Food Additive 978-953-51-0067-6

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FOOD ADDITIVE Edited by Yehia El-Samragy

Food Additive Edited by Yehia El-Samragy

Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2012 InTech All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. As for readers, this license allows users to download, copy and build upon published chapters even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Romana Vukelic Technical Editor Teodora Smiljanic Cover Designer InTech Design Team First published February, 2012 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from [email protected]

Food Additive, Edited by Yehia El-Samragy p. cm. ISBN 978-953-51-0067-6

Contents Preface IX Chapter 1

Food Additive 1 R. M. Pandey and S. K. Upadhyay

Chapter 2

The Safety Assessment of Food Additives by Reproductive and Developmental Toxicity Studies 31 Cansın Güngörmüs and Aysun Kılıç

Chapter 3

Production and Functional Properties of Dairy Products Containing Lactophorin and Lactadherin 49 Mizuho Inagaki, Xijier, Yoshitaka Nakamura, Takeshi Takahashi, Tomio Yabe, Toyoko Nakagomi, Osamu Nakagomi and Yoshihiro Kanamaru

Chapter 4

Emerging Preservation Methods for Fruit Juices and Beverages 65 H. P. Vasantha Rupasinghe and Li Juan Yu

Chapter 5

Biosynthesis, Purification and Biotechnological Use of Exopolysaccharides Produced by Lactic Acid Bacteria 83 María Laura Werning, Sara Notararigo, Montserrat Nácher, Pilar Fernández de Palencia, Rosa Aznar and Paloma López

Chapter 6

Guar Foaming Albumin – A Foam Stabilizer 115 Ami Shimoyama and Yukio Doi

Chapter 7

Utilization of Aspergillus niger Phytase Preparation for Hydrolysis of Phytate in Foods 125 Akiko Matsuo and Kenji Sato

Chapter 8

Biotechnological Production of Xylitol from Agro-Industrial Wastes 139 José Manuel Domínguez, José Manuel Salgado, Noelia Rodríguez and Sandra Cortés

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Chapter 9

Rosemary Compounds as Nutraceutical Health Products 157 Silvia Moreno, Adriana María Ojeda Sana, Mauro Gaya, María Verónica Barni, Olga A. Castro and Catalina van Baren

Chapter 10

Potential of Probiotic Lactobacillus Strains as Food Additives 175 Sheetal Pithva, Padma Ambalam, Jayantilal M. Dave and Bharat Rajiv Vyas

Chapter 11

Earth Food Spirulina (Arthrospira): Production and Quality Standarts 191 Edis Koru

Chapter 12

Aluminum in Food – The Nature and Contribution of Food Additives 203 Robert A. Yokel

Chapter 13

The Use of Blood and Derived Products as Food Additives 229 Jack Appiah Ofori and Yun-Hwa Peggy Hsieh

Preface Food additives are natural or manufactured substances, which are added to foods to restore colors lost during processing, provide sweetness, prevent deterioration during storage and guard against food poisoning (preservatives). A food additive is defined as “any substance not normally consumed as a food in itself and not normally used as a characteristic ingredient of food, whether or not it has nutritive value, the intentional addition of which to food for a technological purpose in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food results, or may be reasonably expected to result, in it or its by-products becoming directly or indirectly a component of such foods.” (Food Safety Authority of Ireland, 2011). This book provides a review of traditional and non-traditional food preservation approaches, including physical methods such as non-thermal pasteurization, chemical methods using natural food preservatives and their combinations for extension of shelf life of fruit juices and beverages. Evaluation of agro-industrial wastes, considering their great potential for the production of industrially relevant food additives, has been presented. The isolation of an albumin fraction with high foaming ability and foam stabilizing ability from guar meal, and designated guar foaming albumin as food additive was studied. Detailed knowledge about the composition, structure, classification, biosynthesis, genetic determinants, mechanisms of production, secretion, enzymes for production, methods of production, purification, analysis and characterization of exopolysaccharides, as well as the potential applications for production of functional food, e.g. prebiotics and immunomodulators, have been covered. Effects of some food colorings such as tartrazine, as food additives, on hepatic function, lipid profile, and biomarkers of oxidative stress in blood and different tissues, have been evaluated. The problems and control of contamination of other enzymes during phytase preparation have been inspected for the possibility of using fungal phytase preparation as food additive. Exposure of humans to aluminum from different sources, i.e. food, water, airborne dust, antiperspirants etc., and related immunizations, allergy injections, and elevated antacids, were investigated. It was concluded that there is no good evidence of adverse health effects attributed to aluminum from food, that this issue has not been adequately assessed and that those who lack good renal function are at greatest risk of aluminum-induced toxicity, perhaps from food. The assessment of potential reproductive and developmental

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toxicity perspectives of some newly synthesized food additives on the market, i.e. lutein, caramel colors, erythrosine, green S, amaranth, brilliant blue FCF, curcumin, canthaxanthin, aspartame, paraben, coriander essential oil, allyl isothiocyanate, and tricalcium phosphate were overviewed in relevance to the six main categories of traditional food additives. Furthermore, potential utilization of lactophorin and lactadherin from sweet whey, a byproduct of cheese manufacturing against human rotavirus gastroenteritis in infants and young children, was discussed. In the attempt to identify future research areas of interest, it should be mentioned that this book pointed out the fact that more information is needed to explore the possibility of using some other materials as food additives. One of these issues is the full potential of the cell wall degrading enzymes in cereal processing such as biopolishing of cereal grain and pre-degradation for animal feeds using enzymatic polishing technique. More and more information on Spirulina is becoming available, which is essentially an exceptional simple extract of blue-green algae used worldwide as a food product and as a dietary supplement. It also has biological, biotechnological and nutrition applications, as well as some remote applications like bio-fuel production and functioning as a life support system in space studies. This interest will continue without any doubt. Research along these lines will be rewarding both socially and professionally. Another area of microorganism use proves that Lactobacillus strains play an important role in food fermentation processes and L. rhamnosus is a potential candidate for probiotic product preparation or as food additives as proven by its ability to acid-bile tolerance, salt, antimicrobial activity against human pathogens and food spoilage organisms. The characteristics of the antimicrobial protein(s) that are produced at different growth phases of LAB, require more work in the future.

Prof. Dr. Yehia El-Samragy Ain Shams UniversityFaculty of Agriculture Food Science Department Cairo, Egypt

1 Food Additive R. M. Pandey and S. K. Upadhyay Division of Genetics, Plant breeding & Agrotechnology, National Botanical Research Institute, Lucknow, India 1. Introduction Substances which are of little or no nutritive value, but are used in the processing or storage of foods or animal feed, especially in the developed countries; includes antioxidants; food preservatives; food coloring agents; flavoring agents; anti-infective agents; vehicles; excipients and other similarly used substances. Many of the same substances are pharmaceutics aids when added to pharmaceuticals rather than to foods. Food additives are substances added to food to preserve flavor or enhance its taste and appearance. Some additives have been used for centuries; for example, preserving food by pickling with vinegar, salting, as with bacon, preserving sweets or using sulfur dioxide as in some wines. With the advent of processed foods in the second half of the 20th century, many more additives have been introduced, of both natural and artificial origin. It is sometimes wrongly thought that food additives are a recent development, but there has certainly been an increase in public interest in the topic. Not all of this has been well-informed, and there are signs that commercial interests have been influenced by consumer pressure, as well as food producers manipulating the situation by marketing techniques. Various labeling regulations have been put into effect to ensure that contents of processed foods are known to consumers, and to ensure that food is fresh-important in unprocessed foods and probably important even if preservatives are used. In addition, we also need to add some preservatives in order to prevent the food from spoiling. Direct additives are intentionally added to foods for a particular purpose. Indirect additives are added to the food during its processing, packaging and storage. Food Preservatives are the additives that are used to inhibit the growth of bacteria, molds and yeasts in the food. Some of the additives are manufactured from the natural sources such as corn, beet and soybean, while some are artificial, man-made additives. Most people tend to eat the ready-made food available in the market, rather than preparing it at home. Such foods contain some kind of additives and preservatives, so that their quality and flavor is maintained and they are not spoiled by bacteria and yeasts. More than 3000 additives and preservatives are available in the market, which are used as antioxidants and anti-microbial agents. Salt and sugar the most commonly used additives. Some of the commonly used food additives and preservatives are aluminum silicate, amino acid compounds, ammonium carbonates, sodium nitrate, propyl gallate, butylated hydrozyttoluene (BHT), butylated hydroxyanisole (BHA), monosodium glutamate, white sugar, potassium bromate, potassium sorbate, sodium benzoate, etc. Some artificial colors are also added to the foods to give them an appealing

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look. These coloring substances are erythrosine (red), cantaxanthin (orange), amaranth (Azoic red), tartrazine (Azoic yellow) and annatto bixine (yellow orange). When the food is to be stored for a prolonged period, use of additives and preservatives is essential in order to maintain its quality and flavor. The excess water in the foods can cause the growth of bacteria, fungi and yeasts. Use of additives and preservatives prevents spoiling of the foods due to the growth of bacteria and fungi. Additives and preservatives maintain the quality and consistency of the foods. They also maintain palatability and wholesomeness of the food, improve or maintain its nutritional value, control appropriate pH, provide leavening and color, and enhance its flavor. There are even foods products that are made entirely from chemicals. Coffee creamers, sugar substitutes, and candies consist almost completely of artificial ingredients. Such manipulation of our food can have a profound effect on our body’s unique biochemical balance. When we need to store any food for a longer time, it should be properly processed. During this processing, some substances and chemicals, known as additives, are added to the food. Additives consistently maintain the high quality of foods. Vegetable gums

Sweeteners

Stabilizers

Tracer gas

Sequestrants

Thickeners

Seasonings

Propellants

Firming agents

Preservatives

Mineral salts

Food additives

Emulsifiers

Humectants

Glazing agents

Coloring agents

Color retention agents

Flavors

Flavor Enhancers

Flour treatement agents

Food acids

Gelling agents

Color fixative

Bulking agents

Antioxidants

Antifoaming agents

Anti-caking agents

Acidity regulator

Fig. 1. Showing classification of food additives

2. Classification of food additives Additives are classified as antimicrobial agents, antioxidants, artificial colors, artificial flavors and flavor enhancers, chelating agents and thickening and stabilizing agents (Fig. 1). Antimicrobial agents such as salt, vinegar, sorbic acid and calcium propionate are used

Food Additive

3

in the products such as salad dressings, baked goods, margarine, cheese and pickled foods. Antioxidants including vitamin C, E, BHT and BHA are used in the foods containing high fats. Chelating agents such as malic acid, citric acid and tartaric acid are used to prevent the flavor changes, discoloration and rancidity of the foods. These are very important in food manufacturing companies. The food Additives is used to retard spoilage, enhance food flavors, replace nutrient lost in processing and makes the food more visually appealing. 2.1 Acidity regulator Acidity regulators are used to change or otherwise control the acidity and alkalinity of foods. Types of acidity regulator i. acid ii. acidifier iii. acidity regulator iv. alkali v. base vi. buffer vii. buffering agent viii. pH adjusting agent 2.2 Anti-caking agents Anti caking agents, prevents the formation of lumps making these products manageable for packaging, transport, and for use by end consumer. Anticaking Agent is the food additive that prevents agglomeration in certain solids, permitting a free-flowing condition. It reduces the tendency of particles of food to adhere to one another. Types of anti-caking agent i. ii. iii. iv.

Anti-caking agent Anti-stick agent Drying agent Dusting agent

Anti-caking agents consist of such substances as starch, magnesium carbonate, and silica and are added to fine-particle solids, such as food products like table salt, flours, coffee, and sugar. Some of the common examples of foods that contain anti-caking agents include: i. Vending machine powders (coffee, cocoa, soup) ii. Milk and cream powders iii. Grated cheese iv. Icing sugar v. Baking powder vi. Cake mixes vii. Instant soup powders viii. Drinking chocolate ix. Table salt

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2.3 Antifoaming agents Antifoaming agents reduce or prevent foaming in foods. Types of anti- foaming agent i. ii.

Antifoaming agent Defoaming agent

2.4 Antioxidants A food additive, which prolongs the shelf-life of foods by protecting against deterioration caused by oxidation. Antioxidants are used to preserve food for a longer period of time. Antioxidants act as oxygen scavengers as the presence of oxygen in the food helps the bacteria to grow that ultimately harm the food. In the absence of antioxidant food additive oxidation of unsaturated fats takes place rendering to foul smell and discoloration of food. Different kinds of antioxidants foods act in a different ways but the end result is to delay or minimize the process of oxidation in food. Some antioxidants foods additives combine with oxygen to prevent oxidation and other prevent the oxygen from reacting with the food leading to its spoilage. Types of anti-oxidant agent i. Anti-browning agent ii. Antioxidant iii. Antioxidant synergist Some popular antioxidant foods Antioxidant vitamins a) Ascorbic acid- E300 Antioxidant vitamins include Ascorbic acid (vitamin C) this antioxidant vitamin is used in beers, cut fruits, dried potatoes and jams. The antioxidant vitamins in these foods helps in preventing the discoloration of food by preventing the oxidation. It can also act as a substitute of vitamin C in potatoes that is lost during processing. b) Citric acid - E330 It is used in biscuits, jams, tinned fruits, alcoholic drinks, cheese and dried soup. It has many uses like it prevents the discoloration of food, increases the anti-oxidant effect of other substances and regulates pH in jams and jellies. c) Tocopherols – E307 (307a, d-alpha-Tocopherol; 307b, Tocopherol concentrate and 307c, dl-alpha-Tocopherol) This antioxidant food additive is used in the meat pies and oils to reduce the oxidation of fatty acids and vitamins. d) Butylated hydroxyanisole (BHA) - E320 It is used in margarine, oils, crisps and cheese. This antioxidant helps in preventing the reactions leading to the breakdown of fats.

Food Additive

5

Antioxidants benefits There are many benefits of using antioxidant food additives. Antioxidants prevent the blockage of arteries with fatty deposits that prevents the heart-attacks. Also these are associated with the prevention of certain types of cancers, arthritis and more conditions of these kinds. 2.5 Bulking agents A food additive, which contributes to the bulk of a food without contributing significantly to its available energy value. Bulking agents such as starch are additives that increase the bulk of a food without affecting its nutritional value. Types of bulking agents i. ii.

Bulking agent Filler

2.6 Color retention agents A food additive, which stabilizes, retains or intensifies the colour of a food. In contrast to colorings, color retention agents are used to preserve a food's existing color. Types of color retention agents i. ii. iii. iv.

Color adjunct Colour fixative Colour retention agent Colour stabilizer

2.7 Coloring A food additive, which adds or restores colour in a food. Colorings are added to food to replace colors lost during preparation, or to make food look more attractive, more visually appealing. Types of coloring agents i. Colour ii. Decorative pigment iii. Surface colorant Beta carotene, Caramel, Carrot oil, Citrus red # 1, Dehydrated beets, FD&C colors: Blue # 1, 2; Red # 3, 40; Yellow # 5, 6 - used in processed foods, especially sweets and products marketed for children, soft drinks, baked goods, frosting, jams, and margarine. Though there is a growing realization that the color additives should be used to the minimum, the fact is that the food doesn't even look presentable at times without it and appears inedible.

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2.8 Emulsifiers A food additive, which forms or maintains a uniform emulsion of two or more phases in a food. Emulsifiers allow water and oils to remain mixed together in an emulsion, as in mayonnaise, ice cream, and homogenized milk. It stops fats from clotting together. Types of emulsifiers i. ii. iii. iv. v. vi. vii. viii.

Clouding agent Crystallization inhibitor Density adjustment agent (flavouring oils in beverages) Dispersing agent Emulsifier Plasticizer Surface active agent Suspension agent

2.9 Emulsifying salt A food additive, which, in the manufacture of processed food, rearranges proteins in order to prevent fat separation. Types of emulsifying salt i. ii.

Emulsifying salt Melding salt

2.10 Firming agents A food additive, which makes or keeps tissues of fruit or vegetables firm and crisp, or interacts with gelling agents to produce or strengthen a gel. 2.11 Flavors Flavors are additives that give food a particular taste or smell, and may be derived from natural ingredients or created artificially. 2.12 Flavor enhancers Flavor enhancers enhance a food's existing flavors. They may be extracted from natural sources (through distillation, solvent extraction, maceration, among other methods) or created artificially. Types of flavor enhancing agents i. ii.

Flavour enhancer Flavour synergist

Some flavor enhancers are as follows: a) b)

Dioctyl sodium-sulfosuccinate - used in processed foods. Disodium guanylate - used in canned meats, meat based foods.

Food Additive

c) d)

7

Hydrolyzed vegetable - used in mixes, stock, processed meats. Monosodium glutamate (MSG) - used in Chinese food, dry mixes, stock cubes, and canned, processed, and frozen meats.

2.13 Flour treatment agents A food additive, which is added to flour or dough to improve its baking quality or colour. Types of flour treatment agent i. ii. iii. iv. v.

Dough conditioner Dough strengthening agent Flour bleaching agent Flour improver Flour treatment agent

2.14 Food acids Food acids are added to make flavors "sharper", and also act as preservatives and antioxidants. Common food acids include vinegar, citric acid, tartaric acid, malic acid, fumaric acid, and lactic acid. 2.15 Gelling agents Gelling agents are food additives used to thicken and stabilize various foods, like jellies, desserts and candies. The agents provide the foods with texture through formation of a gel. Some stabilizers and thickening agents are gelling agents. 2.16 Glazing agents A food additive, which when applied to the external surface of a food, imparts a shiny appearance or provides a protective coating. Glazing agents provide a shiny appearance or protective coating to foods. Types of glazing agent i. ii. iii. iv. v. vi.

Coating agent Film forming agent Glazing agent Polishing agent Sealing agent Surface-finishing agent

2.17 Humectants A food additive, which prevents food from drying out by counteracting the effect of a dry atmosphere. Types of humectants i. Humectant ii. Moisture/water retention agent iii. Wetting agent

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2.18 Mineral salts Mineral salts are added as nutritional additives though they may have other properties like an anti-oxidant or a preservative. Many of them are essentials that need to be included in our daily diets, as they are the source of important nutrients required for the body. The important natural mineral salts that should be consumed are sodium, phosphorus, potassium, chlorine, sulphur and calcium. While the above mentioned happen to be the macro elements of the natural mineral salts, the micro elements are the ones that are essential nutrients for the human body. The micro elements in the minerals salts consist of iodine, iron, fluoride and zinc. 2.19 Preservatives A food additive, which prolongs the shelf-life of a food by protecting against deterioration caused by microorganisms. It prevents or inhibits spoilage of food due to fungi, bacteria and other microorganisms. It stops microbes from multiplying and spoiling the food. Types of preservatives i. ii. iii. iv. v. vi. vii.

Antimicrobial preservative Antimicrobial synergist Antimould and antirope agent Antimycotic agent Bacteriophage control agent Fungistatic agent Preservative

Some preservatives are: a) b)

c)

Benzoic acid and benzoates - are found in soft-drinks, beer, margarine and acidic foods. They are use to extend shelf life and protect food from fungi and bacteria. Nitrites and nitrates - are found in processed meats, such as sausages, hot dogs, bacon, ham, and luncheon meats, smoked fish. They are used to extend shelf life and protect food from fungi and bacteria; preserve color in meats and dried fruits. Sulfites - are found in dried fruits, shredded coconut, fruit based pie fillings. They are used to extend shelf life and protect food from fungi and bacteria.

2.20 Propellants It helps propel food from a container. 2.21 Seasonings Seasoning is the process of imparting flavor to, or improving the flavor of food. 2.22 Sequestrants A sequestrant is a food additive whose role is to improve the quality and stability of the food products. Sequestrants form chelate complexes with polyvalent metal ions, especially copper, iron and nickel, which serve as catalysts in the oxidation of the fats in the food. Sequestrants are a kind of preservative.

Food Additive

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2.23 Stabilizers A food additive, which makes it possible to maintain a uniform dispersion of two or more components. Stabilizers, like agar or pectin (used in jam for example) give foods a firmer texture. While they are not true emulsifiers, they help to stabilize emulsions. i. ii. iii. iv.

