Food Safety and Toxicity 9781684696000

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Food Safety and Toxicity Charles Lewis

Food Safety and Toxicity

Food Safety and Toxicity

Charles Lewis

Food Safety and Toxicity by Charles Lewis www.whitepressacademics.com

© 2018 White Press Academic

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TABLE OF CONTENTS

Preface

xv

Chapter 1 Fundamental Principle of Microbiology ........................................... 1 1.1 Introduction ......................................................................................... 1 1.1.1 General Microbiology ...................................................................... 3 1.1.2 Why is Microbiology Important? ..................................................... 3 1.2 General Principles ............................................................................... 4 1.3 Common Food-Borne Microorganisms ............................................... 5 1.3.1 Bacteria ............................................................................................. 5 1.3.2 Fungi ................................................................................................. 7 1.3.3 Viruses .............................................................................................. 8 1.3.4 Bacteriophage ................................................................................... 9 1.3.5 Gram-positive Bacteria ..................................................................... 9 1.3.6 Gram-negative Bacteria .................................................................. 10 1.4 Micro-Organisms and Food ............................................................... 12 1.4.1 Food Spoilage/Preservation ............................................................ 12 1.4.2 Food Safety ..................................................................................... 13 1.4.3 Fermentation ................................................................................... 14 1.5 What Causes Microorganisms to Grow ............................................. 14 1.5.1 Extrinsic Factors ............................................................................. 14 1.5.2 Intrinsic Factors .............................................................................. 15 1.5.3 Implicit Factors ............................................................................... 17 1.5.4 Interaction between Growth Factors .............................................. 19 1.6 Effects of Microorganisms on Spoilage ............................................ 19 1.6.1 Physical Changes ............................................................................ 20 1.6.2 Chemical Changes .......................................................................... 20

vi 1.7 Effects of Microorganisms on Foodborne Illness .............................. 21 1.7.1 Foodborne Disease ......................................................................... 21 1.8 Controlling Microbial Growth ........................................................... 21 1.9 Branches of Microbiology ................................................................. 23 1.9.1 Pure Microbiology .......................................................................... 23 1.9.2 Applied Microbiology .................................................................... 25 1.10 Applications of Microbiology ......................................................... 26

Chapter 2 Food Poisoning and Other Food Borne Hazards ........................... 28 2.1 What is Food Poisoning? ................................................................... 28 2.1.1 How does Food Become Contaminated? ....................................... 29 2.1.2 Who is at Risk for Food Poisoning? ............................................... 30 2.1.3 How is Food Poisoning Diagnosed? .............................................. 30 2.1.4 How is Food Poisoning Treated? .................................................... 30 2.2 Bacterial Food Poisoning .................................................................. 31 2.2.1 Food-Borne Diseases and Food Poisoning ..................................... 31 2.2.2 Food Poisoning Bacteria ................................................................. 31 2.3 Causes of Food Poisoning ................................................................. 32 2.3.1 Biological Food Poisoning ............................................................. 32 2.3.2 Bacterial Food Poisoning ............................................................... 33 2.3.3 Chemical Food Poisoning............................................................... 34 2.4 Foodborne Illness .............................................................................. 35 2.5 Causes of Foodborne Illness .............................................................. 35 2.5.1 Bacteria ........................................................................................... 35 2.5.2 Mycotoxins and Alimentary Mycotoxicoses .................................. 38 2.5.3 Viruses ............................................................................................ 40 2.5.4 Parasites .......................................................................................... 40 2.5.5 Natural Toxins ................................................................................ 41 2.5.6 Other Pathogenic Agents ................................................................ 41 2.5.7 “Ptomaine Poisoning” .................................................................... 41 2.6 Foodbrone Mechanism ...................................................................... 42 2.6.1 Incubation Period ........................................................................... 42 2.6.2 Infectious Dose ............................................................................... 42 2.7 Microbiology of Foodborne Illness ................................................... 42 2.8 Prevention of Foodborne Illness ........................................................ 43 Chapter 3 Food Spoilage .................................................................................... 47 3.1 Food Spoilage .................................................................................... 47 3.1.1 Scenario of Food Spoilage Worldwide ........................................... 48 3.1.2 Scenario of Food Spoilage in India ................................................ 50 3.2 Food Spoilage Microorganisms ......................................................... 52 3.2.1 Yeasts .............................................................................................. 52 3.2.2 Molds .............................................................................................. 53

