing Industry. 1985. S. M. Herschdoerfer (ed.), Quality Control in the Food Industry, second edition. Volume 1—1985. Volume 2—1985. Volume 3—1986. Volume 4—1987. F. E. Cunningham and N. A. Cox (eds.), Microbiology of Poultry Meat Products. 1987. Walter M. Urbain, Food Irradiation. 1986. Peter J. Bechtel, Muscle as Food. 1986. H. W.-S. Chan, Autoxidation of Unsaturated Lipids. 1986. Chester O. McCorkle, Jr., Economics of Food Processing in the United States. 1987. Jethro Japtiani, Harvey T. Chan, Jr., and William S. Sakai, Tropical Fruit Processing. 1987. J. Solms, D. A. Booth, R. M. Dangborn, and O. Raunhardt, Food Acceptance and Nutrition. 1987. R. Macrae, HPLC in Food Analysis, second edition. 1988. A. M. Pearson and R. B. Young, Muscle and Meat Biochemistry. 1989. Marjorie P. Penfield and Ada Marie Campbell, Experimental Food Science, third edition. 1990. Leroy C. Blankenship, Colonization Control of Human Bacterial Enteropathogens in Poultry. 1991. Yeshajahu Pomeranz, Functional Properties of Food Components, second edition. 1991. Reginald H. Walter, The Chemistry and Technology of Pectin. 1991. Herbert Stone and Joel L. Sidel, Sensory Evaluation Practices, second edition. 1993. Robert L. Shewfelt and Stanley E. Prussia, Postharvest Handling: A Systems Approach. 1993. R. Paul Singh and Dennis R. Heldman, Introduction to Food Engineering, second edition. 1993. Tilak Nagodawithana and Gerald Reed, Enzymes in Food Processing, third edition. 1993. Dallas G. Hoover and Larry R. Steenson, Bacteriocins. 1993. Takayaki Shibamoto and Leonard Bjeldanes, Introduction to Food Toxicology. 1993. John A. Troller, Sanitation in Food Processing, second edition. 1993. Ronald S. Jackson, Wine Science: Principles and Applications. 1994. Harold D. Hafs and Robert G. Zimbelman, Low-fat Meats. 1994. Lance G. Phillips, Dana M. Whitehead, and John Kinsella, Structure-Function Properties of Food Proteins. 1994. Robert G. Jensen, Handbook of Milk Composition. 1995. Yrjö H. Roos, Phase Transitions in Foods. 1995. Reginald H. Walter, Polysaccharide Dispersions. 1997. Gustavo V. Barbosa-Cánovas, M. Marcela Góngora-Nieto, Usha R. Pothakamury, and Barry G. Swanson, Preservation of Foods with Pulsed Electric Fields. 1999. Ronald S. Jackson, Wine Science: Principles, Practice, Perception, second edition. 2000.

Food Science and Technology: International Series 771

R. Paul Singh and Dennis R. Heldman, Introduction to Food Engineering, third edition. 2001. Ronald S. Jackson, Wine Tasting: A Professional Handbook. 2002. Malcolm C. Bourne, Food Texture and Viscosity: Concept and Measurement, second edition. 2002. Benjamin Caballero and Barry M. Popkin (eds), The Nutrition Transition: Diet and Disease in the Developing World. 2002. Dean O. Cliver and Hans P. Riemann (eds), Foodborne Diseases, second edition. 2002. Martin Kohlmeier, Nutrient Metabolism. 2003. Herbert Stone and Joel L. Sidel, Sensory Evaluation Practices, third edition. 2004. Jung H. Han, Innovations in Food Packaging. 2005. Da-Wen Sun, Emerging Technologies for Food Processing. 2005.