Colloidal stabilizer Emulsion stabilizer Foam stabilizer Stabilizer

2.24 Sweeteners Sweeteners are added to foods for flavoring. Sweeteners other than sugar are added to keep the food energy (calories) low, or because they have beneficial effects for diabetes mellitus and tooth decay and diarrhea. These are the substances that sweeten food, beverages, medications, etc., such as sugar, saccharine or other low-calorie synthetic products. They in general can be termed as sweetening agents. They all are called artificial sweeteners as they are usually not a component of the product they are added to. As per the source, these substances can be classified as natural and artificial sweeteners. Natural sweeteners are obtained from the natural sources like sugarcane and sugar beet and from fruits (fructose) and the artificial ones have a chemical origin. Artificial sweeteners are further of two type namely non-caloric sweeteners and sugar alcohols. Noncaloric sweeteners do not add calories to foods. They are used in snack foods and drinks. Sweeteners like saccharine and aspartame fall under this category. Sugar alcohols are used in chewing gums and hard candies and have almost same calories as sugar. Examples of sugar alcohols are sorbitol and mannitol. Commonly used sweeteners a)

b)

c)

d)

Acesulfame K - It is a 0 calorie sweetener, 130- 200 times sweeter than sucrose. It is not metabolized by the body. The only limitation it has is that if used in large quantities, it has an after taste. It is used in fruit preserves, dairy products and all types of beverages. It is used to reduce the calories of the products. It is heat resistant and enhances flavors. Aspartame - It is a low calorie sweetener about 200% more sweet than the sugar. It is disintegrated into aspartic acid, fenylalanine and methanol in the body on digestion. It's taste is similar to sugar only more sweet. It is used in all types of foods and beverages and medicines. It is found naturally in protein rich foods. Cyclamate - This is a calorie free sweetener 30-50 times sweeter than sugar. It is metabolized in the gut by few individuals and generally expelled as such. It is generally used in combination with other sweeteners. It has a pleasant taste, and is stable at high temperatures and is economical. Saccharin - It is one of the earliest low calorie sweeteners that is 300-500 times more sweet than sugar. It doesn't metabolize and absorption is slow. Owing to this it is expelled as such from the body. Saccharin is the most widely used sweetener. It was earlier banned in certain countries but now is used quite commonly. There are other sweeteners like Stevioside, Alitame, Thaumatin, Sucralose, Neohesperidine DC and Aspartame-Acesulfame Salt. All artificial sweeteners have been approved by the U.S. Food and Drug Administration (FDA). They are considered harmless if taken in limited quantities.

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2.25 Thickeners Thickeners are substances which, when added to the mixture, increase its viscosity without substantially modifying its other properties. Types of thickners i. ii. iii. iv.

Binder Bodying agent Texturizing agent Thickener

2.26 Tracer gas Tracer gas allows for package integrity testing preventing foods from being exposed to atmosphere, thus guaranteeing shelf life. 2.27 Vegetable gums Vegetable gums come from the varied sources that can be on land or in sea. Some of the seaweeds are the excellent sources of food gums in which comes the carrageenan and alginates. Whereas guar, locust bean gum, pectin are obtained from the plants. Xanthan gum is obtained by the process of microbial fermentation. The source of gelatin is animal tissue. Vegetable gums are the polysaccharides that have the natural origin and used to increase the viscosity of the solution or food even if used in a very small concentration. Major Vegetable Gums are: a. b. c. d.

Cellulose Gum Xanthan Gum Locust Bean Gum Pectin

3. ‘E’ numbering To regulate these additives, and inform consumers, each additive is assigned a unique number, termed as "E numbers", which is used in Europe for all approved additives. This numbering scheme has now been adopted and extended by the Codex Alimentarius Commission to internationally identify all additives, regardless of whether they are approved for use. E numbers are all prefixed by "E", but countries outside Europe use only the number, whether the additive is approved in Europe or not. For example, acetic acid is written as E260 on products sold in Europe, but is simply known as additive 260 in some countries. Additive 103, alkanet, is not approved for use in Europe so does not have an E number, although it is approved for use in Australia and New Zealand. Since 1987, Australia has had an approved system of labelling for additives in packaged foods. Each food additive has to be named or numbered. The numbers are the same as in Europe, but without the prefix 'E'. The United States Food and Drug Administration listed these items as "Generally recognized as safe" or GRAS; they are listed under both their Chemical Abstract Services number and Fukda regulation under the US Code of Federal Regulations.

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4. Dangers of food additives and preservatives Although additives and preservatives are essential for food storage, they can give rise to certain health problems. They can cause different allergies and conditions such as hyperactivity and Attention Deficit Disorder in the some people who are sensitive to specific chemicals. The foods containing additives can cause asthma, hay fever and certain reactions such as rashes, vomiting, headache, tight chest, hives and worsening of eczema. Some of the known dangers of food additives and preservatives are as follows: •

• •

•

•

• • •

•

Benzoates can trigger the allergies such as skin rashes and asthma as well as believed to be causing brain damage. Bromates destroy the nutrients in the foods. It can give rise to nausea and diarrhea. Butylates are responsible for high blood cholesterol levels as well as impaired liver and kidney function. Caffeine is a colorant and flavorant that has diuretic, stimulant properties. It can cause nervousness, heart palpitations and occasionally heart defects. Saccharin causes toxic reactions and allergic response, affecting skin, gastrointestinal tract and heart. It may also cause tumors and bladder cancer. Red Dye 40 is suspected to cause certain birth defects and possibly cancer. Mono and di-glycerides can cause birth defects, genetic changes and cancer. Caramel is a famous flavoring and coloring agent that can cause vitamin B6 deficiencies. It can cause certain genetic defects and even cancer. Sodium chloride can lead to high blood pressure, kidney failure, stroke and heart attack.

To minimize the risk of developing health problems due to food additives and preservatives, you should avoid the foods containing additives and preservatives. Before purchasing the canned food, you must check its ingredients. You should buy organic foods, which are free from artificial additives. Try to eat the freshly prepared foods as much as possible rather than processed or canned foods.

5. Effects of food additives Avoiding or minimizing toxins in your diet is an important step toward enhancing your health and lowering your risk of disease. Foods, amongst other things (cosmetics & medications), represent a source of these toxins. Effects of food additives may be immediate or may be harmful in the long run if you have constant exposure. Immediate effects may include headaches, change in energy level, and alterations in mental concentration, behavior, or immune response. Long-term effects may increase your risk of cancer, cardiovascular disease and other degenerative conditions. Although it may seem difficult to change habits and find substitutes for foods you enjoy, remind yourself that you will be adding to your diet some new wholesome foods that you will come to enjoy even more. Look for foods that are not packaged and processed, but enjoy nature’s own bounty of fresh fruits, vegetables, grains, beans, nuts and seeds. Find foods that resemble what they looked like when they were originally grown.

6. Cytotoxic effects of food additives Exposure to non-nutritional food additives during the critical development window has been implicated in the induction and severity of behavioral disorders such as attention

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deficit hyperactivity disorder (ADHD). Although the use of single food additives at their regulated concentrations is believed to be relatively safe in terms of neuronal development, their combined effects remain unclear. The neurotoxic effects of four common food additives in combinations of two (Brilliant Blue and L-glutamic acid, Quinoline Yellow and aspartame) has been assess forpotential interactions. Mouse NB2a neuroblastoma cells were induced to differentiate and grow neurites in the presence of additives. After 24 h, cells were fixed and stained and neurite length measured by light microscopy with computerized image analysis. Neurotoxicity was measured as an inhibition of neurite outgrowth. Two independent models were used to analyze combination effects: effect additivity and dose additivity. Significant synergy was observed between combinations of Brilliant Blue with Lglutamic acid, and Quinoline Yellow with aspartame, in both models. Involvement of Nmethyl-D-aspartate (NMDA) receptors in food additive-induced neurite inhibition was assessed with a NMDA antagonist, CNS-1102. L-glutamic acid- and aspartame induced neurotoxicity was reduced in the presence of CNS-1102; however, the antagonist did not prevent food color-induced neurotoxicity. Theoretical exposure to additives was calculated based on analysis of content in foodstuff, and estimated percentage absorption from the gut. Inhibition of neurite outgrowth was found at concentrations of additives theoretically achievable in plasma by ingestion of a typical snack and drink. In addition, Trypan Blue dye exclusion was used to evaluate the cellular toxicity of food additives on cell viability of NB2a cells; both combinations had a straightforward additive effect on cytotoxicity. These data have implications for the cellular effects of common chemical entities ingested individually and in combination. (Lau et al., 2006) Gallic acid is added to foods to prevent oxygen-induced lipid peroxidation and can be obtained by the hydrolysis of tannic acid which can be found in tea, coffee, red wine, and immature fruits. Tannic acid has also been used as a food additive. The effect of gallic acid on mouse spermatogonia, mouse spermatocytes, and mouse Sertoli cells in vitro was investigated. First, each cell line was cultured with predetermined concentrations of gallic acid for 3 h to access the effects of gallic acid on in vitro growth of testicular cells and MTT cytotoxicity assay was used to measure cell viability. Secondly, intracellular levels of hydrogen peroxide in mouse spermatogonia, mouse spermatocytes, and mouse Sertoli cells treated with gallic acid were analyzed using dihydrorhodamine 123 as a probe to evaluate the pro-oxidative property of gallic acid. The results obtained indicate that gallic acid inhibits the growth and proliferation of testicular cells in a dose-dependent manner and increases the intracellular level of hydrogen peroxide in mouse spermatogonia significantly (p < 0.05). It can be suggested that gallic acid exerts cytotoxic effects on testicular cells by its pro-oxidative activity. In conclusion, gallic acid-induced cytotoxicity in mouse spermatogonia, mouse spermatocytes, and mouse Sertoli cells in vitro may be of toxicological research interest considering the testicular toxic potential of gallic acid. (Park et al., 2008) In recent years, the use of carthami flos (the flowers of Carthamus tinctorius) as a colouring and flavouring agent in foods has increased in Iran. In order to evaluate its safety, the teratogenic effects of carthami flos on central nervous system development in mice were investigated. Furthermore, its cytotoxic effect on a rat nerve cell culture was studied to complete safety evaluations. For teratogenic studies, after natural mating, pregnant mice were divided into test and control groups. The groups were treated with different dosage

Food Additive

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regimens of aqueous carthami flos extract during 0-8 days of gestation. Embryos were then isolated at the 13th day of gestation and evaluated for macroscopic, microscopic and morphometric characteristics. The results showed that at higher doses (1.6 and 2 mg/kg/day) the embryos were absorbed, whereas at a lower dose (1.2 mg/kg/day) changes in external, internal and longitudinal diameters, open neuropore, changes in cellular orientation and cellular degeneration were observed. The results obtained from the cytotoxic assay also demonstrated a concentration-dependent cytotoxic effect of carthami flos extract. It is concluded that the use of carthami flos as a food additive should be reconsidered. (Nobakht et al., 2000) The cytotoxicity of 11 dyes, used as food dyes in Japan, on cultured fetal rat hepatocytes was studied. Xanthene dyes containing halogen atoms in their molecules such as phloxin, rose bengal, and erythrosine were more toxic than other groups of food dyes. The effect of food dyes on the cell growth of hepatocytes was also examined. Phloxin was especially toxic to the cell growth and a dose-response relation was observed between the concentration of phloxin and the cell growth of hepatocytes when the dye was added 3 days after plating. (Sako et al., 1980) The synergistic effect of food additives or food colors on the toxicity of 3-amino-1,4dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was investigated using primary cultured rat hepatocytes. When hepatocytes from rats fed a standard diet were treated with a mixture of four major food additives (sorbitol, sodium 1.(+)-glutamate, benzoic acid, and propylene glycol) or a mixture of six typical artificial food colors (erythrosine, allura red, new coccine, brilliant blue, tertrazine, and fast green), the in vitro treated food-color mixture itself showed cytotoxicity: the reduction of cell viability and decreases in the activities of gluconeogenesis and ureogenesis. The food-color mixture enhanced cytotoxicity of Trp-P-1 obviously. The effects of in vivo-dosed food additives or food colors on Trp-P-1-caused toxicity. Hepatocytes were isolated and cultured from rats fed a diet containing a mixture of food additives or a mixture of food colors with half the amount of their respective acceptable daily intake for 4 wk. Trp-P-1 was administered to the hepatocytes at various concentrations for 12 h. Synergistic effects of in vivo-dosed food additives and food colors were not observed on Trp-P-1-caused cytotoxicity as estimated by a loss of cell viability and the reductions of DNA and protein syntheses. On the contrary, it was observed that in vivo administered food colors synergistically facilitated to reduce the activities of gluconeogenesis and ureogenesis in Trp-P-1-treated hepatocytes. These results suggest that the daily intake of artificial food colors may impair hepatic functions such as gluconeogenesis and ureogenesis, when dietary carcinogens are exposed to the liver cells. (Ashida et al., 2000) Study was conducted to investigate the impact of food additives like boric acid, citric acid and sodium metabisulphite individually, in different concentrations, on root tips of Vicia faba L. Cytological studies revealed significant decrease in mitotic index with an increase in concentration of the food additives. Most frequent cytological abnormalities observed are fragments, disturbed metaphase, C-mitosis, laggards, bridges, stickiness, precocious movement of chromosomes, unequal and late separation of chromosomes. Bridges and fragments were more frequent at anaphase. The percentage of chromosomal aberrations at mitosis increased with an increase in concentration of the food additives. (Pandey and Upadhyay, 2007)

14

Food Additive

a

b

c

d

e

f

g

h

i

Fig. 2. a-i — Showing cytological effects of food additives in Vicia faba L., a. Normal metaphase, b. Normal anaphase, c. Disturbed metaphase, d. C-mitosis, e. Late separation at anaphase, f. Unequal separation, g. Fragmentation at anaphase, h. Chromatid bridge, i. Stickiness and Laggard at anaphase Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes. Citric acid induced a significant increase of chromosomal aberrations (CAs) at all the concentrations and treatment periods tested. Citric acid significantly decreased mitotic index (MI) at 100 and 200 lg ml-1 concentrations at 24 h, and in all concentrations at 48 h. However, it did not decrease the replication index (RI) significantly. Citric acid also significantly increased sister chromatid exchanges (SCEs) at 100 and 200 lg ml-1 concentrations at 24 h, and in all concentrations at 48 h. This chemical significantly increased the micronuclei frequency (MN) compared to the negative control. It also decreased the cytokinesis-block proliferation index (CBPI), but this result was not statistically significant. (Serkan et al., 2008) Ester gum (EG) is used in citrus oil-based beverage flavourings as a weighting or colouring agent. In this study, concentrations of 50, 100 and 150 mg/kg body weight were administered orally to male Swiss albino mice, and sister chromatid exchange and chromosomal aberration were used as the cytogenetic endpoints to determine the genotoxic

Food Additive

15

and clastogenic potential of the food additive. Although EG was weakly clastogenic and could induce a marginal increase in sister chromatid exchange frequencies, it was not a potential health hazard at the doses tested. (Mukherjee et al., 1992) Acesulfame-K, a sweetening agent, was evaluated in vivo for its genotoxic and clastogenic potentials. Swiss albino male mice were exposed to the compound by gavage. Bone marrow cells isolated from femora were analysed for chromosome aberrations. Doses of 15, 30, 60, 450, 1500 and 2250 mg of acesulfame-K/kg body weight induced a positive dose-dependent significant clastogenicity (trend test α < 0.05). These doses were within the no-toxic-effect levels (1.5-3 g/kg body weight in rats) reported by the Joint Expert Committee for Food Additives of the World Health Organization and the Food and Agriculture Organization of the United Nations. In view of the present significant in vivo mammalian genotoxicity data, acesulfame-K should be used with caution. (Mukherjee and Chakrabarti, 1997) Aspartame and acesulfame-K, non-nutritive sweeteners, are permitted individually in diets and beverages. These sweeteners of different classes, used in combination, have been found to possess a synergistic sweetening effect. Whether they also have a synergistic genotoxic effect is unknown. Swiss Albino male mice were exposed to blends of aspartame (3.5, 35, 350 mg/kg body weight) and acesulfame-K (1.5, 15 and 150 mg/kg body weight) by gavage. Bone marrow cells isolated from femora were analyzed for chromosome aberrations. Statistical analysis of the results show that aspartame in combination with acesulfame-K is not significantly genotoxic. (Mukhopadhyay et al., 2000) This work aimed to study some blood indices of rats as affected by saccharin and the therapeutic action of vitamins C and E. The used adult female Rattus norvegicus albino rats in the present study were weighing 100—120 g. Administration of saccharin at a dose of 35 mg kg—1 body weight (b.wt.) day—1 for 35 days significantly decreased serum glucose, triglycerides, cholesterol, total protein and albumin values. These decrements were by 20.16%, 22.76%, 44.92%, 20.16% and 40.44%, respectively, compared to control level (p value < 0.01). But it increased levels of kidney function indices. The effect of saccharin was more pronounced on creatinine. Activities of Alanine aminotranferease (ALT), aspartate aminotransferase (AST) and Alkaline phosphatase (ALP) increased significantly following saccharin treatment to rats. Concerning hematoligical parameters, the more obvious changes were observed in the increment of white blood cell (WBC), mean corpuscular volume (MCV) and platelets (PLT) and the decrease in hematocrit, hemoglobin (Hb) and red blood cells (RBCs) count in response to the administration of saccharin. In general, vitamin C or E (150 mg kg—1 b.wt. day—1 for 35 days) was able to reduce the effects of saccharin intake. Both vitamins, however, generally have beneficial effects in reducing the changes in the studied parameters. (Abdelaziz et al., 2011) Monosodium phosphate (MSP - E339i), disodium phosphate (DSP - E339ii), and trisodium phosphate (TSPE339iii) are used as antimicrobials, pH control agents (buffers), boiler water additives, cleaners, coagulants, dispersants, leavening agents, stabilizers, emulsifers, sequestrants, texturizers, nutrients, and dietary supplements. The effects of these have been studied on root tips of Allium cepa L. Roots of A. cepa were treated with a series of concentrations, ranging from 300 to 500 ppm for 24, 48 and 72 h. The results indicated that these food preservatives reduced mitotic division in A. cepa when compared with the respective control. Mitotic index values were generally decreased with increasing concentrations and longer treatment times. Additionally, variations in the percentage of

16

Food Additive

mitotic stages were observed. The total percentage of aberrations generally increased with increasing concentrations of these chemicals and longer period of treatment. Different abnormal mitotic figures were observed in all mitotic phases. Among these abnormalities were stickiness, anaphase bridges, C-mitosis and micronuclei. These food additives remarkably depressed the DNA content in the root meristems of A. cepa. The interphase nuclear volume (INV) also varied between the untreated (controls) and the treated plants. (Turkoglu Sifa, 2009) Citric acid is used widely as an acidulant, pH regulator, flavour enhancer, preservative and antioxidant synergist in many foods, like soft drinks, jelly sweet, baked nutrients, jam, marmalade, candy, tinned vegetable and fruit food Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes. Citric acid induced a significant increase of chromosomal aberrations (CAs) at all the concentrations and treatment periods tested. Citric acid significantly decreased mitotic index (MI) at 100 and 200 lg ml-1 concentrations at 24 h, and in all concentrations at 48 h. However, it did not decrease the replication index (RI) significantly. Citric acid also significantly increased sister chromatid exchanges (SCEs) at 100 and 200 lg ml-1 concentrations at 24 h, and in all concentrations at 48 h. This chemical significantly increased the micronuclei frequency (MN) compared to the negative control. It also decreased the cytokinesis-block proliferation index (CBPI), but this result was not statistically significant. (Serkan Yılmaz et al., 2008)

7. Food additives and safety With the increasing use of processed foods since the 19th century, there has been a great increase in the use of food additives of varying levels of safety. This has led to legislation in many countries regulating their use. For example, boric acid was widely used as a food preservative from the 1870s to the 1920s, but was banned after World War I due to its toxicity, as demonstrated in animal and human studies. During World War II the urgent need for cheap, available food preservatives led to it being used again, but it was finally banned in the 1950s.[2] Such cases led to a general mistrust of food additives, and an application of the precautionary principle led to the conclusion that only additives that are known to be safe should be used in foods. In the USA, this led to the adoption of the Delaney clause, an amendment to the Federal Food, Drug, and Cosmetic Act of 1938, stating that no carcinogenic substances may be used as food additives. However, after the banning of cyclamates in the USA and Britain in 1969, saccharin, the only remaining legal artificial sweetener at the time, was found to cause cancer in rats. Widespread public outcry in the USA, partly communicated to Congress by postage-paid postcards supplied in the packaging of sweetened soft drinks, led to the retention of saccharin despite its violation of the Delaney clause. In September 2007, research financed by Britain’s Food Standards Agency and published online by the British medical journal The Lancet, presented evidence that a mix of additives commonly found in children’s foods increases the mean level of hyperactivity. The team of researchers concluded that “the finding lends strong support for the case that food additives exacerbate hyperactive behaviors (inattention, impulsivity and overactivity) at least into middle childhood.” That study examined the effect of artificial colors and a sodium benzoate preservative, and found both to be problematic for some

17

Food Additive Colour Erythrosine FD&C Red No. 3

Status worldwide: Banned for use in cosmetics and external drug, but not food and ingested drugs in the U.S.