vii 3.2.3 Bacteria ........................................................................................... 54 3.3 How Food Spoils ............................................................................... 56 3.3.1 Microorganisms .............................................................................. 56 3.3.2 Enzymes ......................................................................................... 57 3.3.3 Air ................................................................................................... 57 3.3.4 Light ............................................................................................... 57 3.3.5 Insects, Rodents, Parasites and Other Creatures ............................ 57 3.3.6 Physical Damage ............................................................................ 57 3.3.7 Temperature .................................................................................... 57 3.3.8 Time ................................................................................................ 58 3.4 Types of Food Spoilage ..................................................................... 58 3.4.1 Microbial Spoilage ......................................................................... 58 3.4.2 Physical Spoilage ........................................................................... 58 3.4.3 Chemical Spoilage .......................................................................... 59 3.4.4 Appearance of Spoiled Food .......................................................... 59 3.5 Spoilage of Fruits and Vegetables ..................................................... 59 3.6 Spoilage of Dairy Products ................................................................ 61 3.7 Prevention from Food Spoilage Microorganism ............................... 62 3.7.1 Chemical Preservatives .................................................................. 63 3.7.2 Use of Food Additives .................................................................... 64 3.8 Factors Affecting Food Spoilage ....................................................... 65 3.8.1 Water Content ................................................................................. 65 3.8.2 Environmental Conditions .............................................................. 66 3.8.3 Packaging and Storage ................................................................... 66

Chapter 4 Principle of Safe Food Storage ........................................................ 67 4.1 Food Storage ...................................................................................... 67 4.2 General Principles of Storage ............................................................ 68 4.2.1 Temperature .................................................................................... 68 4.2.2 Humidity ......................................................................................... 68 4.2.3 Rotation .......................................................................................... 68 4.2.4 Position ........................................................................................... 68 4.2.5 Labelling ......................................................................................... 69 4.3 Domestic Food Storage ..................................................................... 69 4.3.1 Food Storage Safety ....................................................................... 69 4.3.2 Storing Oils and Fats ...................................................................... 70 4.3.3 Dry Storage of Foods ...................................................................... 70 4.3.4 Food Rotation ................................................................................. 72 4.3.5 For Emergency Preparation ............................................................ 72 4.4 Commercial Food Logistics ............................................................... 73 4.5 Storing Canned Food ......................................................................... 73 4.6 Food Preservation .............................................................................. 74

viii 4.6.1 Why Preservation? .......................................................................... 75 4.6.2 How Long to Preserve? .................................................................. 76 4.6.3 For Whom to Preserve? .................................................................. 76 4.6.4 Importance of Food Preservation ................................................... 78 4.6.5 Principles of Food Preservation ..................................................... 79 4.7 Traditional Techniques ...................................................................... 80 4.7.1 Curing ............................................................................................. 80 4.7.2 Cooling ........................................................................................... 80 4.7.3 Freezing .......................................................................................... 80 4.7.4 Boiling ............................................................................................ 81 4.4.5 Heating ........................................................................................... 81 4.7.6 Sugaring .......................................................................................... 81 4.7.7 Pickling ........................................................................................... 81 4.7.8 Lye .................................................................................................. 82 4.7.9 Canning........................................................................................... 82 4.7.10 Jellying ......................................................................................... 82 4.7.11 Jugging.......................................................................................... 83 4.7.12 Burial ............................................................................................ 83 4.7.13 Fermentation ................................................................................. 83 4.8 Modern Industrial Techniques ........................................................... 84 4.8.1 Pasteurization ................................................................................. 84 4.8.2 Vacuum Packing ............................................................................. 84 4.8.3 Artificial Food Additives ................................................................ 84 4.8.4 Irradiation ....................................................................................... 84 4.8.5 Pulsed Electric Field Electroporation ............................................. 85 4.8.6 Modified Atmosphere ..................................................................... 85 4.8.7 Non-thermal Plasma ....................................................................... 86 4.8.8 High-pressure Food Preservation ................................................... 86 4.8.9 Biopreservation ............................................................................... 87 4.8.10 Hurdle Technology ....................................................................... 87

Chapter 5 Design of Food Processing Equipment ........................................... 88 5.1 Introduction ....................................................................................... 88 5.2 Legislation ......................................................................................... 89 5.3 Basic Hygienic Requirements ........................................................... 92 5.4 Materials of Construction .................................................................. 95 5.4.1 General Recommendations ............................................................. 95 5.4.2 Use of Metals and Alloys ............................................................... 95 5.4.3 Use of Plastics ................................................................................ 96 5.4.4 Use of Rubbers ............................................................................... 97 5.4.5 Other Materials ............................................................................... 98 5.5 Surface Finish .................................................................................... 99

ix 5.6 Hygienic Design of Open Equipment for Processing of Food .......... 99 5.6.1 Permanent and Dismountable Joints .............................................. 99 5.6.2 Hygienic Design of Process Vessels, Containers, Bins, etc. ........ 103 5.6.3 Equipment Framework ................................................................. 113 5.6.4 Feet ............................................................................................... 114 5.6.5 Castors .......................................................................................... 116 5.6.6 Belt Conveyor ............................................................................... 119 5.6.7 Covers and Guards ....................................................................... 122 5.6.8 Maintenance Enclosures ............................................................... 123 5.7 Hygienic Design Closed Equipment for Processing of Liquid Food ....................................................................................................... 125 5.7.1 Process and Utility Lines .............................................................. 125 5.7.2 Hoses ............................................................................................ 129 5.7.3. Pipe Joints .................................................................................... 129 5.7.4 Hygienic Design of Pumps ........................................................... 132 5.7.5 Sensors and Instrumentation ......................................................... 133 5.7.6 Hygienic Design of Valves ........................................................... 136 5.8 Installation of the Food Processing Equipment in the Food Factory139 5.8.1 Clearance with Respect to the Floor, Walls and Adjacent Equipment .............................................................................................. 139 5.8.2 Raised Walkways and Stairs ......................................................... 140 5.9 Hygiene Practices during Maintenance Operations in the Food Industry ........................................................................................ 141 5.9.1 Maintenance and Repair, a Necessary Evil .................................. 141 5.9.2 Scheduled Preventive Maintenance .............................................. 141

Chapter 6 Basic of Toxicology Related to Food ............................................. 149 6.1 Introduction ..................................................................................... 149 6.2 Basic Principles ............................................................................... 151 6.3 Branches of Toxicology ................................................................... 152 6.4 Types of Toxicology ........................................................................ 152 6.4.1 Medical Toxicology ...................................................................... 152 6.4.2 Clinical Toxicology ...................................................................... 153 6.4.3 Computational Toxicology ........................................................... 153 6.5 Testing Methods .............................................................................. 154 6.5.1 Non-human Animals ..................................................................... 154 6.5.2 Alternative Testing Methods ......................................................... 154 6.6 Dose-Response ................................................................................ 155 6.7 Potency ............................................................................................ 158 6.8 Hormesis .......................................................................................... 158 6.9 Margin of Safety .............................................................................. 159 6.10 Biologic Factors That Influence Toxicity ...................................... 161

x 6.11 Absorption ..................................................................................... 162 6.12 Biological Determination of Toxicants .......................................... 165 6.12.1 Acute Toxicity............................................................................. 165 6.12.2 Genetic Toxicity ......................................................................... 165 6.12.3 Bioassay ...................................................................................... 166 6.12.4 Metabolism ................................................................................. 168 6.12.5 Subchronic Toxicity.................................................................... 168 6.12.6 Teratogenesis .............................................................................. 169 6.12.7 Chronic Toxicity ......................................................................... 171 6.13 Toxicology as a Profession ............................................................ 172