Tartrazine (E102) FD&C Yellow No. 5, Quinoline yellow (E104)

Banned in Norway and Austria.

* Banned in Australia, Japan, Norway and the U.S. Restricted to max. permitted levels in U.K. Banned in Norway, Sweden Sunset and Finland. Restricted to yellow maximum permitted levels (E110)* Yellow FCF in U.K. Orange Yellow S

Carmosine (E122)*

Allura red (E129)* FD&C Red No. 3

Banned in Canada, Japan, Norway, Austria, Sweden and the U.S. Restricted to maximum permitted levels in U.K. Banned in Denmark, Belgium, France, Germany, Switzerland, Sweden, Austria and Norway

Ponceau 4R Banned in US, Canada, Norway, Sweden and (E124)* Conchineal Japan. Restricted to maximum permitted levels in the UK Banned in the U.S. Amaranth (E123) Wine

Indigo Carmine (E132)*

Banned in the US, Japan, Australia and Norway. UK use restricted to maximum permitted levels

Brilliant Blue (E133)*

Banned in Austria, Belgium, France, Norway, Sweden, Switzerland and Germany. Restricted to maximum permitted levels in U.K.

Table 1. Colour additives to avoid

Where found: Cocktail, canned fruits salads confections dairy products snack foods. Ice cream Carbonated drinks Fish sticks

Possible negative effects: Cancer

References The Washington Post, February 7, 1990 CBS News, June 3, 2008

Hyperactivity, asthma, skin rashes, and migraine headaches.

Soft drinks Ice creams Candies Cosmetics medications

Asthma, rashes and hyperactivity. Potential carcinogen in animals: implicated in bladder and liver cancer. Altered reproduction in animals. AVOID in allergies & asthma. Cancer – DNA damage, increases tumors in animals. Growth retardation and severe weight loss in animals. DNA damage and tumours in animals.

UK Food Guide. http://www.ukfoo dguide.net/e102.ht m. Retrieved 2007 FDA, 2007 efsa.europa.eu EFSA updates safety advice on six food colours 091112

Sweets Snack foods Ice-creams, Yoghurts Drinks

Yoghurts Sweets

Carbonated drinks Bubble gum, snacks, Sauces, preserves, Soups, wine, cider, etc. Carbonated drinks Ice-creams Confectioneries Desserts Alcoholic drinks Fish roe

Ice-creams Sweets Baked goods Confectionery items Biscuits Dairy products Sweets Drinks

091113 efsa.europa.eu doi:10.1016/S01406736(07)

Food additives CBC News. 29 September 2008

UK Food Guide, a British food additives website. Last retrieved 20 May 2007 Cancer - DNA damage and Food And Drug Administration tumours in animals. Can Compliance produce bad reactions in Program Guidance asthmatics Manual p.10 May worsen or induce FDA/CFSAN Food asthma, allergies or hives. Compliance Program: Domestic Food Safety Program United States Food May cause nausea, vomiting, skin rashes, and and Drug Administration brain tumors. DNA damage and tumors in animals. FDA, 1993 Hyperactivity and skin rashes. Listed as human carcinogen by the US EPA. Causes DNA damage and tumors in animals May worsen or induce asthma, rhinitis (including hayfever), or urticaria (hives).

18

Food Additive

children. Further studies are needed to find out whether there are other additives that could have a similar effect, and it is unclear whether some disturbances can also occur in mood and concentration in some adults. In the February 2008 issue of its publication, AAP Grand Rounds, the American Academy of Pediatrics concluded that a low-additive diet is a valid intervention for children with ADHD: “Although quite complicated, this was a carefully conducted study in which the investigators went to great lengths to eliminate bias and to rigorously measure outcomes. The results are hard to follow and somewhat inconsistent. For many of the assessments there were small but statistically significant differences of measured behaviors in children who consumed the food additives compared with those who did not. In each case increased hyperactive behaviors were associated with consuming the additives. For those comparisons in which no statistically significant differences were found, there was a trend for more hyperactive behaviors associated with the food additive drink in virtually every assessment. Thus, the overall findings of the study are clear and require that even we skeptics, who have long doubted parental claims of the effects of various foods on the behavior of their children, admit we might have been wrong.” In 2007, Food Standards Australia New Zealand published an official shoppers' guidance with which the concerns of food additives and their labeling are mediated. There has been significant controversy associated with the risks and benefits of food additives. Some artificial food additives have been linked with cancer, digestive problems, neurological conditions, ADHD, heart disease or obesity. Natural additives may be similarly harmful or be the cause of allergic reactions in certain individuals. For example, safrole was used to flavor root beer until it was shown to be carcinogenic. Due to the application of the Delaney clause, it may not be added to foods, even though it occurs naturally in sassafras and sweet basil. Blue 1, Blue 2, Red 3, and Yellow 6 are among the food colorings that have been linked to various health risks. Blue 1 is used to color candy, soft drinks, and pastries and there has been some evidence that it may cause cancer. Blue 2 can be found in pet food, soft drinks, and pastries, and has shown to cause brain tumors in mice. Red 3, mainly used in cherries for cocktails has been correlated with thyroid tumors in rats and humans as well. Yellow 6, used in sausages, gelatin, and candy can lead to the attribution of gland and kidney tumors and contains carcinogens, but in minimal amounts. Some studies have linked some food additives to hyperactivity in children. A recent British study found that children without a history of any hyperactive disorder showed varying degrees of hyperactivity after consuming fruit drinks with various levels of additives. Among those that were studied were: Sodium benzoate (E211), Tartrazine (E102), quinoline yellow (E104), Sunset yellow (E110), Carmosine (E122), Allura red (E129). See tables below for more information.

8. Joint FAO/WHO Expert Committee on Food Additives (JECFA) The Joint FAO/WHO Expert Committee on Food Additives (JECFA) is an international scientific expert committee that is administered jointly by the Food and Agriculture Organization of the United Nations FAO and the World Health Organization WHO. It has been meeting since 1956, initially to evaluate the safety of food additives. Its work now also includes the evaluation of contaminants, naturally occurring toxicants and residues of veterinary drugs in food.

19

Food Additive

ii) Food preservatives to avoid: Sodium benzoate (E211)*

Sulphur Dioxide (E220)*

Not banned anywhere.

Sodium metabisulphite

Food Standards Agency issues revised advice on certain artificial colours, 2007

Carbonated drinks Pickles Sauces Certain medicines (even some “natural and homeopathic” medications for kids)

Aggravates asthma and suspected to be a neurotoxin and carcinogen, may cause fetal abnormalities. Worsens hyperactivity

Carbonated drinks Dried fruit Juices Cordials Potato products

International Chemical May induce gastric irritation, nausea, diarrhea, Safety Card 0074 asthma attacks, skin rashes. Destroys vitamin B1. Causes fetal abnormalities and DNA damage in animals.

Preservative and antioxidant.

May provoke life threatening asthma

http://www.fedupwithf oodadditives.info /factsheets/Factsafeaddi tives.htm

Potassium nitrate (E249)

Not banned anywhere

Cured meats and canned meat products.

May lower oxygen carrying capacity of blood; may combine with other substances to form nitrosamines that are carcinogens; may negatively effect the adrenal gland.

International Chemical Safety Card 1069

Calcium benzoate (E213)

Not banned anywhere

Drinks, low-sugar products, cereals, meat products.

May temporarily inhibit digestive enzyme function and may deplete levels of the amino acid glycine. AVOID with allergies, hives, & asthma.

http://www.fedupwithf oodadditives.info /factsheets/Factsafeaddi tives.htm

Calcium sulphite In the U.S., sulphites (E226) are banned from many foods, including meat

In a vast array of foodsfrom burgers to biscuits, from frozen mushrooms to horseradish. Used to make old produce look fresh.

May cause bronchial problems, flushing, low blood pressure, tingling, and anaphylactic shock. Avoid them if you suffer from bronchial asthma, cardiovascular or respiratory problems and emphysema.

http://www.fedupwithf oodadditives.info /factsheets/Factsafeaddi tives.htm

Butylated Hydroxy-anisole (E320) BHA/BHT

Particularly in fat containing foods, confectionery, meats.

doi:10.1021/jm00191a02 BHA/BHT is may be 0 carcinogenic to humans. BHA also interacts with nitrites to form chemicals known to cause changes in the DNA of cells.

Benzoic acid (E210)

Drinks, low sugar products, cereals, meat products.

International Chemical May temporarily inhibit digestive enzyme function. Safety Card 0103 May deplete glycine levels. AVOID in asthma, or allergies.

Table 2. Food preservatives to avoid

20

Food Additive

iii) Flavourings & sweeteners to avoid: Monosodium Gluatamate MSG (E621)* **

Not banned anywhere

Processed foods & drinks, soup mixes.

Aspartame (E951)*

US Air Force pilots are banned from drinking soft drinks containing aspartame.

200 times sweeter than sugar

Acesulphame K (E950)*

Not banned anywhere.

200 times sweeter than sugar

Saccharine (E954)*

Banned in Germany, Spain, Portugal, Hungary, France, Malaysia, Zimbabwe, Fiji, Peru, Israel, Taiwan.

350 times sweeter than sugar

High Fructose Corn Syrup (HFCS)

Not banned anywhere

Carbonated drinks other sweetened drinks (juices) baked goods candies canned fruits jams & jellies dairy products

Destroys nerve cells in brain and linked with aggravating or accelerating Huntington's, Alzheimer's and Parkinson's diseases. Causes cancer, DNA damage and fetal abnormalities in animals. Increases hyperactivity. May cause neurological damage, especially in younger children where brain is still developing. Breaks down in the body to phenylalanine (neurotoxin may cause seizures), aspartic acid (damages developing brain) and methanol (converts to formaldehyde). Crosses the placental barrier from mother to baby, even in small doses. Implicated in diseases such as MS and NonHodgkin's Lymphoma. May contribute to obesity. Causes cancer in animals. Linked to hypoglycemia, lung tumours, increased cholesterol and leukemia. May contribute to obesity May interfere with blood coagulation, blood sugar levels and digestive function. Causes cancer of the bladder, uterus, ovaries, skin and blood vessels in animals. Linked to DNA damage and congenital abnormalities in animals. May contribute to Obesity.

doi:10.1111/j.13652222.2009.03221.x

Obesity Accelerated aging Insulin resistance Diabetes mellitus Complications of diabetes Fatty liver Increased triglycerides Increased uric acid Chronic diarrhea Irritable bowel syndrome Hives

The American Medical Association, 2007

FDA Consumer Magazine, 1999

British Pharmacopoeia Commission Secretariat, 2009 USDA, 1972

*All of these additives are considered the “Dirty Dozen Food Additives” and are prohibited in the UK for foods marketed for children less than 36 months. **MSG-intolerant people can develop MSG symptom complex, which is characterized by one of more of the following: • A burning sensation in the back of the neck, forearms and chest. • Numbness in the back of the neck, radiating to the arms and back. • A tingling, warmth and weakness in the face, temples, upper back, neck and arms. • Facial pressure or tightness, swelling of lips/face • Chest pain, rapid heartbeat • Headache, nausea, drowsiness • Bronchospasm (difficulty breathing) in MSG-intolerant people with asthma.

Table 3. Flavourings & sweeteners to avoid

21

Food Additive

JECFA has evaluated more than 1500 food additives, approximately 40 contaminants and naturally occurring toxicants, and residues of approximately 90 veterinary drugs. The Committee has also developed principles for the safety assessment of chemicals in food that are consistent with current thinking on risk assessment and take account of recent developments in toxicology and other relevant sciences. Codex Alimentarius

Issues & Priorities

WHO, FAO Member states

Guidelines

Call of Data

JECFA Meeting

Publications

Experts

Fig. 3. Showing flow chart which shows the process flow for JECFA The above flow chart shows the process flow for JECFA. A brief summary of the purpose, history, and workings of the Joint FAO/WHO Expert Committee on Food Additives.

9. Sites related to JECFA •

•

JMPR The Joint FAO/WHO Meeting on Pesticide Residues is the related expert committee for pesticide residues in food. It is responsible for reviewing and evaluating toxicological residue and analytical aspects of the pesticides under consideration. Codex Alimentarius The Codex Alimentarius Commission was created in 1963 by FAO and WHO to develop food standards, guidelines and related texts such as codes of practice under the Joint FAO/WHO Food Standards Programme. The main purposes of this Programme are protecting health of the consumers and ensuring fair trade practices in the food trade, and promoting coordination of all food standards work undertaken by international governmental and non-governmental organizations. Codex standards are based on scientific advice as provided by JECFA and JMPR.

10. General information related to chemical risks in food Chemicals are the building blocks of life and are important for many, if not all, aspect of human metabolism. However, human exposure to chemicals at toxic levels, as well as

22

Food Additive

nutritional imbalances, are known or suspected to be involved in causing cancer, cardiovascular disease, kidney and liver dysfunction, hormonal imbalance, reproductive disorders, birth defects, premature births, immune system suppression, musculoskeletal disease, impeded nervous and sensory system development, mental health problems, urogenital disease, old-age dementia, and learning disabilities. Possibly a significant part of these disorders and diseases can be attributed to chemical exposure, and for many (environmental) chemicals food is the main source of human exposure. Consequently, the protection of our diet from these hazards must be considered one of the essential public health functions of any country. Under the World Trade Organization's Agreement on Sanitary and Phytosanitary Measures, food traded internationally must comply with Codex Standards that are established to protect health of consumers on basis of a sound risk assessment. Such independent international risk assessments are performed by Joint FAO/WHO Expert Committee on Food Additives (JECFA) and Joint FAO/WHO Meeting on Pesticide Residues (JMPR) as well as ad hoc expert meetings to address specific and emerging issues. The experts estimate a safe level of exposure (acceptable or tolerable daily intake ADI, TDI) and estimate the exposure to chemicals from the diet and from specific foods. Such exposure assessments often are based on national data or international data from the WHO Global Environment Monitoring System - Food Contamination Monitoring and Assessment Programme (GEMS/Food). Based on the risk assessments provided through these expert meetings, the Codex Alimentarius Commission can recommend specific measures, such as maximum limits in foods to assure that exposure do not exceed the acceptable/tolerable level of intake. Other measure can be the development of 'Codes of Practices' to reduce levels of contaminants in food. Also, levels of use for food additives can be recommended or maximum residue levels for pesticides or veterinary drug residues when applied in accordance with good practices. The scientific advice provided through these expert meetings also often serves directly as the basis for national food safety standards.

11. Conclusion Food additives preserve the freshness and appeal of food between the times it is manufactured and when it finally reaches the market. Additives may also improve nutritional value of foods and improve their taste, texture, consistency or color. All food additives approved for use in the United States are carefully regulated by federal authorities to ensure that foods are safe to eat and are accurately labeled. Food additives have been used by man since earliest times. Today, food and color additives are more strictly regulated than at any time in history. Additives may be incorporated in foods to maintain product consistency, improve or maintain nutritional value, maintain palatability and wholesomeness provide leavening or control acidity/alkalinity, and/or enhance flavor or impart desired color. The Food and Agriculture Organization (FAO), however, recognizes additives as any substance whose intended use will affect, or may reasonably be expected to affect, the characteristics of any food. FAO law prohibits the use of any additive that has been found to cause cancer in humans or animals. To market a new food or color additive, a manufacturer must first petition the FAO for its approval. FAO regulations require evidence

Food Additive

23

that each substance is safe at its intended levels of use before it may be added to foods. In deciding whether an additive should be approved, the agency considers the composition and properties of the substance, the amount likely to be consumed, its probable long-term effects and various safety factors. Some food additives like boric acid, citric acid and sodium metabisulphite showed mitotoxicity and genotoxicity having potential risks for human health. Thus, all additives are subject to ongoing safety review as scientific understanding and methods of testing continue to improve.

12. Acknowledgements Authors are thankful to Dr. C. S. Nautiyal, Director, CSIR-National Botanical Research Institute, Lucknow for encouragement. Thanks are also due to Ms. Rekha Singh, Project Assistant, Cytogenetics Laboratory, CSIR-NBRI for help.

13. References Added use level data, SPET calculations and indication of food categories with highest SPET as used in exposure estimations of flavourings at the 73rd JECFA meeting (2010), Complete Table Ashida H., Hashimoto T., Tsuji S. , Kanazawa K., Dasno G.I. (2000) Synergistic effects of food colors on the toxicity of 3-amino-1,4-dimethyl-5h-pyrido[4,3-b]indole (Trp-P-1) in primary cultured rat hepatocytes. Journal of nutritional science and vitaminology vol. 46, no3, pp. 130-136 Assessment of technologies for determining cancer risks from the environment. Darby, PA, USA: DIANE publishing. 1981. pp. 177. ISBN 142892437X. Bucci, Luke (1995). Nutrition applied to injury rehabilitation and sports medicine. Boca Raton: CRC Press. pp. 151. ISBN 0-8493-7913-X. Codex Alimentarius. "Class Names and the International Numbering System for Food Additives.". http://www.codexalimentarius.net/download/standards/7/CXG_036e.pdf. Evaluation of the carcinogenic hazards of food additives (Fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 220, 1961 (out of print). Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 228, 1962 (out of print). Evaluation of food additives: specifications for the identity and purity of food additives and their toxicological evaluation: some extraction solvents and certain other substances; and a review of the technological efficacy of some antimicrobial agents. (Fourteenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 462, 1971 (out of print). Evaluation of food additives: some enzymes, modified starches, and certain other substances: Toxicological evaluations and specifications and a review of the technological efficacy of some antioxidants (Fifteenth report of the Joint

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Food Additive

FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 488, 1972. Evaluation of certain food additives and the contaminants mercury, lead, and cadmium (Sixteenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 505, 1972, and corrigendum (out of print). Evaluation of certain food additives (Eighteenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 557, 1974, and corrigendum (out of print). Evaluation of certain food additives: some food colours, thickening agents, smoke condensates, and certain other substances. (Nineteenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 576, 1975 (out of print). Evaluation of certain food additives (Twentieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 599, 1976. English français español Evaluation of certain food additives (Twenty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 617, 1978. Evaluation of certain food additives and contaminants (Twenty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 631, 1978 (out of print). Evaluation of certain food additives (Twenty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 648, 1980, and corrigenda. Evaluation of certain food additives (Twenty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 653, 1980. Evaluation of certain food additives (Twenty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 669, 1981. Evaluation of certain food additives and contaminants (Twenty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 683, 1982. Evaluation of certain food additives and contaminants (Twenty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 696, 1983, and corrigenda (out of print). Evaluation of certain food additives and contaminants (Twenty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 710, 1984, and corrigendum. Evaluation of certain food additives and contaminants (Twenty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 733, 1986, and corrigendum. Evaluation of certain food additives and contaminants (Thirtieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 751, 1987. Evaluation of certain food additives and contaminants (Thirty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 759, 1987, and corrigendum.

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Evaluation of certain veterinary drug residues in food (Thirty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 763, 1988. Evaluation of certain food additives and contaminants (Thirty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 776, 1989. Evaluation of certain veterinary drug residues in food (Thirty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 788, 1989. Evaluation of certain food additives and contaminants (Thirty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 789, 1990, and corrigenda. Evaluation of certain veterinary drug residues in food (Thirty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 799, 1990. Evaluation of certain food additives and contaminants (Thirty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 806, 1991, and corrigenda. Evaluation of certain veterinary drug residues in food (Thirty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 815, 1991. Evaluation of certain food additives and naturally occurring toxicants (Thirty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series No. 828, 1992. Evaluation of certain veterinary drug residues in food (Fortieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 832, 1993. Evaluation of certain food additives and contaminants (Forty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 837, 1993. Evaluation of certain veterinary drug residues in food (Forty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 851, 1995. Evaluation of certain veterinary drug residues in food (Forty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 855, 1995, and corrigendum. Evaluation of certain food additives and contaminants (Forty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 859, 1995. Evaluation of certain veterinary drug residues in food (Forty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 864, 1996. Evaluation of certain food additives and contaminants (Forty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 868, 1997.

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Evaluation of certain veterinary drug residues in food (Forty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 876, 1998. Evaluation of certain veterinary drug residues in food (Forty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 879, 1998. Evaluation of certain food additives and contaminants (Forty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 884, 1999. Evaluation of certain veterinary drug residues in food (Fiftieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 888, 1999. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 891, 2000. Evaluation of certain veterinary drug residues in food (Fifty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 893, 2000. Evaluation of certain food additives and contaminants (Fifty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 896, 2000. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 900, 2001. Evaluation of certain food additives and contaminants (Fifty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 901, 2001. Evaluation of certain mycotoxins (Fifty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 906, 2002. Evaluation of certain food additives and contaminants (Fifty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 909, 2002. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 911, 2002. Evaluation of certain food additives (Fifty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 913, 2002. Evaluation of certain veterinary drug residues in food (Sixtieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series No. 918, 2003. Evaluation of certain food additives and contaminants (Sixty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 922, 2004.

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Evaluation of certain veterinary drug residues in food (Sixty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 925, 2004. Evaluation of certain food additives (Sixty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 928, 2005. Evaluation of certain food contaminants (Sixty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 930, 2006. Evaluation of certain food additives (Sixty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 934, 2006. Evaluation of certain veterinary drug residues in food (Sixty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 939, 2006. Evaluation of certain food additives and contaminants (Sixty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 940, 2007. Evaluation of certain food additives and contaminants (Sixty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 947, 2007. Evaluation of certain food additives (Sixty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 952, 2009. Evaluation of certain veterinary drug residues in food (Seventieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 954, 2009. Evaluation of certain food additives (Seventy-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 956, 2010 Evaluation of certain contaminants in food (Seventy-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 959, 2011 Evaluation of certain food additives and contaminants (Seventy-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 960, 2011 Fennema, Owen R. (1996). Food chemistry. New York, N.Y: Marcel Dekker. pp. 827. ISBN 08247-9691-8. Fumiyo Sako, Noriko Kobayashi, Hiroyuki Watabe and Naoyuki Taniguchi (1980) Cytotoxicity of food dyes on cultured fetal rat hepatocytes Toxicology and Applied Pharmacology Volume 54, Issue 2, Pages 285-292 General principles governing the use of food additives (First report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 129, 1957 (out of print). International Organization for Standardization. "67.220: Spices and condiments. Food additives".

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Ismael Abdelaziz, Abd El Rahiem,A Ashour (2011) Effect of saccharin on albino rats’ blood indices and the therapeutic action of vitamins C and E. Hum Exp Toxicol vol. 30 no. 2 129-137) Karen Lau, W. Graham McLean, Dominic P. Williams, and C. Vyvyan Howard (2006) Synergistic Interactions between Commonly Used Food Additives in a Developmental Neurotoxicity Test. TOXICOLOGICAL SCIENCES 90(1), 178–187 McCann, D; Barrett, A; Cooper, A; Crumpler, D; Dalen, L; Grimshaw, K; Kitchin, E; Lok, K et al. (2007). "Food additives and hyperactive behaviour in 3-year-old and 8/9-yearold children in the community: a randomised, double-blinded, placebo-controlled trial.". Lancet 370 (9598): pp. 1560–7. doi:10.1016/S0140-6736(07)61306-3. PMID 17825405. Mukherjee A., K. Agarwal and J. Chakrabarti (1992) Genotoxicity studies of the food additive ester gum. Food and Chemical Toxicology Volume 30, Issue 7, Pages 627630 Mukherjee A. and J. Chakrabarti (1997) In vivo cytogenetic studies on mice exposed to acesulfame-K—A non-nutritive sweetener. Food and Chemical Toxicology Volume 35, Issue 12, Pages 1177-1179 Mukhopadhyay K M., A. Mukherjee and J. Chakrabarti (2000) In vivo cytogenetic studies on blends of aspartame and acesulfame Food and Chemical Toxicology Volume 38, Issue 1, Pages 75-77 Nobakht, M., Fattahi, M., Hoormand, M., Milanian, I., Rahbar, N., Mahmoudian (2000) A study on the teratogenic and cytotoxic effects of safflower extract. M. Journal of Ethnopharmacology Vol. 73 No. 3 pp. 453-459 Pandey Ram Milan and Santosh Upadhyay, (2007) Impact of Food Additives on Mitotic Chromosomes of Vicia faba L. CARYOLOGIA Vol. 60, no. 4: 309-314 Procedures for the testing of intentional food additives to establish their safety for use (Second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 144, 1958 (out of print). Rev. Lyman Abbott (Ed.) (1900). The Outlook (Vol. 64). Outlook Co. pp. 403. Serkan Yılmaz, Fatma Ünal, Deniz Yüzbaşıoğlu and Hüseyin Aksoy (2008) Clastogenic effects of food additive citric acid in human peripheral lymphocytes. Cytotechnology 56:137–144) Specifications for identity and purity of food additives (antimicrobial preservatives and antioxidants) (Third report of the Joint FAO/WHO Expert Committee on Food Additives). These specifications were subsequently revised and published as Specifications for identity and purity of food additives, Vol. I. Antimicrobial preservatives and antioxidants, Rome, Food and Agriculture Organization of the United Nations, 1962 (out of print). Specifications for identity and purity of food additives (food colours) (Fourth report of the Joint FAO/WHO Expert Committee on Food Additives). These specifications were subsequently revised and published as Specifications for identity and purity of food additives, Vol. II. Food colours, Rome, Food and Agriculture Organization of the United Nations, 1963 (out of print).

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Specifications for the identity and purity of food additives and their toxicological evaluation: emulsifiers, stabilizers, bleaching and maturing agents (Seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 281, 1964 (out of print). English, pp. 1-100; English, pp. 101-189; Specifications for the identity and purity of food additives and their toxicological evaluation: food colours and some antimicrobials and antioxidants (Eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 309, 1965 (out of print). Specifications for the identity and purity of food additives and their toxicological evaluation: some antimicrobials, antioxidants, emulsifiers, stabilizers, flourtreatment agents, acids, and bases (Ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 339, 1966 (out of print). Specifications for the identity and purity of food additives and their toxicological evaluation: some emulsifiers and stabilizers and certain other substances (Tenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 373, 1967 (out of print). Specifications for the identity and purity of food additives and their toxicological evaluation: some flavouring substances and non-nutritive sweetening agents (Eleventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 383, 1968 (out of print). Specifications for the identity and purity of food additives and their toxicological evaluation: some antibiotics (Twelfth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 430, 1969 (out of print). Specifications for the identity and purity of food additives and their toxicological evaluation: some food colours, emulsifiers, stabilizers, anticaking agents, and certain other substances (Thirteenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 445, 1970 (out of print). Toxicological evaluation of certain food additives with a review of general principles and of specifications (Seventeenth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 539, 1974, and corrigendum (out of print). Turkoglu Si. fa (2009) Genotoxic effects of mono-, di-, and trisodium phosphate on mi¬totic activity, DNA content, and nuclear volume in Allium cepa L. CARYOLOGIA Vol. 62, no. 3: 171-179 U.S. Food and Drug Administration. (1993). Everything Added to Food in the United States. Boca Raton, FL: C.K. Smoley (c/o CRC Press, Inc.). Wansu Park, Mun Seog Chang, Hocheol Kim, Ho Young Choi, Woong Mo Yang, Do Rim Kim, Eun Hwa Park and Seong Kyu Park (2008) Cytotoxic effect of gallic acid on testicular cell lines with increasing H2O2 level in GC-1 spg cells. Toxicology in Vitro Volume 22, Issue 1, Pages 159-163

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Online References http://www.ncbi.nlm.nih.gov/pubmed/17825405?ordinalpos=7&itool=EntrezSystem2.PEn trez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDoc Sum. http://www.foodstandards.gov.au/newsroom/publications/choosingtherightstuff/. http://www.webmd.com/diet/features/the-truth-about-seven-common-food-additives http://www.sixwise.com/newsletters/06/04/05/12-dangerous-food-additives-the-dirtydozen-food-additives-you-really-need-to-be-aware-of.htm http://www.iso.org/iso/iso_catalogue/catalogue_ics/catalogue_ics_browse.htm?ICS1=67 &ICS2=220.

2 The Safety Assessment of Food Additives by Reproductive and Developmental Toxicity Studies Cansın Güngörmüş* and Aysun Kılıç* Hacettepe University/Department of Biology, Ankara Turkey 1. Introduction The overall consumption of food additives is 139 lbs/year/person. If the common additives like spices, sugars, salt, honey, pepper, mustard, dextrose etc. are excluded, the consumption decreases to 5 lbs/year. Due to widespread consumption, it is necessary to evaluate the implications for the health of consumers because of the presence of newly synthesized food additives before commence production according to accepted guidelines such as Food and Drug Administration (FDA), U.S. Environmental Protection Agency (EPA) and European Food Safety Authority (EFSA). “Redbook 2000” is one of the revised form of Redbook II guideline published in 1993 by FDA; also defined as “Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food”. This document is a guidance for determining toxicity studies, for designing and reporting the results of toxicity studies, conducting statistical analyses of data, the review of histological data and the submission of this information to FDA. The toxicological testing should provide not only information relevant to the average consumer, but also relevant to those population groups whose pattern of food consumption, physiological or health status may make them vulnerable such as young age, pregnancy and other metabolic disorders. Possible toxicological effects due to additive consumption should be tested especially in reproduction and developmental studies which are designed to evaluate effects on sexuality and fertility of males and females, developing organisms (mortality, structural abnormality and functional deficiencies). Besides, multigenerational reproductive toxicity studies provide information about the effects of a test substance on gonadal function, estrous cycle, mating behavior, lactation and development of the offspring. For new food additives, a safety evaluation is obtained generally from experimental data derived from investigations in laboratory animals. Although it may be possible to use human data derived from medical use, occupational epidemiology or from volunteers, the obtained data would be limited. Therefore, the likely effects on man can be estimated by intentive extrapolation from laboratory animals. The end points and the indices obtained must provide sufficient information and statistical power to permit FDA to determine whether the additive is associated with changes in reproduction and fertility. * Both authors have equal contribution in the chapter.

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This chapter will highlight the possible health effects of newly synthesized food additives on market and focus on their reproductive and developmental toxicity perspectives. Considering that food technology is a complex area in which even the simplest additive interacts with all the others to produce qualified food; we are expecting that this chapter will help to examine food additives that can probably affect the human life.

2. Overview of food additives Advances in food technology have resulted in an increased number of modified foods and additives in 20th century. An additive is a substance which may intentionally become a component of food or affect its characteristics. There are about 3000 different food additives defined up to date. Food additives may be divided in several groups; although there is some overlap between them. Main six categories of food additives are classified as preservatives, nutritional supplements, flavoring agents, colorings, texturing agents and miscellaneous. According to the functional classes, definitions and technological functions, food additives are summarized in Figure 1 (COABISCO, 2011).

Fig. 1. Six main categories of food additives Each food additive is assigned a unique E number, which have been assessed for use within the European Union (EU) to inform consumers (Figure 2). E numbers for European countries are all prefixed by “E”; on the other hand non-European countries do not use this prefix. E letter stands for the approval of the food additive in Europe. The numbering scheme fallows that of the International Numbering System (INS) as determined by Codex Alimentarius Committee (Codex Alimentarius, 2009). Though, only a subset of INS additives are approved for use in the EU. The United States Food and Drug Administration listed these items as “generally recognized as safe” or GRAS. As an example, additive E 341 (Tricalcium phosphate) is approved by US so has an “E” prefix and 341 numbering which stands for 340-349 subset known as “phosphates” under Antioxidants and Acidity Regulators group.

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Fig. 2. Classification of additives by numeric range In the EU, to authorize a substance as a food additive, a reasonable case of technological need, no hazard to consumers at level of proposed use and no misguidance to consumers should be demonstrated. To evaluate whether the newly released food additive has an effect on health; The European Commission is required a consultancy from Scientific Committee on Food (SCF). In this context, SCF deals with questions relating to the toxicology and hygiene in the entire food production chain for consumer health and food safety issues. For submission of a new food additive, the evaluation process by the SCF requires administrative, technical, toxicological data and references (European Commission Health & Consumer Protection Directorate, 2011). Among these, toxicological data obtained from experimental studies have a crucial role for consumers’ health due to the presence of any additive in food. During the general toxicological evaluation of food additives, the SCF first issued Guidelines for the Safety Assessment of Food Additives in 1980 (Scientific Committee for Food, 1980). However, new guidance documents have been published as Joint FAO/WHO Expert Committee on Food Additives (JECFA) (IPCS/JECFA, 1987), SCF guideline is still applicable. The aim of toxicological testing should provide sufficient information relevant to average consumer and vulnerable populations such as young age, pregnancy, diabetes, etc. The testing conditions depend on the chemical structure, proposed levels of use in food. The human data is derived from occupational epidemiology, medical use and volunteers but; for newly submitted food additives experimental data is commonly derived from laboratory animals. For evaluation of the safety of food additives, core studies are required such as; metabolism/toxicokinetics, subchronic toxicity, genotoxicity, chronic toxicity, carcinogenicity, reproduction and developmental toxicity. In this chapter, we are going to discuss the significant role of reproductive and developmental toxicity studies for evaluating new food additives.

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3. Traditional and newly released food additives “Toxicological Principles for the Safety Assessment of Food Ingredients” (Redbook 2000) is the new name of Redbook I which was previously published in draft form in 1983. Redbook 2000 provides information about toxicological data of food ingredients which are submitted to Center for Food Safety and Applied Nutrition and Office of Food Additive Safety for industry and other stakeholders. Food and color additives, food contact substances (which are also defined as indirect food additives) and substances classified as generally recognized as safe (GRAS) are the components of food ingredients (U.S. Food and Drug Administration, 2007). At the end of the toxicological studies data derived from animal studies can be used to extrapolate to give information on human exposure. Therefore, defining the Acceptable Daily Intake (ADI), described as the dose level at which the additive causes effects on the health of the animals, is important. The highest level at which no adverse effect on the health of the animals is observed is called the NOAEL (No-Observed-Adverse-Effect-Level). An ADI is derived by dividing the NOAEL obtained from these studies, by an appropriate ‘uncertainty’ factor, which is intended to take account of differences between the animals on which the additive was tested and humans, in order to reduce further possibility of risk to humans. This uncertainty factor is commonly 100 (assuming that human beings are 10 times more sensitive than test animals and that the different levels of sensitivity within the human population is in a 10 fold range), but may be as much as 1,000 (if, for example, the toxic effect in animals is found to be particularly severe) or as low as 10 (where it has been found that humans are less likely than animals to be affected, based on actual data on the additive in humans) (Food Safety Authority of Ireland, 2011).

4. Assessment of potential reproductive and developmental toxicity of recently used food additives Before a new food additive is introduced to the market, it should be tested if it causes any reproductive and developmental toxicity (EFSA, 2010a). To observe the potential effects, multigeneration reproduction studies have to be conducted. Laboratory species such as mouse, rabbit and especially rat are used at least for two generations and one litter per generation (Scientific Committee for Food, 1980). The test substance should be administrated in normal diet. On the other hand, two laboratory species, usually a rodent and a non-rodent should be used in developmental toxicity studies. The test substance should either be in normal diet or administered by oral gavage during whole gestation period in order to detect the potential toxicological effects. In addition to a multigenerational and/or developmental toxicity study; in order to provide the possible effects after postnatal development and function (such as neurological function and behavior), examinations should be continued from the beginning of embryogenesis through to weaning. 4.1 Reproductive toxicity studies The aim of a reproductive toxicity study is to ensure data about effects on the sexuality and fertility of males and females. These include reproductive behavior, pregnancy carriage

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ability, pre-postnatal survival rate, reproductive ability-capacity of the offspring and to examine major target organs for toxicity including reproductive organs in both parents and offspring histopathologically (Scientific Committee for Food, 1980). Besides, multigeneration reproductive toxicity studies ensure information about the effects on gonadal function, estrous cycles, conception, parturition, lactation (Joint FAO/WHO Expert Committee, 2000). Reproductive studies constitutively target multigenerational studies as a result of human exposure to most food additives and preservatives during the whole lifetime. Studies performed with multigeneration enable researchers to detect any potential effect of a specific additive on each litter per generation. The administration of the test substance to parental and offspring generations should be continuous via the diet. The end point evaluated in the indices calculated must provide sufficient information and statistical power to permit FDA whether the additive has effects on reproduction and fertility (U.S. Food and Drug Administration, 2007). The minimal reproduction study should consist at least two generations with one litter per generation. The reproduction study generation number should be expanded if the developmental toxicity effect of food additive is observed. A brief summary of the recommended reproduction study design according to FDA was given in Figure 3. In a basic two generation reproduction and teratology study, the first step is to find the appropriate dose range of the compound in order to conduct the main study. The second step, includes the selection of experimental animal species due to its’ life span, body size, breeding conditions, gestation length, high fertility rate etc. 5-9 weeks of aged animals, preferably rats are typically chosen. Each test and control group should include approximately 20 males and 20 pregnant females with uniform weight and age. For the detection of dose-related responses, minimum three doses of the test substance should be used: high, intermediate and low doses. The administration of the test substance may be via diet, drinking water or by gavage. In the first parental group (F0), males should be administered for the duration of spermatogenesis and epididymal transit before and throughout the mating period. First parental females should be administered for the same length of time as males and through pregnancy to the weaning of the F1a litter. Litters should be exposed throughout their entire lives. A female should be mated with a single randomly selected male from the same dose group until the pregnancy occurs or three weeks have elapsed. Each morning all females should be examined for the presence of vaginal plug or sperm in the vaginal lavage which is considered as “day zero” of gestation. Each animal should be observed twice each day at predefined time intervals. All animals should be weighted before administration, once weekly thereafter and at necropsy. During necropsy, the organs of reproductive system belonging to weanlings and parental animals (males-females) should be examined histopathologically. Uterus and ovaries for females; testis, seminal vesicle, prostate, epididymis for males should be weighted and evaluated separately. Brain, thymus and spleen tissues should also be examined for weanlings. Organ weights should be recorded both as absolute and relative weights. Indices which are the animal number responding to the test substance during conception until weaning period should be calculated for each reproduction study. To evaluate the endpoints of male reproductive toxicity, apart from counting testicular spermatid numbers, minimum 200 sperm per sample from cauda epididymis or proximal vas deferens should be examined. Acquired data from control and test groups of animals should be compared statistically using suitable statistics program.