Chapter 7 Food Additives ................................................................................. 174 7.1 Introduction ..................................................................................... 174 7.2 Regulations ...................................................................................... 177 7.3 Preservatives .................................................................................... 180 7.3.1 Benzoic Acid ................................................................................ 181 7.3.2 Sorbic Acid and Potassium Sorbate .............................................. 183 7.3.3 Hydrogen Peroxide ....................................................................... 184 7.3.4 AF-2 [2-(-furyl)-3-(5-nitro-2-furyl)acrylamide] ........................... 184 7.4 Antioxidants ..................................................................................... 185 7.4.1 L-Ascorbic Acid (Vitamin C) ....................................................... 186 7.4.2 dl-a-Tocopherol (Vitamin E) ........................................................ 186 7.4.3 Propyl Gallate ............................................................................... 187 7.4.4 Butylated Hydroxyanisol and Butylated Hydroxytoluene ........... 187 7.5 Sweeteners ....................................................................................... 188 7.5.1 Saccharin and Sodium Saccharin ................................................. 188 7.5.2 Sodium Cyclamate ........................................................................ 189 7.6 Colouring Agents ............................................................................. 190 7.6.1 Amaranth (FD&C Red No. 2) ...................................................... 191 7.6.2 Tartrazine (FD&C Yellow No. 4) ................................................. 192 7.7 Flavouring Agents ........................................................................... 193 7.7.1 Methyl Anthranilate ...................................................................... 194 7.7.2 Safrole (1-Allyl-3,4- Methylenedioxybenzene) ........................... 194 7.7.3 Diacetyl (2, 3-butane dione) ......................................................... 195 7.8 Flavour Enhancers ........................................................................... 195 Chapter 8 Toxicants Formed during Food Processing .................................. 196 8.1 Introduction ..................................................................................... 196 8.2 Polycyclic Aromatic Hydrocarbons (PAHs) .................................... 197 8.2.1 Occurrence .................................................................................... 197 8.2.2 Benzo[a]pyrene (BP) .................................................................... 199 8.3 Maillard Reaction Products ............................................................. 202

xi 8.4 Polycyclic Aromatic Amines (PAA) ................................................ 203 8.4.1 Occurrence .................................................................................... 203 8.4.2 Toxicity ......................................................................................... 205 8.5 N-Nitrosamines ................................................................................ 206 8.5.1 Precursors ..................................................................................... 206 8.5.2 Occurrence in Various Foods ........................................................ 207 8.5.3 Toxicity ......................................................................................... 208 8.5.4 Mode of Toxic Action ................................................................... 209 8.5.5 General Considerations ................................................................ 210 8.6 Acrylamide ...................................................................................... 211 8.6.1 Formation Mechanisms of Acrylamide in Foods ......................... 211 8.6.2 Toxicity ......................................................................................... 213 8.6.3 Mode of Action ............................................................................. 213 8.6.4 General Considerations ................................................................ 214 8.7 Food Irradiation ............................................................................... 215