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Fig. 3. A reproduction study design by FDA According to the recommendations of FDA (U.S. Food and Drug Administration, 2007), a toxicity study should be conducted according to Good Laboratory Practice Regulations. The animals used in the study should be well cared and housed according to the recommendations of Guide for the Care and the Use of Laboratory Animals. All test animals should be categorized according to their species, strain, sex and weight or age. 4.2 Pre- and post-natal developmental toxicity studies Developmental toxicity studies assess the effects of the test substance on the developing organism including the death, structural abnormalities, altered growth and functional deficiencies (Joint FAO/WHO Expert Committee, 2000). Lethal, teratogenic and other toxic effects on the embryo and fetus are examined under the consideration of prenatal developmental toxicity studies (Scientific Committee for Food,

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1980). Besides, postnatal developmental toxicity studies deal with the observed side effects caused by maternal or maternal milk way exposure. Embryonic and fetal resorptions, death, fetal weight, sex ratio, external and visceral skeletal morphology are in the context of prenatal developmental toxicity. The major objectives of postnatal developmental studies are physical, functional and behavioral development in animals exposed from embryogenesis through to weaning. These tests include neurological function, behavior during both early postnatal and adulthood phases, the measurement of vaginal opening in female pups, etc. 4.3 Current studies about recently used food additives All food additives prior to their authorization should be evaluated for their safety by SCF or EFSA. According to the international legislations, all food additives must be kept under continuous observations and must be re-evaluated in the light of new scientific techniques. This part will be an overview of commonly used food additives listed below: 4.3.1 Lutein (E 161b) Food colors are the first evaluated additives whose data are old. As soon as new available studies are conducted, the results should be renewed with the old ones. This is the reason of the evaluation priority of food color additives. Lutein (E 161b) is a natural carotenoid dye which is approved as a food additive by the EU. In a 90 day rat study, according to NOAEL (No Observed Adverse Effect Level) of 200 mg/kg bw/day, which was defined by EFSA Panel, there were no developmental toxicity effects observed at dose levels up to 1000 mg/kg bw/day, the highest dose tested. Additionally, available reported data showed no effects on reproductive organs in oral 90day studies. Due to the fact that lutein is a normal constituent of diet, ADI can be altered. Lutein is found as a non-genotoxic additive; but the absence of multigeneration reproductive toxicity and chronic toxicity/carcinogenicity studies caused EFSA to get the decision for ADI of lutein as 1 mg/kg bw/day with the uncertainty factor of 200 (EFSA, 2010a). 4.3.2 Caramel colours (E 150a,b,c,d) Caramel colours are colouring substances approved as food additives according to the reactants used in their manufacture. E 150a is Class I Plain Caramel or Caustic Caramel, E 150b is Class II Caustic Sulphite Caramel, E 150c is Class III Ammonia Caramel and E 150d is Class IV Sulphite Ammonnia Caramel. EFSA determined the NOAEL as 30 g/kg bw/day with the uncertainty factor of 100 and ADI as 300 mg/kg bw/day for caramel colours. Up to date, there are no reproductive and/or developmental study of Class I and II caramels. JECFA states that there are only three developmental studies on Class III caramels. For Class III and IV caramels, studies with pregnant CD1 mice showed that, after treatment on days 615 of gestation, there were no effects on the number of implantation sites and resorption numbers, maternal-fetal survival and fetal skeletal defects (Morgareidge, 1974a; 1974b). In other studies conducted with pregnant Wistar rats and Dutch-belted rabbits, no treatmentrelated effects of Class III caramels were seen in the dams and fetal parameters (Morgareidge, 1974a). In the studies which were conducted on Wistar rats for Class IV caramels, no adverse effect was seen on female fertility, litter size, number of implantation

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sites or sex ratio of the pups (Til and Spanjers, 1973). Available reproduction and developmental studies, although limited, do not reveal any effects of concern. The studies also did not reveal any effects on reproductive organs. Lymphocytopenia is the only concern reported in short term studies with caramel colours (EFSA, 2010b). 4.3.3 Erythrosine (E 127) Erythrosine is a xanthene-dye which was evaluated by JECFA with the ADI of 0-0.1 mg/kg bw/day. It is used especially for cocktail and candied cherries. In rats (Collins et al., 1993a; 1993b) and rabbits (Burnett et al., 1974) exposed to erythrosine by gavage or in drinking water, on day 0-19 of gestation, it was found neither fetotoxic nor teratogenic. Vivekanandhi et al. conducted a study with Swiss male Albino mice in which 64, 128 and 256 mg/kg bw/day erythrosine were administered daily by gavage resulted in decreased sperm motility and increased sperm abnormalities in dose dependent manner (Vivekanandhi et al., 2006). 4.3.4 Green S (E 142) Green S is a triarylmethane dye authorized as a food additive in the EU. JECFA has previously established an ADI of 25 mg/kg bw/day in 1975; however, this was re-evaluated and later considered to be 5 mg/kg bw/day by SCF. In a previous study established by rats, the NOAEL of Green S was derived as 500 mg/kg bw/day, based on the results as increased spleen and kidney weight. At the high dose treatment (1000 mg/kg bw/day), there was evidence that the amniotic membranes had a green colouring. However, intra-uterine and post-natal development was not affected. Within the light of these results, the NOAEL is concluded as 1000 mg/kg bw/day for fetal development. In an additional study, 15 male and 15 female Wistar rats were administrated Green S in the diet at dose levels of 250, 500 and 1500 mg/kg bw/day for 13 weeks. When compared with controls, increases in mean body weight were observed in all treatment groups (BIBRA, 1978; Clode et al., 1987). Also, adequate reproduction and embryotoxicity studies including teratology are still requested (EFSA, 2010c). 4.3.5 Amaranth (E 123) Amaranth (E 123) is an azo dye approved as a food additive in the EU. The ADI of Amaranth was established as 0-0.5 and 0.08 mg/kg bw/day by JECFA and SCF respectively. There are several studies which examined the reproductive and developmental toxicity of Amaranth. When all of these studies are taken into account, NOAELs for Amaranth is as follows: mouse 100 mg/kg bw/day, rat 15 mg/kg bw/day and rabbit 15 mg/kg bw/day (EFSA, 2010d). According to the results of Shtenberg and Gavrilenko, oral Amaranth exposure at doses of 1.5 and 15 mg/kg bw/day for 12-14 months in parental generation causes a significantly higher percentage of unsuccessful pregnancies with no live born born pups and increases percentages of stillborns in rats (Shtenberg and Gavrilenko, 1970). In the study, 1.5 and 15 mg/kg bw/day administration of Amaranth in drinking water was reported to cause increased rate of post-implantation death with increased mortality at post-partum in both treatment groups and a higher incidence of stillbirth at 15 mg/kg bw/day group rats. Khera et al. administrated Amaranth to Wistar rats at dose levels of 15, 30, 100, 200 mg/kg bw/day on Days 0-18 of

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gestation, either by gavage or in diet (Khera et al., 1974). According to the results, all dams were killed on day 19. The Panel considered that the NOAEL of this study is 200 mg/kg bw/day Amaranth, which is the highest dose tested. 4.3.6 Brilliant Blue FCF (E 133) Brilliant Blue FCF (E 133) is another commonly used triarylmethane dye which is authorized as a food additive by the EU. Both JECFA and SCF established an ADI of 12.5 mg/kg bw/day. Depending on the newly conducted long-term studies, the ADI was revised to 10 mg/kg bw/day by SCI in 1984. The NOAEL was assigned as 2500 mg/kg bw/day by JECFA with uncertainty factor of 200. Afterwards, SCF established Brilliant Blue NOAEL as 1073 mg/kg bw/day in male and female rats with uncertainty factor of 100 (EFSA, 2010e). Among few chronic toxicity studies, the lowest NOAEL came from the most recent toxicity study (IRCD, 1981; Borzelleca, 1990). The Panel agreed with the authors and considered that the new NOAEL as 631 mg/kg bw/day. With uncertainty factor 100, the new established ADI for Brilliant Blue is 6 mg/kg bw/day. Data available up to date shows Brilliant Blue is poorly absorbed by the body and it is mainly excreted as unchanged in faeces. In addition, there have been several rat studies up to date; however in none of the studies treatmentrelated abnormalities were observed. 4.3.7 Curcumin (E 100) Curcumin (E 100) is a dicinnamoylmethane dye consisting of three principal colouring components. It is also approved by the EU for the use as a food additive. JECFA allocated an ADI dose of 0-3 mg/kg bw/day and a NOAEL dose of 250-320 mg/kg bw/day with uncertainty factor of 100 depending on the results of a multigeneration study which is conducted by Ganiger et al. In the study, rats were fed with Curcumin for 24 weeks at doses of 250-320 mg/kg bw/day and 960-100 mg/kg bw/day (Ganiger et al., 2007). In the high dose group, there was a decrease in body weight gain. Garg conducted a multigeneration study in Wistar rats according to OECD Testing guideline administering curcumin (Garg, 1974). Rats were fed with diets containing 0, 1500, 3000, and 10000 mg/kg bw/day curcumin. At the end of the study, it was reported that there was a dose-related decrease on body weight gain in the dams of the parental generation during days 10-15 of gestation. However, no other effects were observed. According to its’ chemical composition, Curcumin is a rapidly metabolized dye which is later excreted with faeces (EFSA, 2010f). 4.3.8 Canthaxanthin (E 161 g) Canthaxanthin is a carotenoid pigment which is authorized by the EU as a food additive. It is mainly composed of all-trans β-carotene-4,4’-dione with other minor carotenoids. The ADI dose of canthaxanthin is established as 0.03 mg/kg bw/day. There is no data reporting adverse effects of xanthaxanthin on reproductive system or on the developing fetus in high doses up to 1000mg/kg bw/day in rats and in high doses up to 400 mg/kg bw/day in rabbits. Hoffmann-La Roche reported that there were no adverse effects on fertility, litter size, the number of young weaned and their weights after 0 (placebo) and 0.1% canthaxanthin exposure in rats (Hoffmann – La, 1990). In a three-generation reproduction study by Buser, male and female rats were fed with diet including 0, 250, 500 and 1000 mg/kg bw/day canthaxanthin (Buser, 1987). The results indicated that there were no

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treatment-related effects in reproductive system. The EFSA Panel evaluated the highest dose tested of 1000 mg/kg bw/day as NOAEL for reproductive system, embryotoxicity and teratogenicity (EFSA, 2010g). 4.3.9 Aspartame Nowadays, aspartame (APM) is the most commonly used artificial sweetener in the world (Hazardous Substances Data Bank, 2005). APM is approved by both FDA and the EU for the use in all foods (FDA, 1996; EFSA, 2006h). According to long term studies, ADI of APM is 2.5-5 mg/kg bw/day (Butchko et al., 2002). Long term carcinogenicity bioassays performed on rat and mice indicated that APM is a high effective carcinogenic additive causing lymphomas, leukemias and neoplastic lesions in females and Schwannomas in males (Soffritti et al., 2006). However, recently conducted lifespan studies with Swiss mice at dose levels of 0, 2000, 8000, 16000, 32000 ppm resulted that it does not affect the daily feed consumptions, mean body weights and the survival of males-females (Soffritti et al., 2010). 4.3.10 Paraben Parabens have wide range of use in different industrial areas. One of them is its’ use in food ingredients as an anti-microbial agent (Hossani et al., 2000). Parabens are lately reported to act as xenoestrogens which are a class of endocrine disruptors due to the lengths of their alkyl side chains (Okubo et al., 2001). It is known that parabens have an effect on reproductive tissues, induce aberrant estrogenic signaling in cells, cause changes in the expression patterns of multiple genes in rat fetal reproductive system (Naciff et al., 2003). Despite the potential health effects, parabens are approved as food additives by the EU with ADI dose of 0-10 mg/kg bw/day (Ishiwatari et al., 2007) and with NOAEL as 1000 mg/kg bw/day (Boberg et al., 2010). In a developmental study conducted by Thuy et. al, during juvenile-peripubertal period, female rats were administered with methyl-, ethyl-, propyl-, isopropyl-, butyl- and isobutylparabens at doses of 62.5, 250, 1000 mg/kg bw/day (Thuy et al., 2010). Their results showed that in the highest dose group there was a significant delay in the date of vaginal opening, a decrease in length of estrous cycle, morphological changes in the uterus and increased number of cystic follicles in ovaries. Additionally, after 10, 100, 1000 mg/kg bw/day polyparaben treatment, decreases in daily testis sperm production and in serum testosterone levels in a dose-dependent manner were observed in all doses (Oishi, 2002). 4.3.11 Coriander essential oil Coriander essential oil is obtained by steam distillation of the dried fruits (seeds) of Coriandrum sativum L. In the food industry, coriander oil is used as a flavoring agent and adjuvant. Coriander oil is both approved for the use as food additive by FDA and The EU (Vollmuth et al., 1990). While maternal NOAEL of coriander oil was determined as 250 mg/kg bw/day, developmental NOAEL was established as 500 mg/kg bw/day (FFHPVC, 2002). Vollmuth et al. administered 250, 500, 100 mg/kg bw/day coriander oil to pregnant pregnant Crl CD rats (7 day before cohabitation, during gestation, 4 day post-parturition). At the highest dose significant decreases in gestation index, length of gestation, viability of pups and litter size were noted.

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4.3.12 Allyl isothiocyanate (AITC) Allyl isothiocyanate (AITC) is used both as a food additive and a flavouring agent. It can occur naturally in certain vegetables such as cabbage, mustard and horseradish (EFSA 2010i). EFSA Panel (2010) established the ADI dose as 0.02 mg/kw bw/day with an uncertainty factor of 500 (EFSA, 2010i). The Panel regarded a Low Observed Adverse Effect Level (LOAEL) as 12 mg/kw bw/day rather than the NOAEL in order to cover uncertainties resulting from extrapolation. Oral doses of AITC up to 18.5, 23.8 and 12.3 mg/kg bw/day did not cause any developmental toxicity in pregnant rats, hamsters and rabbits and may be fetotoxic to mouse at doses higher than 6 mg/kg bw/day without any teratogenic effects (EFSA, 2010i). 4.3.13 Tricalcium phosphate (E 341) Tricalcium phosphate (E 341) is a commonly used flavor preservative, anti-caking, stabilizing and anti-souring food additive. JECFA reported the ADI dose of E 341 as 70 mg/kg bw/day (JECFA, 2001). In a recently published study, Wistar rats were treated with 175 and 350 mg/kg bw/day E 341 during gestation days 0-20. Decrease in placental weights and skeletal morphometry in fetus were observed in both doses (Güngörmüş et al., 2010). There was also a decrease in trans-umblical cord lengths in treatment group. According to these results, it was concluded that rat prenatal development during gestation is sensitive to E 341 exposure.

5. Alternative methods To support and explore in more depth of results obtained from fundamental studies; immunotoxicity, allergenicity, neurotoxicity, genotoxicity, human volunteer studies, predictive mechanistic and special studies may also be helpful. Another recent method for developmental toxicity testing is in vitro studies, which are not based on the use of animals. In vitro studies provide sufficient data by using cellular and subcellular systems to predict the mechanisms involved in early stage of development. As a result of ethical issues about animal use, in vitro testing mechanisms will come into prominence in upcoming years. Over the past few decades, one of the most notable revolutionized technologies is the application of nanotechnology in food sector. Therefore, in this part, concerns and health implications on the application of nanotechnology in food will be discussed. The possible effects on reproductive system due to consumption of foods involving nanoparticles are evaluated. 5.1 Approaches of in vitro studies for assessing food additives Commonly, the toxicological risk to humans from exposure to an individual chemical is evaluated using animal data from long-term or acute in vivo toxicity studies. Over the last 20 years, there has been a clear tendency for increased use of in vitro methods in toxicology as supplements to animal tests. Although such studies have previously been considered during the hazard characterization of many compounds they generally have had no direct influence on the calculation of ADI values. In vitro studies may be a useful perspective in bridging the gap between a test species and the human situation, thereby providing a more scientific

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basis for the use of a specific data. In vitro testing systems are increasingly becoming an essential tools as part of integrated toxicology testing strategy and scientific progress in the fields of cellular and molecular biology. These studies are used in a wide range of processes including the determination of ADI, for reporting suggestions in safety approaches, metabolism pathways of specific compounds. According to the limited number of toxicological studies, in vitro applications are useful for the prospective toxicological classification and characterization of food additives. In vitro studies have the potential for calculation and detection of both inter-species and inter-individual variability in toxicokinetics and toxicodynamics of food additives (Walton, 1999). 5.2 Nanoparticle based applications of food additives in food industry Public interest in the subject of nanotechnology in the food industry is growing. It opens up many new possibilities which are of interest to the food industry. Nanofood market potential was predicted as 20.4 billion US dollars for the year 2010 (IFST, 2006). More than 200 companies worldwide are already believed to be involved in this sector, especially in the USA, Japan and China. Several companies are investigating encapsulation technology for the delivery of active ingredients in food products (e.g. flavouring agents, vitamins, fatty acids). Nanotechnology in the food industry is a sensitive subject. Manufacturers fear a blanket rejection of products containing nanomaterials, similar to what has happened with genetic engineering. Food products naturally contain nano-sized ingredients. These are different from synthetically manufactured nanomaterials. Food proteins can be mentioned as examples of natural nanostructures whose size can vary between several hundred nanometers such as milk proteins and casein. With encapsulation (Figure 4), in which active agents and substances can be encapsulated in nanostructured materials, the purpose is to enhance solubility (e.g. of colouring agents), facilitate controlled release (e.g. only in certain parts of the alimentary tract, for instance in order to prevent the bad taste of an ingredient which in itself is beneficial such as omega-3 fatty acids in fish oils), improve bioavailability, i.e. the amount of a nutritional ingredient which is actually absorbed by the body (e.g. vitamins, minerals), protect micronutrients and bioactive compounds during manufacture, storage and retail. The most important nanostructured materials are currently nano-capsules (micelles, liposomes) and nanoemulsions (Greßler et al., 2010). Nanocarrier systems can be used to mask the unpleasant tastes and flavours of ingredients and additives such as fish oils, to protect the encapsulated ingredients from degradation during processing and storage, as well as to improve dispersion of water-insoluble food ingredients. However, current studies on the application of nanoencapsulation mainly address its potential for target delivery of active ingredients of functional food and nutraceuticals (Hsieh and Ofori, 2007).

Food ingredients or additives Nanoparticles

Fig. 4. Schematic diagram of nanoencapsulation (Centre for Food Safety, Hong Kong, 2010)

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Experimental data demonstrated that the distribution of nanoparticles after oral administration is dependent upon particle size. Smaller-sized nanoparticles have a more widespread tissue distribution in organs like kidney, liver, lungs and brain while the bigger particles (28 nm and 58 nm) remain almost solely inside the gastrointestinal tract (The Government of the Hong Kong Centre for Food Safety Food and Environmental Hygiene Department, 2010). Studies have been performed on the ability of nanoparticles to penetrate the placental barrier. There is also information that certain nanomaterial (C60 fullerene) can pass across the placenta. However, due to the inconsistent results of some in vitro and animal studies, no general conclusion on the penetration power of nanoparticles across the placental barrier can be made. There is no information on whether nanomaterials are transferred into milk (Tsuchiya et al., 1996; EFSA, 2010j). Because nanoparticle food industry is a recently developing field, the reproductive and developmental toxicity studies are rare. In one of the few studies conducted by Durnev et al., silicon crystal 2-5 nm nanoparticles in the form of 1-5 μ granules in water suspension were injected intraperitoneally in a single dose to male F1(CBA×C57Bl/6) mice or to outbred albino rats on days 1, 7, and 14 of gestation (Durnev et al., 2010). It was reported that injection of 50 mg/kg dose of silicon crystal nanoparticles reduced body weight gain in pregnant rats and newborn rats at different stages of the experiment, but had no effect on other parameters of physical development of rat progeny and caused no teratogenic effects. In a recent study it was also reported that nanosized silicon materials are generally nontoxic and biodegradable (Fucikova et al., 2011). Nowadays, in comparison to other materials currently used in medicine applications, nanosized Si based materials are the only one showing complete biodegrability and nontoxicity without any significant inflammatory reactions.

6. Discussion Food additives are natural or manufactured substances, which are added to foods for restoring colors lost during processing, providing sweetness, preventing deterioration during storage and guarding with preservatives against food poisoning. A food additive is defined as a substance not normally consumed as a food in itself and not normally used as a characteristic ingredient of food whether or not it has nutritive value, the intentional addition of which to food for a technological purpose in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food results, or may be reasonably expected to result, in it or its by-products becoming directly or indirectly a component of such foods (Food Safety Authority of Ireland, 2011). All food additives undergo a safety assessment that may be used in the manufacture or preparation of foodstuffs in the European Union. Up to 2002, this safety assessment was carried out by the EU SCF but since 2003, the responsibilities of the SCF have been taken by EFSA. The safety evaluation of a food additive involves examination of the chemical structure and characteristics, including its specifications, its impurities and potential breakdown products. Toxicological data is essential to identify and characterize the possible health hazards of an additive and to allow extrapolation of the findings in animals and other test systems to humans. In these studies, the additive is administered to laboratory animals.

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Such tests are designed to give information on any possible effects from short-term or longterm exposure to the additive, including whether it may have any potential to cause cancer (carcinogenicity), or to affect male or female reproduction or the development of the embryo or the fetus if consumed by a pregnant woman (reproductive or developmental toxicity). Other effects include the genotoxicity potential of the compound; which is the ability to cause the development of cancer or adverse effects in future generations. This chapter is an overview of the developmental and reproductive studies conducted by the administration of commonly used food additives. Recent studies show that there is a lot of concern about the safety of food additives in toxicological manner. With the increasing amount of progression in food additive industry, people who need more information about popular additives admit to being confused with the possible health effects. There are both safety evaluations and regulations of newly released food additives. As a matter of fact, it is inevitable to use food additives with the increasing demand of high-quality food. Excessive parental exposure of food additive throughout lifespans makes reproductive and developmental endpoints remarkable for investigating. This chapter strived to focus on the fine prints of toxicological assessments, especially the possible reproductive and developmental effects of food additives and to give perspectives for new approaches in the evaluations of food additives in concordance with the improvement of food industry.

7. Acknowledgements The authors would like to thank Prof. Dr. Dürdane Kolankaya and Prof. Dr. M. Turan Akay for their improving suggestions and advices as supervisors.

8. References British Industrial Biological Research Association (BIBRA). (1978). Report No 196/1/78. Short term toxicity of Green S in rats. Clode, SA, Gaunt, IF, Hendy, RJ, Cottrell, RC, Gangolli, SD. Boberg, J., Camilla, T., Christiansen, S. & Ulla., Hass. (2010). Possible endocrine disrupting effects of parabens and their metabolites. Reproductive Toxicology, Vol.30, pp.301– 312. Borzelleca, J.F., Depukat, K. & Hallagan, J.B. (1990). Lifetime toxicity/carcinogenicity studies of FD & C Blue No. 1 (Brilliant blue FCF) in rats and mice. Food and Chemical Toxicology. Vol.28, pp.221-234. Burnett, C.M., Agersborg, H., Borzelleca, J., Eagle, E., Ebert, A., Pierce, E., Kirschman, J. & Scala, R. (1974). Teratogenic studies with certified colors in rats and rabbits. Toxicology and Applied Pharmacology. Vol.25, pp.121-128. Buser, S. (1987). Canthaxanthin in a three-generation study in rats. Unpublished Report No. HLR 138/86755 of Huntingdon Research Centre Ltd. Submitted by Hoffmann-La Roche & Co., Basel, Switzerland. Butchko, H.H., Stargel, W.W., Comer, C.P., Mayhew, D.A., Benninger, C., Blackburn, G.L., De Sonneville, L.M.J., Geha, R.F., Herteley, Z., Kostner, A., Leon, A.S., Liepa, G.U., McMartin, K.E., Mendnhall, C.L., Munro, I.C., Novotny, E.J., Renwick, A., Gi

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Schiffman, S.S., Schomer, D.L., Shaywitz, B.A., Spiers, P.A., Tephly, J.A. & Trefz, F.K. (2002). Intake of aspartame vs the acceptable daily intake. Regulative Toxicology and Pharmacology. Vol.35, pp.13-16. CAOBISCO Website. (2011). www.caobisco.com/doc_uploads/legislation/XOT4-EN.pdf, Belgium. Clode, S.A., Gaunt, I.F., Hendy, R.J., Contrell, R.C. & Gangolli, S.D. (1987). Short-term toxicity of Green S in rats. Food and Chemical Toxicology. Vol.25, pp.969-975. Codex Alimentarius (2009). “Noms de Categorie et Systeme International de Numerotation des Additifs Alimentaries” Collins, T.F., Black, T.N., O'Donnell, M.W.J., Shackelford, M.E. & Bulhack, P. (1993a). Teratogenic potential of FD & C Red no. 3 when given in drinking water. Food and Cosmetics Toxicology. Vol.31, pp.161-167. Collins, T.F., Black, T.N. & Ruggles, D.I. (1993b). Teratogenic potential of FD&C Red No. 3 when given by gavage. Toxicology and Industrial Health. Vol.9, No.4, pp.605-16. Durnev, A.D., Solomina, A.S., Daugel-Dauge, N.O., Zhanataev, A.K., Shreder, E.D., Nemova, E.P., Shreder, O.V., Veligura, V.A., Osminkina, L.A., Timoshenko, V.Y. & Seredenin, S.B. (2010) Evaluation of Genotoxicity and Reproductive Toxicity of Silicon Nanocrystals. Bulletin of Experimental Biology and Medicine. Vol.149, No.4, pp.445-449. EFSA. (2010). Scientific Opinion on the safety of allyl isothiocyanate for the proposed uses as a food additive. EFSA Journal. Vol.8, No.12, pp.1943. EFSA. (2010). Scientific Opinion on the re-evaluation of Amaranth (E 123) as a food additive. EFSA Journal. Vol.8, No.7, pp.1649. EFSA. (2010). Scientific Opinion on the re-evaluation of Brilliant Blue FCF (E 133) as a food additive. EFSA Journal. Vol.8, No.11, pp.1853. EFSA. (2010). Scientific Opinion on the re-evaluation of Canthaxanthin (E 161 g) as a food additive. EFSA Journal. Vol.8, No.10, pp.1852. EFSA Scientific opinion on the re-evaluation of Caramel colours (E 150 a,b,c,d) as food additives (2011) EFSA Journal, Vol.9, No.3, pp.2004. EFSA. (2010). Scientific Opinion on the re-evaluation of Curcumin (E 100) as a food additive. EFSA Journal. Vol.8, No.9, pp.1679. EFSA. (2010). Scientific Opinion on the re-evaluation of Green S (E 142) as a food additive. EFSA Journal. Vol.8, No.11, pp.1851. EFSA. (2010). Scientific opinion on the re-evaluation of Lutein (E161b) as a food additive. EFSA Journal. Vol.8, No.7, pp.1678. EFSA. (2009). Scientific Opinion of the Scientific Committee on the Potential Risks Arising from Nanoscience and Nanotechnologies on Food and Feed Safety. European Commission Health & Consumer Protection Directorate-General. (2011). Guidance on Submissions for Food Additive Evaluations by the Scientific Committee on Food. European Food Safety Authority (EFSA). (2006). Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC) on a request from the Commission related to a new long-term carcinogenicity study on aspartame. EFSA Journal. Vol.356, pp.1-44.

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FFHPVC. (2002). The terpene consortium test plan for terpenoid tertiary alcohols and related esters. In: Report Submitted to the EPA Under the HPV Challenge Program, The Flavor and Fragrance High Production Volume Consortia, Washington, DC. Food and Drug Administration (FDA). (1996). Food additives permitted for direct addition to food for human consumption: Aspartame. Federal Regulations. Vol.61, pp.3365433656. Food Safety Authority of Ireland. (2011) Guidance on Food Additives, Revision 1. Fucikova, A., Valenta, J., Pelant, I., Kusova, K. & Brezina, V. (2011). Nanocrystalline silicon in biological studies. Physica Status Solidi. Vol.8, No.3, pp.1093–1096. Ganiger, S., Malleshappa, H.N., Krishnappa, H., Rajahekhar, G.V., Ramakrishna, R. & Sullivan, F. (2007). A two generation reproductive toxicity study with curcumin, turmeric yellow, in Wistar rats. Food and Chemical Toxicology. Vol.45, pp.64-69. Garg, S.K. (1974) Effects of Curcuma longa on fertility. Planta medica. Vol.26, pp.225-227. Greßler, S., Gazsó, A., Simkó, M., Nentwich, M., Fiedeler, U. (2010). Nanoparticles and nanostructured materials in the food industry. Nano Trust Dossiers. No.004en. Güngörmüş, C., Kılıç, A., Akay, M.T. & Kolankaya, D. (2010). The effects of maternal exposure to food additive E341 (tricalcium phosphate) on foetal development of rats. Environmental Toxicology and Pharmacology. Vol.29, pp.111–116. Hazardous Substances Data Bank. (2005). TOXNET: Toxicological Data Network. Available: http://www.toxnet.net.nlm.nih.gov Hoffmann-La, R. (1990). Canthaxanthin. Unpublished report submitted to WHO by F. Hoffmann - La Roche & Co., Basle, Switzerland (as referred to by JECFA). Hossaini, A., Larsen, J.J. & Larsen, J.C. (2000) Lack of oestrogenic effects of food preservatives (parabens) in uterotrophic assays. Food and Chemical Toxicology. Vol.38, pp.319–23. Hsieh, Y.H.P. & Ofori, J.A. (2007). Innovation in food technology for health. Asia Pacific Journal of Clinical Nutrition. Vol.16, No.Supplemental1, pp.65-73. Institute of Food Science & Technology (IFST). (2006) Nanotechnology Information Statement. IPCS/JECFA. (1987) Environmental Health Criteria 70: Principles for the Safety Assessment of Food Additives and Contaminants in Food. World Health Organization, Geneva. International Research & Development Corporation (IRCD). (1981). Longt-term dietary toxicity/carcinogenicity study in rats. Unpublished report. Ishiwatari, S., Suzuki, T., Hitomi, T., Yoshino, T., Matsukuma, S. and Tsuji, T. (2007) Effects of methyl paraben on skin keratinocytes. Journal of Applied Toxicology. Vol.27, pp.1– 9. JECFA. (2001). http://www.inchem.org/documents/jecfa/jeceval/jec 2306.htm. Joint FAO/WHO Expert Committee. (2000). Guidelines for the preparation of toxicological working papers on food additives. Geneva. Khera, K.S., Przybylski, W. & McKilnley, W.P. (1974). Implantation and embryonic survival in rats treated with Amaranth during gestation. Food and Cosmetics Toxicology. Vol.12, pp.507-510. Morgareidge, K. (1974a). Teratologic evaluation of FDA 71-82 (caramel, bakers and confectioners)in mice, rats and rabbits. Unpublished report No. PB 234-870 from

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Food and Drug Research Laboratories Inc., Waverly, NY; USA (as referred to by JECFA, 1987). Morgareidge, K., (1974b). Teratologic evaluation of FDA 71-83 (caramel beverage) in mice, rats and rabbits. Unpublished report No. PB 234-867 from Food and Drug Research Laboratories Inc., Waverly, NY; USA. Naciff, J.M., Overmann, G.J., Torontali, S.M., Carr, G.J., Tiesman, J.P. & Richardson, B.D. (2003) Gene expression profile induced by 17 alpha-ethynyl estradiol in the prepubertal female reproductive system of the rat. Toxicological Sciences Vol.72, pp.314–30. Oishi, S. (2002) Effects of propyl paraben on the male reproductive system. Food and Chemical Toxicology. Vol.40, No.12, pp.1807–1813. Okubo, T., Yokoyama, Y., Kano, K. & Kano, I. (2001). ER-dependent estrogenic activity of parabens assessed by proliferation of human breast cancer MCF-7 cells and expression of ERalpha and PR. Food and Chemical Toxicology. Vol.39, pp.1225–1232. Scientific Committee for Food. (1980). Guidelines for the Safety Assessment of Food Additives. Reports of the Scientific Committee for Food (Tenth series). Commission of the European Communities, Luxembourg EUR 6892. Shtenberg, A.I. & Gavrilenko, E.V. (1970). Effects of Amaranth food dye on reproductive function and progeny development in experiments with albino rats. Voprosy Pitaniia. Vol.29, pp.66. Soffritti, M., Belpoggi, F., Degli Esposti, D., Lambertini, L., Tibaldi, E. & Rigano, A. (2006). First experimental demonstration of the multipotential carcinogenic effects of aspartame administered in the feen to Spraguq-Dawley rats. Environmental Health Perspective Vol.114, pp.379-385. Soffritti, M., Belpoggi, F., Manservigi, M., Tibaldi, E., Lauriola, M., Falcioni, L. & Bua, L. (2010). Aspartame administered in feed, beginning prenatally through life span, induces cancers of the live and lung in male swiss mice. American Journal of Industrial Medicine. Vol.53, No.12, pp.1197-1206. The Government of the Hong Kong Centre for Food Safety Food and Environmental Hygiene Department Special Administrative Region. (2010). Nanotechnology and Food Safety, Risk Assessment Studies Report No. 41. Thuy, T.B., Vo, Y.M.Y., Kyung-Chul, C. & Eui-Bae, J. (2010) Potential estrogenic effect(s) of parabens at the prepubertal stage of a postnatal female rat model. Reproductive Toxicology. Vol.29, pp.306–316. Til, H.P. & Spanjers, M. (1973). Reproduction study in rats with six different Ammonia Caramels. Unpublished report No. R 4068 from Centraal Instuut voor Voedingsonderzoek (CIVO/TNO), Zeist, The Netherlands. Tsuchiya, T., Oguri, I., Yamakoshi, Y.N. & Miyata, N. (1996). Novel harmful effects of fullerene on mouse embryos in vitro and in vivo. FEBS Letters Vol.393, No.1, pp.139-45. U.S. Food and Drug Administration. (2007). Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000. Vivekanandhi, J., Devi, C.P.A., Jayaraman, K. & Raghavan, L. (2006). Effect of Erythrosine on testicular function in mice. Toxicology International. Vol.13, pp.119-125.

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3 Production and Functional Properties of Dairy Products Containing Lactophorin and Lactadherin Mizuho Inagaki1, Xijier2, Yoshitaka Nakamura3, Takeshi Takahashi3, Tomio Yabe1,2, Toyoko Nakagomi4, Osamu Nakagomi4 and Yoshihiro Kanamaru1,2 1Department of Applied Life Science, Gifu University Graduate School of Agricultural science, Gifu University 3Food Science Institute, Division of Research and Development, Meiji Co., Ltd. 4Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences and Global Center of Excellence, Nagasaki University Japan 2United

1. Introduction In this chapter, we introduce the possible protective utilization of cow milk proteins, lactophorin (LP) and lactadherin (also known as periodic acid Schiff 6/7 (PAS6/7)), against human rotavirus (HRV) gastroenteritis. Milk is the natural food of the newborn mammal, and it is endowed with protective components against pathogens, such as antibodies. Our previous studies have demonstrated that the 2 proteins, LP and lactadherin, exhibit potent inhibitory activity against HRV. HRV is the single most important etiologic agent of severe gastroenteritis in infants and young children. To determine whether cow’s milk could serve as a protective food additive effective against HRV infection, this chapter discusses the potential utilizations of LP and lactadherin from normal cow’s milk to protect against HRV gastroenteritis, focusing in particular on sweet whey, a byproduct of industrial-scale cheese manufacturing.

2. Rotavirus gastroenteritis Infectious gastroenteritis is distinguished between bacterial and viral origin, depending on pathogenesis. Rotavirus, adenovirus, and norovirus are well-known infectious gastroenteritis pathogens of viral origin. HRV was first discovered by Ruth Bishop et al. in 1973, and was recognized as a major cause of childhood diarrheal morbidity and mortality worldwide (Bishop et al., 1973; Bishop, 2009). The virus is transmitted by the fecal-oral route. It infects the enterocytes of the villi of the small intestine and causes gastroenteritis. The incubation period of rotavirus infection is 2-4 days, and once diarrhea occurs, recovery usually requires approximately 1 week. By the age of 5

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years, nearly every child in the world has been infected with rotavirus at least once (Velázquez et al., 1996). The estimated annual incidence of rotavirus gastroenteritis is approximately 114 million episodes requiring home care and 600,000 deaths in children worldwide (Dennehy, 2008). More than 85% of these deaths occur in developing countries, South Asia, and subSaharan Africa (Naghipour et al., 2008; Centers for Disease Control and Prevention, 2011). In the absence of vaccination, rotavirus gastroenteritis has been estimated to cause 87,000 hospitalizations in Europe (Soriano-Gabarro et al., 2006), 55,000–70,000 hospitalizations in the USA (Parashar et al., 2006), and 78,000 hospitalizations in Japan (Nakagomi et al., 2005) among children below 5 years of age. Thus, rotavirus gastroenteritis causes large human costs in developing countries and large public medical burdens in developed countries. In general, vaccination is the most effective method for protection against viral diseases. To reduce the aforementioned global burden posed by rotavirus gastroenteritis, the 2 oral rotavirus vaccines Rotarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium) and RotaTeq® (Merck and Co., Whitehouse station, NJ) have been licensed for use in more than 100 countries worldwide (Tate et al., 2010). Large-scale trials in Europe and North and Latin America demonstrated that these vaccines are safe and effictive (Ciarlet & Schödel, 2009; O'Ryan & Linhares, 2009). Clinical studies are ongoing in Asia and Africa to assess the safety and efficacy of the vaccines in these populations (Zaman et al., 2010; Armah et al., 2010). However, to reduce the risk of intussusceptions, the first doses of both vaccines are strictly limited between the age of 6-15 weeks, and full doses of vaccines need to be completed by ages 6-8 months (Cortese et al., 2009). Therefore, prophylactic options against HRV infection are needed. Young mammals depend on passive immunity obtained via breast-feeding for resistance against infectious diseases, because their immature immune systems cannot produce antibodies immediately after birth. The mother is able to produce antibodies against infectious agents, and they are passively transmitted to the offspring via milk. It has been proposed that passive protection against HRV infection could be achieved by using immunoglobulin G (IgG) from the colostrum of cows hyper-immunized with rotavirus (Ebina et al., 1992; Sarker et al., 1998). Unfortunately, the clinical use of bovine colostrum from hyper-immunized cows has been limited because of difficulties in largescale production. Recently, skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6-7 days after parturition exhibited high potency in inhibiting human rotaviral replication in vitro and in vivo (Inagaki et al., 2010a), indicating that SCBLC is likely to play an alternative role to colostrum of cows hyper-immunized with rotavirus. Furthermore, studies of milk components exhibiting inhibitory activity against rotavirus have also been reported. For example, supplemental dietary whey protein concentrate (WPC) (Wolber et al., 2005; Pérez-Cano et al., 2008) and macromolecular bovine whey protein fraction (MMWP) (Kvistgaard et al., 2004; Bojsen et al., 2007) exhibited protective efficacy against simian rotavirus and murine rotavirus infection in vivo. The inhibitory mechanism of WPC remains to be elucidated, whereas Bojsen et al. found that mucin 1 and immunoglobulins were the major rotavirus inhibitors in MMWP (2007).

3. Inhibitory activity of LP and lactadherin against HRV infection Recently, we identified LP16 (16 kDa LP fragment) and bovine lactadherin (PAS6/7) as human rotavirus inhibitors in bovine milk (Inagaki et al., 2010b). In this section, we will describe the anti-viral properties of these components.

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3.1 Anti-HRV activity of LP LP was initially found to be a glycoprotein in the heat- and acid-stable proteose peptone (PP) fraction and was referred to as PP component 3 (Girardet et al., 1996). LP is found in bovine, cameline (Girardet et al., 2000), caprine (Sørensen et al., 1997), and ovine milk (Sørensen et al., 1997), but not in human milk (Sørensen et al., 1997). LP is present at an average concentration of 0.3 g/l in normal bovine milk (Koletzko et al., 2005). LP consists of 2 major glycopeptides; 28 kDa (LP28) and 18 kDa (LP18) (Girardet et al., 1996). LP28 contains 5 partial phosphorylation sites (Ser29, Ser34, Ser38, Ser40, and Ser46), 3 O-glycosylation sites (Thr16, Thr60, (Kjeldsen et al., 2003), and Thr86), and 1 Nglycosylation site (Asn77) (Girardet & Linden, 1996). It exists in various molecular forms formed via posttranslational modification (Kanno, 1989a, 1989b). LP18 has an amino acid sequence corresponding to the 54-135 C-terminal portion of LP28, and this sequence is thought to occur as a proteolytic degradation product of LP28 (Girardet & Linden, 1996). Thus far, emulsification and inhibition of lipolytic activity have been reported as the characters and functions of LP (Kanno, 1989a; Girardet et al., 1993). Recently, LP has been found to stimulate immunoglobulin production in human hybridoma cells and human peripheral blood lymphocytes (Sugahara et al., 2005). However, its biological function remains unclear. The inhibitory activity of LP against HRV infection was identified as follows. Previously, Kanamaru et al. (1999) reported that high-Mr glycoprotein fraction (F1) from cow milk whey potently inhibited HRV infection in vitro. They reported that F1 formed a complex with various proteins but failed to identify the inhibitory entity in F1. Ten years later, LP was identified as one of the inhibitory components of HRV replication in F1 (Inagaki et al., 2010b). In brief, F1 was initially heated at 95°C for 30 min, rendering milk antibodies inert, and then subjected to ammonium sulfate fractionation. The component with a molecular size of 16 kDa, found in a certain fraction from ammonium sulfate fractionation, exhibited inhibitory activity against HRV replication. Sequencing analysis of this substance resulted in the first 7 N-terminal amino acid residues of ILKEKHL, which is consistent with the sequence of residues 69-75 of bovine LP. Thus, LP16 exhibited a strong inhibitory activity against HRV replication. Furthermore, a preliminary experiment revealed that LP28 and LP18 potently inhibited HRV infection, suggesting that the consensus structure of LP28 and LP18 (i.e., sequence of residues 54-135 C-terminal portion of LP) was involved in their inhibitory activities (Inagaki et al., unpublished observation). Further studies are in progress for detailed elucidation of the HRV inhibitory mechanism of LP. 3.2 Anti-HRV activity of lactadherin Lactadherin is a major milk fat globule membrane component in milk. Lactadherin in bovine milk is also known as PAS6/7. Lactadherin consists of 2 N-terminal epidermal growth facter (EGF)-like domains followed by 2 repeated C domains with homology to the C1 and C2 domains of blood clotting factors V and VIII (Mather, 2000). Interestingly, lactadherin has first EGF-like domain containing glycosylation sites, whereas human lactadherin has defects in this domain (Mather, 2000).

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Lactadherin binds to integrins αvβ3 (Taylor et al., 1997; Andersen et al., 2000; Hanayama et al., 2002) and αvβ5 (Andersen et al., 2000), which are expressed by endothelial cells. However, the physiological function of lactadherin in milk is little known. The inhibitory activity against HRV infection of human lactadherin was first identified by Yolken et al. (1992). Furthermore, a previous clinical study indicated a correlation between human lactadherin in breast milk and morbidity due to rotavirus gastroenteritis in young children (Newburg et al., 1998). These reports led us to investigate non-immunoglobulin component(s) of rotavirus inhibitor in bovine milk. One report indicated that bovine lactadherin did not have anti-HRV activity (Kvistgaard et al., 2004). The study was performed using the human Wa strain rotavirus infected to Caco-2 cells and a short-term (1 h) incubation of cells with lactadherin. On the contrary, the inhibitory activity of bovine lactadherin against HRV infection was reported by Inagaki et al. (2010b). The study was performed using the human MO strain rotavirus infected to MA104 cells and demonstrated that long-term (22 h) incubation of cells with lactadherin resulted in significant antiviral effects. The reasons for the inconsistent results are unclear because of distinct experimental conditions. However, it has been reported that lactadherin binds to MA104 cells via integrin αvβ3 (Taylor et al., 1997; Andersen et al., 2000; Hanayama et al., 2002), which is known as one of the cell receptors for rotavirus (Guerrero et al., 2000). Therefore, the interaction between lactadherin and cell surface components is likely important for its antiviral activity. Thus, the inhibitory mechanisms of lactadherin remain controversial.