Chapter 9 Personal Hygiene and Sanitary Food Handling........................... 216 9.1 Personal Hygiene ............................................................................. 216 9.1.1 Employee Hygiene ....................................................................... 216 9.1.2 Skin ............................................................................................... 217 9.1.3 Fingers .......................................................................................... 219 9.1.4 Fingernails .................................................................................... 219 9.1.5 Jewelry .......................................................................................... 219 9.1.6 Hair ............................................................................................... 219 9.1.7 Eyes .............................................................................................. 220 9.1.8 Mouth ........................................................................................... 220 9.1.9 Nose, Nasopharynx, and Respiratory Tract .................................. 220 9.1.10 Excretory Organs ........................................................................ 221 9.1.11 Personal Contamination of Food Products ................................. 222 9.1.12 Hand Washing ............................................................................. 223 9.1.13 Methods of Disease Transmission .............................................. 226 9.1.15 Requirements for Hygienic Practices ......................................... 227 9.2 Sanitary Food Handling................................................................... 228 9.2.1 Role of Employees ....................................................................... 229 9.2.2 Required Personal Hygiene .......................................................... 229 9.2.3 Facilities ....................................................................................... 229 9.2.4 Employee Supervision .................................................................. 230 9.2.5 Employee Responsibilities ........................................................... 230 Chapter 10 Quality Assurance for Sanitation .................................................. 231 10.1 The Role of Total Quality Management ........................................ 232 10.2 Quality Assurance for Effective Sanitation ................................... 233

xii 10.2.1 Major Components of Quality Assurance .................................. 233 10.3 Organization for Quality Assurance .............................................. 234 10.3.1 Major Responsibilities of a Sanitation Quality Assurance Programme ............................................................................................. 235 10.3.2 The Role of ISO Accreditation ................................................... 236 10.3.3 The Role of Management in Quality Assurance ......................... 236 10.3.4 Quality Assurance and Job Enrichment ...................................... 237 10.3.5 Quality Assurance Programme Structure ................................... 237 10.4 Establishment of a Quality Assurance Programme ....................... 239 10.4.1 Elements of a Total Quality Assurance System .......................... 240 10.4.2 New Employee Training ............................................................. 240 10.4.3 Hazard Analysis Critical Control Point (HACCP) Approach..... 240 10.4.4 Programme Evaluation ............................................................... 241 10.4.5 Assay Procedures for Evaluation of Sanitation Effectiveness .... 241 10.4.6 Importance of a Monitoring Programme .................................... 242 10.4.7 Auditing Considerations ............................................................. 243 10.4.8 Preparation for an Audit ............................................................. 244 10.4.9 Recall of Unsatisfactory Products .............................................. 244 10.4.10 Sampling for a Quality Assurance Programme ........................ 245 10.4.11 Sampling Procedures ................................................................ 246 10.4.12 Basic QA Tools ......................................................................... 247 10.4.13 Role of Statistical Quality Control ........................................... 248 10.4.14 Central Tendency Measurements .............................................. 248 10.4.15 Displaying Data ........................................................................ 249 10.4.16 Control Charts .......................................................................... 250 10.4.17 Attribute Control Charts ........................................................... 253 10.4.18 Explanation and Definition of Statistical Quality Control Programme Standards ............................................................................ 256 10.4.19 Cumulative Sum (CUSUM) Control Charts ............................. 257

Chapter 11 Environmental Toxicology ............................................................. 258 11.1 Environmental Toxicology ............................................................ 258 11.2 Sources of Environmental Toxicity ............................................... 259 11.2.1 PCBs ........................................................................................... 259 11.2.2 Heavy Metals .............................................................................. 259 11.2.3 Pesticides .................................................................................... 260 11.3 Environmental Contaminants and Food Safety ............................. 261 11.4 Pollution from Food Processing Factories and Environmental Protection ............................................................................................... 262 11.5 Pollution from Food Processing .................................................... 263 11.5.2 Meat, Poultry, and Seafood Sector ............................................. 264 11.5.3 Beverage and Fermentation Sector ............................................. 265 11.5.4 Dairy Sector ................................................................................ 265

xiii 11.5.5 Can Cooker Products .................................................................. 266 11.5.6 Wastewater from Food Processing Factories .............................. 266 11.5.7 Defining Load Using BOD5 and COD ....................................... 267 11.5.8 Relating COD to BOD5 .............................................................. 267 11.6 Environmental Protection .............................................................. 267 11.6.1 Source Reduction ........................................................................ 267 11.6.2 Management Alternatives ........................................................... 268 11.6.3 Clean Technology Developments ............................................... 269 11.7 Future Trends ................................................................................. 272