4. Utilization of sweet whey proteins against HRV gastroenteritis Sweet whey is manufactured as a byproduct of cheese production. Thus, its production increases as the consumption of cheese expands. However, its routine disposal will become a significant problem in the dairy industry. Therefore, the extended utilization of sweet whey should be pursued. Based on the above findings that LP and lactadherin exhibit inhibitory activities against HRV infection, we attemped to investigate the potential utilization of sweet whey as a protective food additive against HRV gastroenteritis. 4.1 Microfiltration retentate fraction (MFRF) from sweet whey To concentrate LP and lactadherin, we attempted to examine the presence of both inhibitory components in sweet whey, which was produced during cheese manufacturing. As shown in Fig. 1, the concentrate was collected as the MFRF. Then, it was pasteurized by a hightemperature short-time method sterilization (HTST) method consisting of heating at 72°C for 15 s, followed by spray drying (referred to as Dried MFRF). Fig. 2A shows the result of two-dimensional electrophoresis of Dried MFRF stained with Coomassie Brilliant Blue. Dried MFRF contains α-lactalbumin (α-LA, Mw: 14,100 Da) and β-lactoglobulin (β-LG, Mw: 18,200 Da) as the major protein components. The existence of LP and lactadherin could be observed, although not as obviously as the major components, indicating that the inhibitory components appeared to be contained in Dried MFRF. When immunochemical detection using the specific monoclonal antiserum for each protein (Aoki et al., 1994) was performed, as shown in Fig. 2B and 2C, LP and lactadherin could certainly be detected in Dried MFRF.

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Bovine milk Pasteurisaton Acidification Coagulation (addition of rennet) Cheese

Sweet whey Microfiltration (MF) Defatted whey

Microfiltration retentate fraction (MFRF) Pasteurization (72 ºC, 15 sec) Spray drying

Pasteurized Microfiltration retentate fraction (Dried MFRF)

Fig. 1. Flow chart for the production of Dried MFRF (kDa) 97 66 45 31 21 14 6

(A)

(kDa)

(kDa)

97 66 45 31

97 66 45 31

21

21

14 6

14 6

(B)

(C)

Fig. 2. Dried MFRF contains LP and Lactadherin. A. Two-dimensional protein profile of Dried MFRF. The horizontal dimension was isoelectric focusing (pI, 3.0-10.0), and the second dimension was 15% polyacrylamide gel electrophoresis (PAGE). The gel was stained with Coomassie Brilliant Blue. The molecular weights of the standards (broad range, Bio-Rad) are indicated in kDa on the left. B. Immunochemical detection of LP. Two-dimensional PAGE was performed as in panel A, and then samples were transferred onto a polyvinylidene difluoride (PVDF) membrane and immunostained for LP using the monoclonal anti-LP 1C10 primary antibody (Aoki et al., 1994), followed by horse-radish peroxidase (HRP)-conjugated goat antimouse IgG secondary antibody. The molecular weights of the standards (broad range, BioRad) are indicated in kDa on the left. C. Immunochemical detection of lactadherin. Twodimensional PAGE and western blotting were performed as in panel B. Samples were immunostained for lactadherin with the monoclonal anti-lactadherin 3F12 primary antibody (Aoki et al., 1994), followed by HRP-conjugated goat anti-mouse IgG secondary antibody. The molecular weights of the standards (broad range, Bio-Rad) are indicated in kDa on the left

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4.2 Inhibitory activity of Dried MFRF against HRV infection Next, we investigated the inhibitory activity of Dried MFRF against HRV infection. A replication inhibition (neutralization) assay for HRV was performed using MA104 cells (African rhesus monkey kidney cell line) following a procedure described previously (Inagaki et al., 2010b) with slight modifications. Our previously published focus reduction assay for rotaviral infection was performed using a suspension of MA104 cells, and a preincubated virus/milk sample mixture was incubated further for 22 h with the cells before fixation. In this study, a confluence monolayer of MA104 cells was established in wells of a glass slide, and a virus/milk sample mixture was inoculated for 1 h and removed from the monolayer before further advancing the viral infection to exclude the influence of the milk sample on MA104 cells by prolonged incubation. As shown in Fig. 3, MFRF was found to potently inhibit the replication of HRV MO strain (serotype G3P[8]) with an MIC of 3.1 μg/ml. Furthermore, even after pasteurization by HTST method, the neutralizing activity of Dried MFRF remained, with an MIC of 4.7 μg/ml. This activity might also be attributed largely to the heat-resistant character of LP. The colostrums whey from the hyper-immunization of pregnant cows with human rotavirus (rotawhey) was used as a positive control. Rotawhey contains a high level of specific antihuman rotavirus antibodies, and it exhibited a robust inhibitory activity, with an MIC of 0.012 μg/ml (Fig. 3). Bovine lactoferrin also exhibited an inhibitory activity, although weak, with an MIC of 180 μg/ml (Fig. 3). The MIC value of Dried MFRF indicated that it has great potential as a protective food additive against HRV infection. 100

% inhibition

80

60

40

MFRF Dried MFRF rotawhey

20

bovine lactoferrin 0 0.0001

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protein concentration (μg/ml) Fig. 3. MFRF exhibits inhibitory activity after pasteurization treatment MA104 cells were plated into the wells of a 24-well heavy Teflon (HT)-coated slide (AR Brown, Tokyo) and grown to full confluence. A suspension containing infectious virus at a titer of 1 × 105 - 1 × 106 fluorescent cell focus-forming units (FCFU)/ml was treated with 20 μg/ml trypsin (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C. After appropriate dilution

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with Eagle’s minimum essential medium (E-MEM) containing 2% fetal calf serum to give a titer of approximately 103 FCFU per 100 μl, aliquots were mixed with equal volumes (100 μl) of one-half serially diluted samples in microtubes for 1 h at 37ºC. The diluted mixtures (20 μl/well) were added to the confluent monolayer of MA104 cells. The control produced approximately 100 infected foci per well without the test samples of milk. The cells were further cultured for 1 h at 37°C in an atmosphere of 5% CO2. After removal of the inoculums, the cells were washed once with E-MEM to remove unbound virus, followed by incubation at 37°C in an atmosphere of 5% CO2. After 17 h of incubation, the cells were fixed with cold methanol for 10 min. Infected cells were detected by an indirect immunofluorescence assay using the PO-13 monoclonal anti-pigeon rotavirus antibody (Minamoto et al., 1993) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG serum. The foci numbers of infected cells were measured by observation of fluorescence microscopy. Neutralizing activity was expressed as the percentage reduction in the foci numbers of infected cells as compared with infected cells without milk sample. The minimum inhibitory concentration (MIC), the minimum concentration inducing a 50% reduction in infected cells, was calculated for each sample from a logarithmic regression of the concentration-dependent percentage focus reduction. The inhibitory activity of each sample is expressed as a percentage of infected cells as compared to control cells (100%). The experiments were performed in triplicate at least 3 times, and representative results for each sample are given as the mean (SD). 4.3 Analysis of the protective components in Dried MFRF To verify and further characterize the effective components of Dried MFRF regarding protection against HRV infection, we attempted to fractionate Dried MFRF by size exclusion chromatography on Sephacryl S-500 HR. As shown in Fig. 4, 3 fractions were collected according to the elution pattern of Dried MFRF.

Absorbance at 280 nm (mAU)

600

F1

F2

400

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F3

500 400 300 200 100 0 0

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800

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Elution volume (ml) Fig. 4. Fractionation of Dried MFRF by Size Exclusion Chromatography on Sephacryl S-500 HR

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The column (60 × 5.0 cm, GE Healthcare UK Ltd., Little Chalfont, UK) was equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl, 2 mM EDTA, and 0.02% NaN3. Dried MFRF was dissolved in elution buffer at a concentration of 5 mg/ml, and 30 ml were added to a Sephacryl S-500 HR column. The flow rate was 10 ml/min. Eluted fractions were freeze-dried after dialysis against distilled water. Next, to investigate the protein components in the fractions, we attempted to resolve the fractions by two-dimensional PAGE. The results are shown in Fig. 5. We confirmed by immunoblot analysis that only F1 contained lactadherin (result not shown). LP was mainly detected in F2 and slightly present in F1. Although 2 major whey proteins, α-LA and β-LG, were detected in each of the three fractions, the vast majority of them detected in F3. Lactadherin and LP28 were present as minor components in F1 and F2, respectively. α-LA and β-LG were present as major components in F3. (kDa)

F1

(kDa)

F2

(kDa)

97 66 45

97 66 45

97 66 45

31

31

31

21

21

21

14 6

14 6

14 6

F3

Fig. 5. Protein profiles of size exclusion chromatography fractions. Two-dimensional PAGE profiles of each fractions. The horizontal dimension was isoelectric focusing (pI, 3.0-10.0), and the second dimension was 15% PAGE. The gel was stained with Coomassie Brilliant Blue. The molecular weights of the standard (broad standard, Bio-Rad) are indicated in kDa on the left As mentioned above, IgG was identified as a rotavirus inhibitor in bovine milk (Ebina et al., 1992; Sarker et al., 1998). Accordingly, to address the contribution of IgG to the anti-HRV activity of Dried MFRF, we attempted to separate IgG in each fraction from other components by using affinity chromatography on a HiTrap Protein G HP column (5 ml, GE Healthcare UK Ltd.). A typical elution pattern of F3 is shown in Fig. 6. The bound fraction was IgG, and the unbound fractions from each fraction were collected as F1′, F2′, and F3′. We found that IgG was removed from F2 and F3, although a small portion remained, as shown in Fig. 6. Conversely, we did not observe the elution of IgG from F1 (results not shown). These results indicated that IgG might represent a minor component in Dried MFRF. In this manner, we obtained 4 fractions: F1′ (fraction containing lactadherin), F2′ (fraction containing LP28), F3′ (fraction containing α-LA and β-LG as major components) and IgG collected from F3. F3 was dissolved in 20 mM sodium phosphate buffer (pH 7.0) at a concentration of 1 mg/ml. The column was equilibrated with the same buffer, and the column was connected with and controlled by the ÄKTA prime system (GE Healthcare UK Ltd.). The flow rate was 2 ml/min. Proteins were monitored at 280 nm (solid line). The unbound fraction was collected as F3′. The bound fraction, IgG, eluted with a step 100% elution buffer (0.1 M glycine-HCl, pH 2.7) (dotted line). The eluted IgG fractions were neutralized with 1 M TrisHCl (pH 9.0). Each fraction was freeze-dried after dialysis against distilled water.

Production and Functional Properties of Dairy Products Containing Lactophorin and Lactadherin F3’

100

80 100 60

40 50

Elution buffer (%)

Absorbance at 280nm (mAU)

150

57

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IgG 0

0 0

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Elution volume (ml)

Fig. 6. Fractionation of F3 by Affinity Chromatography on HiTrap Protein G HP column In the following focus reduction assay, IgG exhibited inhibitory activity against HRV MO infection, with an MIC of 0.27 μg/ml (Fig. 7). F2′ exhibited similar inhibitory activity level as IgG, with an MIC of 0.32 μg/ml (Fig. 7). F1′ exhibited slightly weaker inhibitory activity than did F2′, with an MIC of 1.2 μg/ml (Fig. 7). Although F3, before proteinG affinity chromatography, exhibited a strong inhibitory activity (result not shown), F3′ lost this activity after chromatography, resulting in an MIC of 20,000 μg/ml (Fig. 7). Taken together, the inhibitory components in Dried MFRF should include at least lactadherin, LP, and IgG. These components exhibited very similar activity, although the former 2 could not be

100

% inhibition

80 60

Dried MFRF F1’

40

F2‘ F3’ IgG

20 0 0.001

0.01

0.1

1

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protein concentration (μg/ml)

Fig. 7. The Fractions Obtained by Protein G Affinity Chromatography and Their in Vitro Inhibitory Activity against HRV MO Strain

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Food Additive

purified from Dried MFRF in this study. Our previous study demonstrated that the MICs of lactadherin and LP16 in pure form were 0.016 and 1.8 μg/ml, respectively (Inagaki et al., 2010b). As the precise content of these active components in Dried MFRF is at present not clear, we conclude that their contribution to the inhibitory efficacy against HRV infection of Dried MFRF is likely to be comparable. Inhibitory activity was determined as described in the legend to Fig. 3. The inhibitory activity of each sample was expressed as the percent decrease in foci numbers of infected cells as compared to the foci numbers of control cells, which were treated with PBS in place of the milk sample (100%). The experiments were performed in triplicate at least 3 times, and representative results for each sample are given as the mean (SD). 4.4 Inhibitory activity of Dried MFRF against various types of HRV Furthermore, we investigated the protective efficacy of Dried MFRF against other types of HRV besides the MO strain. Rotavirus has two independent serotypes (G and P types), and they are defined by VP7 and VP4, respectively. Epidemiological studies on rotavirus showed that strains with G-types of G1, G2, G3, and G4 and those with P-types of P[4] and P[8] are the most prevalent causes of rotavirus gastroenteritis in humans (Gentsch et al., 2005; Santos and Hoshino, 2005; McDonald et al., 2009). Furthermore, the rotavirus G/Ptype distribution varies from year-to-year (O'Ryan, 2009). As shown in Fig. 8, Dried MFRF also exhibited inhibitory activity against the Wa strain (serotype G1P[8]) and the Hochi strain (serotype G4P[8]), with MICs of 2.8 and 3.2 μg/ml, respectively. Therefore, Dried MFRF can be concluded to have potential as a protective food additive against several serotypes of HRV.

Fig. 8. Dried MFRF Exhibits Inhibitory Activities against Various Types of HRV Inhibitory activity was determined as described in the legend to Fig. 3. The inhibitory activity of each sample was expressed as the percent decrease in foci numbers of infected cells as compared to the foci numbers of control cells, which were treated with PBS in place of the milk sample (100%). The experiments were performed in triplicate at least 3 times, and representative results for each sample are given as the mean (SD).

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4.5 Protective efficacy of Dried MFRF against HRV-induced diarrhea in suckling mice Finally, we investigated whether a single administration of Dried MFRF exhibits prophylactic efficacy against HRV-induced diarrhea in vivo. As shown in Fig. 9, in the PBS group, 10 of the 11 mice developed diarrhea 48 h post inoculation (hpi), and all mice recovered from diarrhea by 96 hpi. In the Dried MFRF (2.5 mg) group, only 2 of 16 mice developed diarrhea at 48 hpi, and all mice recovered from symptoms by 72 hpi. In the Dried MFRF (1.0 mg) group, 4 of 11 mice developed diarrhea at 48 hpi, and all mice recovered by 72 hpi. This result clearly indicated that Dried MFRF is a promising candidate for a prophylactic food additive against HRV infection.

Fig. 9. Dried MFRF Exhibits Preventive Efficacy against HRV-Induced Diarrhea in Suckling Mice Pregnant BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). Litters of 5day-old mice were orally administered with PBS (n = 11), 1.0 mg of Dried MFRF (n = 11), or 2.5 mg of Dried MFRF (n = 16) for 60 min before inoculation with 2.5 ×105 FCFU of the HRV MO strain. Stools were examined daily to assess diarrhea for 4 days after viral inoculation. Liquid-like mucous yellow stool was considered diarrhea.

5. Summary Milk contains essential components for child growth. In this chapter, we introduced the inhibitory activity of LP and lactadherin against HRV infection, and examined the possibility of MFRF, which is obtained as a byproduct of cheese manufacturing, as an alternative therapeutic option against HRV gastroenteritis. Dried MFRF exhibited inhibitory activity against several types of HRV in vitro. Furthermore, we demonstrated that prophylactic oral administration of Dried MFRF once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. Finally, we concluded that Dried MFRF contained LP, lactadherin, and IgG as rotavirus inhibitors. As the anti-HRV activity of LP was not affected by heating at 95°C for 30 min (Inagaki et al., result not shown), the anti-HRV activity of MFRF would be stable to partial heat sterilization.

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Food Additive

Recently, it was reported that one-third of all pediatric rotavirus gastroenteritis patients are children between 3 and 6 years of age, an age group outside the primary target of rotavirus vaccine in Japan (Ito et al., 2011; Nakanishi et al., 2009). Thus, these epidemiological studies indicated the need for not only vaccination but also alternative preventive procedures against HRV infection. In conclusion, Dried MFRF, in which the non-immunoglobulin components including LP and lactadherin are concentrated, is a promising candidate prophylactic food additive against HRV infection. Dried MFRF was also found to be a potent inhibitor of several types of bovine rotavirus derived from field breeds (Inagaki et al., results not shown). Rotavirus gastroenteritis is an important issue in livestock animals as well. Taken together, Dried MFRF is very useful as a protective food additive against rotaviral infection.

6. Acknowledgements We thank Dr. Nobuyuki Minamoto and Dr. Makoto Sugiyama for kindly providing the PO13 monoclonal antibody, and Dr. Tsukasa Matsuda for kindly providing the 1C10 and 3F12 monoclonal antibodies. We also express our thanks to Mr. Kengo Kishita and Mr. Tomohiro Katsura for their technical assistance. This research was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-Oriented Industry.

7. References Andersen, M. H., Graversen, H., Fedosov, S. N., Petersen, T. E., & Rasmussen, J. T. (2000). Functional analyses of two cellular binding domains of bovine lactadherin. Biochemistry, Vol. 39, No. 20, (April 2000), pp. 6200–6206, ISSN 1520-4995 Aoki, N., Kuroda, H., Urabe, M., Taniguchi, Y., Adachi, T., Nakamura, R., & Matsuda, T. (1994). Production and characterization of monoclonal antibodies directed against bovine milk fat globule membrane (MFGM). Biochimica et biophysica acta, Vol. 1199, No. 1, (January 1994), pp. 87-95, ISSN 006-3002 Armah, G. E., Sow, S. O., Breiman, R. F., Dallas, M. J., Tapia, M. D., Feikin, D. R., Binka, F. N., Steele, A. D., Laserson, K. F., Ansah, N. A., Levine, M. M., Lewis, K., Coia, M. L., Attah-Poku, M., Ojwando, J., Rivers, S. B., Victor, J. C., Nyambane, G., Hodgson, A., Schödel, F., Ciarlet, M., & Neuzil, K. M. (2010). Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised, double-blind, placebo-controlled trial. The Lancet, Vol. 376, No. 9741, (August 2010), pp. 606-614, ISSN 0099-5355 Bishop, R. F., Davidson, G. P., Holmes, I. H., & Ruck, B. J. (1973). Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. The Lancet, Vol. 302, No. 7841, (December 1973), pp. 1281-1283, ISSN 0099-5355 Bishop, R. F. (2009). Discovery of rotavirus: Implications for child health. Journal of gastroenterology and hepatology, Vol. 24, No. s3, (October 2009), pp. S81-S85, ISSN 1440-1746 Bojsen, A., Buesa, J., Montava, R., Kvistgaard, A. S., Kongsbak, M. B., Petersen, T. E., Heegaard, C. W., & Rasmussen, J. T. (2007). Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo. Journal of dairy science, Vol. 90, No. 1, (January 2007), pp. 66-74, ISSN 0022-0302

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Centers for Disease Control and Prevention (CDC) (2011). Rotavirus surveillance --worldwide, 2009. MMRW. Morbidity and mortality weekly report, Vol. 60, No. 16, (April 2009), pp. 514-516, ISSN 1545-861X Cortese, M. M., Parashar, U. D., & Centers for Disease Control and Prevention (CDC) (2009). Prevention of rotavirus gastroenteritis among infants and children: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports / Centers for Disease Control, Vol. 58, No. RR-2, (February 2009), pp. 1-25, ISSN 1545-8601 Ciarlet, M., & Schödel, F. (2009). Development of a rotavirus vaccine: clinical safety, immunogenicity, and efficacy of the pentavalent rotavirus vaccine, RotaTeq. Vaccine, Vol. 27, No. suppl 6, (December 2009), pp. G72-G81, ISSN 0264-410X Dennehy, P. H. (2008). Rotavirus vaccine: an overview. Clinical microbiology reviews, Vol. 21, No. 1, (January 2008), pp. 198-208, ISSN 0893-8512 Ebina, T., Ohta, M., Kanamaru, Y., Yamamoto-Osumi, Y., & Baba, K. (1992). Passive immunizations of suckling mice and infants with bovine colostrum containing antibodies to human rotavirus. Journal of medical virology, Vol. 38, No. 2, (October 1992), pp. 117-123, ISSN 0146-6615 Guerrero, C. A., Méndez, E., Zárate, S., Isa, P., López, S., & Arias, C. F. (2000). Integrin alpha(v)beta(3) mediates rotavirus cell entry. Proceeding of the National Academy of Sciences of the United States of America, Vol. 97, No. 26, (December, 2000), pp. 1164414649, ISSN 1091-6490 Gentsch, J. R., Laird, A.R., Bielfelt, B., Griffin, D. D., Banyai, K., Ramachandran, M., Jain, V., Cunliffe, N. A., Nakagomi, O., Kirkwood, C. D., Fischer T. K., Parashar U. D., Bresee J. S., Jiang, B., and Glass, R. I. (2005). Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. The Journal of infectious diseases, Vol. 192, No. Suppl 1, (September 2005) pp. 146-159. ISSN 0022-1899 Girardet, J. M., Linden, G., Loye, S., Courthaudon, J. L., & Lorient, D. (1993). Study of mechanism of lipolysis inhibition by bovine milk proteose-peptone component 3. Journal of dairy science, Vol. 76, No. 8, (August 1993), pp. 2156-2163, ISSN 0022-0302 Girardet, J. M., & Linden, G. (1996). PP3 component of bovine milk: a phosphorylated whey glycoprotein. The Journal of dairy research, Vol. 63, No. 2. (May 1996), pp. 333-350, ISSN 0022-0299 Girardet, J. M., Saulnier, F., Gaillard, J. L., Ramet, J. P., & Humbert, G. (2000). Camel (Camelus dromedarius) milk PP3: evidence for an insertion in the amino-terminal sequence of the camel milk whey protein. Biochemistry and cell biology, Vol. 78, No. 1, (January 2000), pp. 19-26, ISSN 0829-8211 Hanayama, R., Tanaka, M., Miwa, K., Shinohara, A., Iwamatsu, A., & Nagata, S. (2002). Identification of a factor that links apoptotic cells to phagocytes. Nature, Vol. 417, No.6885, (May 2002), pp. 182-187, ISSN 1476-4687 Inagaki, M., Yamamoto, M., Xijier, Cairangzhouma, Uchida, K., Yamaguchi, H., Kawasaki, M., Yamashita, K., Yabe, T., & Kanamaru, Y. (2010a). In vitro and in vivo evaluation of the efficacy of bovine colostrum against human rotavirus infection. Bioscience, biotechnology, and biochemistry, Vol. 74, No. 3, (March 2010), pp. 680-682, ISSN 09168451

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Inagaki, M., Nagai, S., Yabe, T., Nagaoka, S., Minamoto, N., Takahashi, T., Matsuda, T., Nakagomi, O., Nakagomi, T., Ebina, T., & Kanamaru, Y. (2010b). The bovine lactophorin C-terminal fragment and PAS6/7 were both potent in the inhibition of human rotavirus replication in cultured epithelial cells and the prevention of experimental gastroenteritis. Bioscience, biotechnology, and biochemistry, Vol. 74, No. 7, (July 2010), pp. 1386-1390, ISSN 0916-8451 Ito, H., Trabe, O., Katsumi, Y., Matsui, F., Kidowaki, S., Mibayashi, A., Nakagomi, T., & Nakagomi, O. (2011). The incidence and direct medical cost of hospitalization due to rotavirus gastroenteritis in Kyoto, Japan, as estimated from a retrospective hospital study. Vaccine, in press, (August 2011), ISSN 1873-2518 Kanamaru, Y., Etoh, M., Song, X-G., Mikogami, T., Hayakawa, H., Ebina, T., & Minamoto, N. (1999). A high-Mr glycoprotein fraction from cow’s milk potent in inhibiting replication of human rotavirus in vitro. Bioscience, biotechnology, and biochemistry, Vol. 63, No. 1, (January 1999), pp. 246-249, ISSN 0916-8451 Kanno, C. (1989a). Purification and separation of multiple forms of lactophorin from bovine milk whey and their immunological and electrophoretic properties. Journal of dairy science, Vol. 72, No. 4, (April 1989), pp. 883-891, ISSN 0022-0302 Kanno, C. (1989b). Characterization of multiple forms of lactophorin isolated from bovine milk whey. Journal of dairy science, Vol. 72, No. 7, (July 1989), pp. 1732-1739, ISSN 0022-0302 Kjeldsen, F., Haselmann, K. F., Budnik, B. A., Sørensen, E. S., & Zubarev, R. A. (2003). Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage. Analytical chemistry, Vol. 75, No. 10, (May 2003), pp. 2355-2361, ISSN 0003-2700 Koletzko, B., Baker. S., Cleghorn, G., Neto, U. F., Gopalan, S., Hernell, O., Hock, Q. S., Jirapinyo, P., Lonnerdal, B., Pencharz, P., Pzyrembel, H., Ramirez-Mayans, J., Shamir, R., Turck, D., Yamashiro, Y., & Zong-Yi, D. (2005). Global standard for the composition of infant formula: recommendations of an ESPGHAN coordinated international expert group. Journal of pediatric gastroenterology and nutrition, Vol. 41, No. 5, (November 2005), pp. 584-599, ISSN 1536-4801 Kvistgaard, A. S., Pallesen, L. T., Arias, C. F., López, S., Petersen, T. E., Heegaard, C. W., & Rasmussen, J. T. (2004). Inhibitory effects of human and bovine milk constituents on rotavirus infections. Journal of dairy science, Vol. 87, No. 12, (December 2004), pp. 4088-4096, ISSN 0022-0302 McDonald, S. M., Matthijnssens, J., McAllen, J. K., Hine, E., Overton, L., Wang, S., Lemey, P., Zeller, M., Van Ranst, M., Spiro, D. J., & Patton, J. T. (2009). Evolutionary dynamics of human rotaviruses: balancing reassortment with preferred genome constellations. PLoS Pathogens, Vol. 5, No. 10, (October 2009), pp. e1000634, ISSN 1553-7374 Mather, I. H. (2000). A review and proposed nomenclature for major proteins of the milk-fat globule membrane. Journal of dairy science, Vol. 83, No. 2, (February 2000), pp. 203247, ISSN 0022-0302 Minamoto, N., Sugimoto, O., Yokota, M., Tomita, M., Goto, H., Sugiyama, M., & Kinjo, T. (1993). Antigenic analysis of avian rotavirus VP6 using monoclonal antibodies. Archives of virology, Vol. 131, No. 3-4, (February 1993), pp. 293-305, ISSN 0304-8608

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Nakanishi, K., Tsugawa, T., Honma, S., Nakata, S., Tatsumi, M., Yoto, Y., & Tsutsumi, H. (2009). Detection of enteric viruses in rectal swabs from children with acute gastroenteritis attending the pediatric outpatient clinics in Sapporo, Japan. Journal of Clinical Virology: the official publication of the Pan American Society for Clinical Virology, Vol. 46, No. 1, (September 2009), pp. 94-97, ISSN 1873-5967 Nakagomi, T., Nakagomi, O., Takahashi, Y., Enoki, M., Suzuki, T., & Kilgore, P. E. (2005). Incidence and burden of rotavirus gastroenteritis in Japan, as estimated from a prospective sentinel hospital study. The Journal of infectious diseases, Vol. 192, No. Suppl 1, (September 2005), pp. S106-S110, ISSN 0022-1899 Naghipour, M., Nakagomi, T., and Nakagomi, O. (2008). Issues with reducing the rotavirusassociated mortality by vaccination in developing countries. Vaccine, Vol. 26, No. 26, (June 2008) pp. 3236-3241. ISSN 1873-2518 Newburg, D. S., Peterson, J. A., Ruiz-Palacios, G. M., Matson, D. O., Morrow, A. L., Shults, J., Guerrero, M. L., Chaturvedi, P., Newburg, S. O., Scallan, C. D. , Taylor, M. R., Ceriani, R. L., & Pickering, L. K. (1998). Role of human-milk lactadherin in protection against symptomatic rotavirus infection. Lancet, Vol. 351, No. 9110, (April 1998), pp. 1160-1164, ISSN 0140-6736 O'Ryan, M. (2009). The ever-changing landscape of rotavirus serotypes. The Pediatric infectious disease journal, Vol. 28, No. 3 Suppl, (March 2009), pp. S60-62, ISSN 15320987 O'Ryan, M., & Linhares, A. C. (2009). Update on rotarix: an oral human trotavirus vaccine. Expert review of vaccines, Vol. 8, No. 12, (December 2009), pp. 1627-1641, ISSN 17448395 Parashar, U. D., Alexander, J. P., & Glass, R. I. (2006). Prevention of rotavirus gastroenteritis among infants and children. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports / Centers for Disease Control, Vol. 55, No. RR-12, (August 2006), pp. 1-13, ISSN 1545-8601 Pérez-Cano, F. J., Marín-Gallén, S., Castell, M., Rodríguez-Palmero, M., Rivero, M., Castellote, C., & Franch, A. (2008). Supplementing suckling rats with whey protein concentrate modulates the immune response and ameliorates rat rotavirus-induced diarrhea. The Journal of nutrition, Vol. 138, No. 12, (December 2008), pp. 2392-2398, ISSN 0022-3166 Santos, N., & Hoshino, Y. (2005). Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Reviews in medical virology, Vol. 15, No. 1, (January/February 2005) pp. 2956. ISSN 1099-1654 Sarker, S. A., Casswall, T. H., Mahalanabis, D., Alam, N. H., Albert, M. J., Brüssow,.H., Fuchs, G. J., & Hammerström, L. (1998). Successful treatment of rotavirus diarrhea in children with immunoglobulin from immunized bovine colostrum. The Pediatric infectious disease journal, Vol. 17, No. 12, (December 1998), pp. 1149-1154, ISSN 08913668 Soriano-Gabarro, M., Mrukowicz, J., Vesikari, T., & Verstraeten, T. (2006). Burden of rotavirus disease in European Union countries. The Pediatric infectious disease journal, Vol. 25, No. 1 Suppl, (January 2006), pp. S7–S11, ISSN 1532-0987

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Sørensen, E. S., Rasmussen, L. K., Møller, L., & Petersen, T. E. (1997). The localization and multimeric nature of component PP3 in bovine milk: purification and characterization of PP3 from caprine and ovine milks. Journal of dairy science, Vol. 80, No. 12 (December 1997), pp. 3176-3681, ISSN 0022-0302 Tate, J. E., Patel, M. M., Steele, A. D., Gentsch, J. R., Payne, D. C., Cortese, M. M., Nakagomi, O., Cunliffle, N. A., Jiang, B., Neuzil, K. M., de Oliveira, L. H., Glass, R. I., & Parashar, U. D. (2010). Global impact of rotavirus vaccines. Expert review of vaccines, Vol. 9, No. 4, (April 2010), pp. 395-407, ISSN 1744-8395 Taylor, M. R., Couto, J. R., Scallan, C. D., Ceriani, R. L., & Peterson, J. A. (1997). Lactadherin (formerly BA46), a membrane-associated glycoprotein expressed in human milk and breast carcinomas, promotes ArgGlyAsp (RGD)-dependent cell adhesion. DNA and cell biology, Vol. 16, No. 7, (July 1997), pp. 861–869, ISSN 1044-5498 Wolber, F. M., Broomfield, A. M., Fray, L., Cross, M. L., & Dey, D. (2005). Supplemental dietary whey protein concentrate reduces rotavirus-induced disease symptoms in suckling mice. The Journal of nutrition, Vol. 135, No. 6, (January 2005), pp. 1470-1474, ISSN 1541-6100 Yolken, R. H., Peterson, J. A., Vonderfecht, S. L., Fouts, E. T., Midthum, K., & Newburg, D. S. (1992). Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. The Journal of clinical investigation, Vol. 90, No. 5, (November 1992), pp. 1984-1991, ISSN 0021-9738 Zaman, K., Dang, D. A., Victor, J. C., Shin, S., Yunus, M., Dallas, M. J., Podder, G., Vu, D. T., Le, T. P., Luby, S. P., Le, H. T., Coia, M. L., Lewis, K., Rivers, S. B., Sack, D. A., Schödel, F., Steele, A. D., Neuzil, K. M., & Ciarlet, M. (2010). Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double-blind, placebo-controlled trial. Lancet, Vol. 376, No. 9741, (August 2010), pp. 615-623, ISSN 1474-547X

4 Emerging Preservation Methods for Fruit Juices and Beverages H. P. Vasantha Rupasinghe and Li Juan Yu Nova Scotia Agricultural College Canada

1. Introduction This chapter provide a review of traditional and non-traditional food preservation approaches including physical methods (non-thermal pasteurization), chemical methods (natural food preservatives) and their combinations for extension of the shelf life of fruit juices and beverages. Traditionally, the shelf-life stability of juices has been achieved by thermal processing. Low temperature long time (LTLT) and high temperature short time (HTST) treatments are the most commonly used techniques for juice pasteurization. However, thermal pasteurization tends to reduce the product quality and freshness. Therefore, some non-thermal pasteurization methods have been proposed during the last couple of decades, including high hydrostatic pressure (HHP), high pressure homogenization (HPH), pulsed electric field (PEF), and ultrasound (US). These emerging techniques seem to have the potential to provide “fresh-like” and safe fruit juices with prolonged shelf-life. Some of these techniques have already been commercialized. Some are still in the research or pilot scale. The first part of the chapter will give an update of these emerging non-thermal techniques. Apart from thermal pasteurization, some chemical preservatives are also widely used for the extension of the shelf-life of fruit juices and beverages. Two of the most commonly used preservatives are potassium sorbate and sodium benzoate. However, consumer demand for natural origin, safe and environmental friendly food preservatives has been increasing since 1990s. Natural antimicrobials such as bacteriocins, organic acids, essential oils and phenolic compounds have shown considerable promise for use in some food products. The second part of the chapter will comprise of applications of these natural antimicrobials in fruit juice preservation. From scientific literature, it is apparent that some individual non-thermal methods as well as natural antimicrobials are effective to inactivate microorganisms or reduce the log colony forming units (CFU) while not adversely affecting the sensory and nutritional quality. Moreover, the combination of these techniques could also provide synergistic effects on prolonging the shelf-life of fruit juices and beverages and potentially could become replacements for traditional pasteurization methods. The third part of the chapter will provide recent progresses of these combined techniques in fruit juice shelf-life extension.

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2. Traditional thermal pasteurization Thermal processing is the most widely used technology for pasteurization of fruit juices and beverages. Juice pasteurization is based on a 5-log reduction of the most resistant microorganisms of public health significance (USFDA 2001). The process could be accomplished by different time-temperature combinations. 2.1 Low temperature long time (LTLT) Fruit juice has been traditionally pasteurized by batch heating at 63-65°C for relatively long time (D'Amico et al. 2006). This method has been replaced by high temperature short time treatment due to the undesirable quality changes during this process. 2.2 High temperature short time (HTST) HTST treatment could minimise those undesirable quality changes made by batch heating due to the much less duration of heat treatment. Currently, HTST pasteurization is the most commonly used method for heat treatment of fruit juice. For example, orange juice is processed by HTST at 90 to 95°C for 15 to 30 s (Braddock 1999). And apple juice is treated by HTST at 77 to 88°C for 25 to 30 s (Moyer & Aitken 1980).

3. Non-traditional method Thermal processing has been proven to be effective for preservation of fruit juice and beverages. However, thermal treatment tends to reduce the product quality and freshness. Nowadays, consumer demand for natural, healthy and convenient food products is fast growing, which leads to the innovation of novel food preservation technologies. Based on the literature, these novel technologies can be generally divided into physical methods (mostly non-thermal methods) and chemical approaches. 3.1 Physical methods (Non-thermal pasteurization) Some non-thermal pasteurization methods have been proposed during the last couple of decades, including high hydrostatic pressure (HHP), high pressure homogenization (HPH), pulsed electric field (PEF), and ultrasound (US). These emerging techniques seem to have the potential to provide “fresh-like” and safe fruit juices with prolonged shelf-life. 3.1.1 High hydrostatic pressure (HHP) High hydrostatic pressure (HHP) processing uses pressures up to 1000 MPa, with or without heat, to inactivate harmful microorganisms in food products (Ramaswamy et al. 2005). High hydrostatic pressure has traditionally been used in non-food areas such as ceramic and steel production. The application of HHP in food area started from 1900s when Hite and other researchers applied HHP on the preservation of milk, fruits and vegetables. However, it takes a long time for the commercial products to emerge in the market. In 1990, the first HHP processed fruit jams were sold in the Japanese market. Subsequently, HHP processed commercial products including fruit juices and beverages, vegetable products, among others, have been produced in North America, Europe, Australia, and Asia (Balasubramaniam et al. 2008).

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Generally, there are two principles that govern the behaviour of foods under pressure: the Le Chatelier-Braun principle and the Isostatic principle. The Le Chatelier-Braun principle indicates that any phenomenon (such as phase transition, change in molecular configuration, chemical reaction, etc.) accompanied by a decrease in volume is enhanced by the increase in applied pressure. The isostatic principle means that the distribution of pressure into the sample is uniform and instantaneous. Thus, the process time is independent of sample size and shape (Ramaswamy et al. 2005). HPP is proven to meet the FDA requirement of a 5-log reduction of microorganisms in fruit juices and beverages without sacrificing the sensory and nutritional attributes of fresh fruits (San Martín et al. 2002). Compared with thermal processing, HHP has many advantages. It can provide safe product with reduced processing time. It can maintain maximum fresh-like flavor and taste in the product due to the lower processing temperatures. Moreover, it is environmentally friendly since it requires only electrical energy and no waste by-products generated (Ramaswamy et al. 2005, Toepfl et al. 2006). Due to these advantages, HHP has been widely used in food product preservation including fruit and beverages in the areas of microbial inactivation (Table 1) and shelf-life extension (Table 2). Foods

Microorganism

Treatment parameters

Log reduction

Sources

Orange juice

Escherichia coli O157: H7 Listeria monocytogenes Escherichia coli 29055 Escherichia coli, Listeria innocua, Salmonella Escherichia coli, Listeria innocua

550 MPa, 30ºC, 5 min 200 MPa, 20ºC, 5 min 400 MPa, 25ºC

6

Linton et al. 1999

2.8 >5

545 MPa, 1min

5

Préstamo et al. 1999 Ramaswamy et al. 2003 Avure Technologies

241 MPa, 3 min

5

Apple jam Apple juice Apple juice Orange juice

Guerrero-Beltran et al. 2011

Table 1. Examples of HHP inactivation of microorganisms in fruit products Foods Blueberry juice

Treatment parameters 200 MPa, 15 min

Blueberry juice

400-600 MPa, 15 min

Blood orange 400-600 MPa, juice 15 min

Storage conditions Tested right after treatment

Quality changes

Total phenolic and anthocyanin content increased, whereas no changes in antioxidant capacity, pH, °Brix and Colors Tested right Total phenolic and anthocyanin after content increased; no changes in treatment pH, °Brix and Colors; but antioxidant capacity decreased 4ºC for 93.4% retention rate of anthocyanin; 10 days 85% retention rate of ascorbic acid

Table 2. Examples of HHP effect on quality attributes of fruit products

Sources Barba et al. 2011

Barba et al. 2011

Torres et al. 2011

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3.1.2 Pulsed electric field (PEF) Pulsed electric field processing (PEF) applies short bursts of high voltage electricity for microbial inactivation and causes no or minimum effect on food quality attributes. Briefly, the foods being treated by PEF are placed between two electrodes, usually at room temperature. The applied high voltage results in an electric field that causes microbial inactivation. The applied high voltage is usually in the order of 20-80 kV for microseconds. The common types of electrical field waveform applied include exponentially decaying and square wave (Knorr et al. 1994, Zhang et al. 1995, Barbosa-Cánovas et al. 1999). The principles of PEF processing have been explained by several theories including the trans-membrane potential theory, electromechanical compression theory and the osmotic imbalance theory. One of the most accepted theories is associated with the electroporation of cell membranes. It is generally believed that electric fields induce structural changes in the membranes of microbial cells based on generation of pores of the cell membrane, leading consequently to microbial destruction and inactivation (Tsong 1991, Barbosa-Cánovas et al. 1999). Compared with thermal processing, PEF processing has many advantages. It can preserve the original sensory and nutritional characteristics of foods due to the very short processing time and low processing temperatures. Energy savings for PEF processing are also important compared with conventional thermal processing. Moreover, it is environmentally friendly with no waste generated (Toepfl et al. 2006). Due to these advantages, PEF processing has been widely used in food product preservation including fruit and beverages in the areas of microbial inactivation (Table 3) and shelf-life extension (Table 4). Foods

Microorganism

Treatment parameters Apple E. coli 8739, 30kV/cm, juice Escherichia coli O157: H7 172µs,