Chapter 12 Food Laws and Food Standards .................................................... 274 12.1 Food Laws ..................................................................................... 275 12.1.1 Prevention of Food Adulteration Act .......................................... 275 12.1.2 The Fruit Products Order ............................................................ 276 12.1.3 Meat Products Order ................................................................... 276 12.2 ISI Standards ................................................................................. 277 12.3 The Agmark Standard .................................................................... 277 12.4 Export Inspection Council ............................................................. 277 12.5 Standards of Weights and Measures .............................................. 278 12.6 Food Adulteration .......................................................................... 278 12.6.1 Common Adulterants and Their Ill-effects ................................. 278 12.6.2 Incidental Adulterants ................................................................. 279 12.6.3 Metallic Contamination .............................................................. 279 12.6.4 Contamination by Pests and Pesticide Residues ........................ 280 12.6.5 Packaging Hazards ..................................................................... 280 12.66 Health Hazards due to Consuming Exposed Snacks ................... 281 12.6.7 Simple Physical Tests for Detection of Food Adulterants .......... 281 12.6.8 Simple Laboratory Chemical Tests ............................................. 282 12.7 Consumer Protection ..................................................................... 282 12.7.1 Municipal Laboratories .............................................................. 283 12.7.2 Food and Drug Administration ................................................... 283 12.7.3 The Central Food Testing Laboratory ........................................ 284 12.7.4 Export Inspection Council Laboratory ....................................... 284 12.7.5 Central Grain Analysis Laboratory............................................. 284 12.8 Voluntary Agencies ........................................................................ 284 12.8.1 Quality Control Laboratories of Companies .............................. 285 12.8.2 Quality Control laboratories of Consumer Co-operatives .......... 285 12.8.3 Private Testing Laboratories ....................................................... 286 12.8.4 Consumer Guidance Society ...................................................... 286 Bibliography ..................................................................................... 287 Index ................................................................................................. 290

PREFACE

The book starts with an introduction discussing the history of hygiene. This chapter discusses the first origins of hygiene as a concept thousands of years ago. It demonstrates very clearly why hygiene is so important and why, even today, people die because of not complying with basic hygiene requirements. To be able to decide on measures to control product safety, it is essential to understand the risks associated with product safety. Chapter 1 discuss about the fundamental principles of microbiology. This chapter also tells what causes microorganism to grow and their effects. Chapter 2 depicts about the Food Poisoning and Other Food Borne Hazards. This chapter presents the various illness causes by food borne and their prevention. Chapter 3 describes about food spoilage and their prevention from microorganisms. Chapter 4 depicts about the Principle of Safe Food Storage and modern industrial techniques. Chapter 5 discuss about the Design of Food Processing Equipment and Hygiene practices during maintenance operations in the food industry. Chapter 6 tells about the Basic of Toxicology Related to Food. Chapter 7 tells about the Food Additives and their uses. Chapter 8 depicts about the Toxicants Formed During Food Processing. Chapter 9 tells about the Personal Hygiene and Sanitary Food Handling. Chapter 10 describes standards for Quality Assurance of Sanitation. Chapter 11 discuss about Environmental Toxicology. The last chapter is based on various Food Laws and Food Standards.

1 Fundamental Principle of Microbiology

1.1 INTRODUCTION

Microbiology is the study of microscopic organisms and their activities; within this orbit is the study of the distribution of the organisms, their characteristics, and their beneficial and harmful effects, especially in relation to the changes they make in their environment of which food is one particular example. These small living organisms or microorganisms as they are called are capable of individual existence and are not aggregated into tissues or organs as in higher plants and animals. Six major groups of microorganisms are generally recognized, namely bacteria, fungi, viruses, algae, protozoa and rickettsias. The bacteria are the most important in relation to food but fungi also have a significant role. Of less importance are the viruses whilst the remaining groups, algae (simple plants), protozoa (single-celled animals) and rickettsias (specialized insect parasites) are beyond the scope of this book and will not be considered further except in relation to food-borne illnesses. Microorganisms, particularly bacteria, are the most ubiquitous of living organisms. Bacteria occur in the air in varying numbers and up to considerable heights. Large numbers are found in the soil (1 g of typical garden soil will contain many millions of bacteria) whilst considerably smaller numbers are found in fresh water and sea water. Bacteria, as a whole, are extremely adaptable and in view of this flourish in a wide range of environments. One such specialized group, the barophiles, has been isolated from the depths of the sea and from oil wells where pressures in excess of 100 atm may be experienced. Some bacterial species are adapted to live at very high temperatures

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whilst others favour low temperatures. At one extreme bacteria from a volcanic crater in the Bay of Naples are able to grow at 110°C; at the other extreme bacteria, isolated from Antarctic regions, may be incapable of growth above 1 ooc. Certain bacteria are capable of surviving concentrations of injurious chemicals which would be fatal to other forms of life. Bacteria are found on the surfaces and within the alimentary and respiratory tracts of larger animals although tissues of healthy animals and plants are generally free from bacteria. This adaptability of bacteria, enabling them to live in extreme environmental conditions, can often cause unexpected problems in the food industry. Microbiology is the study of microorganisms, which are unicellular or cell-cluster microscopic organisms. This includes eukaryotes such as fungi and protists and prokaryotes such as bacteria and certain algae. Viruses are also included. Microbiology subdivided into divisions including bacteriology, virology, mycology, parasitology and others. A scientist who specializes in the area of microbiology is called a microbiologist. Microbiology can be divided into several subdisciplines, including: • Microbial physiology: The study of how the microbial cell functions biochemically. Includes the study of microbial growth, microbial metabolism and microbial cell structure. • Microbial genetics: The study of how genes are organised and regulated in microbes in relation to their cellular functions. Closely related to the field of molecular biology. • Molecular Microbiology: The study of the molecular biology and genomics of microorganisms. • Medical microbiology: The study of the role of microbes in human illness. Includes the study of microbial pathogenesis and epidemiology and is related to the study of disease pathology and immunology. • Veterinary microbiology: The study of the role in microbes in veterinary medicine or animal taxonomy. • Environmental microbiology: The study of the function and diversity of microbes in their natural environments. Includes the study of microbial ecology, microbially-mediated nutrient cycling, geomicrobiology, microbial diversity and bioremediation. Characterisation of key bacterial habitats such as the rhizosphere and phyllosphere. • Evolutionary microbiology: The study of the evolution of microbes. Includes the study of bacterial systematics and taxonomy. • Industrial microbiology: The exploitation of microbes for use in industrial processes. Examples include industrial fermentation and wastewater treatment. Closely linked to the biotechnology industry. This field also includes brewing, an important application of microbiology. • Aeromicrobiology: The study of airborne microorganisms. • Food Microbiology: The study of microorganisms causing food spoilage.

Fundamental Principle of Microbiology

3

• Pharmaceutical microbiology: The study of microorganisms causing pharmaceutical contamination and spoillage. 1.1.1 GENERAL MICROBIOLOGY • Microbial Biodegradation, Bioremediation and Biotransformation Interest in the microbial biodegradation of pollutants has intensified in recent years as mankind strives to find sustainable ways to cleanup contaminated environments. These bioremediation and biotransformation methods endeavour to harness the astonishing, naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds. • Environmental Microbiology The study of the composition and physiology of microbial communities in the environment i.e. the soil, water, air and sediments covering the planet. Can also include the microorganisms living on or in the animals and plants that inhabit these areas. • Oral Microbiology The study of the microorganisms that inhabit the mouth and in particular those involved in the two major dental diseases: caries and periodontal disease. Oral bacteria include streptococci, lactobacilli, staphylococci, corynebacteria, and various anaerobes in particular bacteroides. • Plant Pathogenic Bacteria Bacteria pathogenic for plants are responsible for devastating losses in agriculture. Plant pathogenic bacteria impact innumerable and valuable agricultural crops, causing hundreds of millions of dollars in damage each year. The use of antibiotics to control such infections is restricted in many countries due to worries over the evolution and transmission of antibiotic resistance. • Microbiology Societies A list of societies relevant to microbiology 1.1.2 WHY IS MICROBIOLOGY IMPORTANT? To the lay person, microbiology means the study of sinister, invisible ‘bugs’ that cause disease. As a subject, it generally only impinges on the popular consciousness in news coverage of the latest ‘health scare’. It may come as something of a surprise therefore to learn that the vast majority of microorganisms coexist alongside us without causing any harm. Indeed, many perform vital tasks such as the recycling of essential elements, without which life on our planet could not continue. Other microorganisms have been exploited by humans for our own benefit, for instance in the manufacture of antibiotics and foodstuffs. To get some idea of the importance of microbiology in the world today, just consider the following list of some of the general areas in which the expertise of a microbiologist might be used: • Medicine • Environmental science • Food and drink production • Fundamental research

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Food Hygiene and Toxicology

• Agriculture • Pharmaceutical industry • Genetic engineering. The popular perception among the general public, however, remains one of infections and plagues. Think back to the first time you ever heard about microorganisms; almost certainly, it was when you were a child and your parents impressed on you the dangers of ‘germs’ from dirty hands or eating things after they’d been on the floor. In reality, only a couple of hundred out of the half million or so known bacterial species give rise to infections in humans; these are termed pathogens, and have tended to dominate our view of the microbial world.

1.2 GENERAL PRINCIPLES

There are two characteristics that hold together the field of microbiology, one fundamental and the other less so. The fundamental characteristic of microorganisms are that they are small. In fact, most of the organisms studied by microbiologists – or, more broadly, introduced by general microbiology texts – are so small that studying individual organisms, other than via microscopy techniques, is technically demanding (as, too, is microscopy, though in this modern age we take for granted its sophistication relative to what was available before good, easy-to-use microscopes became widely available). Other than microscopically, microbiological characterization typically involves the study of bulk properties, especially those of clonal populations rather than study of the properties of individual organisms. The consequence of this analysis of populations rather than of individual organisms is that a key step in microbiological characterization is to bring organisms into what is known as a pure culture, that is, clonal populations. This is equivalent, perhaps, to studying fish properties within an aquarium in which the fish are isolated from other fish. Microorganisms found in pure culture, however, are typically generated by microbial population growth rather than by the mixing together of individuals removed from communities containing other species. Notable additional differences between fish and microbes in this analogy are that fish more typically are sexual in their reproduction and fish also are less likely to be euthanized en masse for the sake of studying their common but nonetheless individual properties, whereas such mass killing of laboratory population is a standard technique within the microbiology laboratory. The second key characteristic that unites much though not all of microbiology is medicine. Thus, there are organisms that are studied within the context of biomedicine that strictly are not microorganisms, but which nevertheless are small, pathogenic, and which can be analyzed via an approximation of typical microbiological techniques. These latter organisms include especially the helminthes, a.k.a., parasitic worms, though perhaps arguably also could include various additional multicellular eukaryotes such as molds or certain algae. Medical microbiology thus tends to encompass a greater as

Fundamental Principle of Microbiology

5

well as different diversity of organisms than does non-medical microbiology, focusing on organisms that are associated with infectious disease rather organisms that more strictly may be described as microorganisms. Thus, though less germane to what exactly a microorganism is, a large fraction of microbiological research is medically oriented. This emphasis is a result of funding priorities. As a consequence, the microbiology literature tends to be dominated by studies that are initiated at least in part from a perspective of health issues. More generally, the types of organisms that microbiologists tend to study are ones that have health or economic relevance, or both. Microbial evolution studies are less bound by this need to be medically or economically relevant. Therefore what is studied and how can be more diverse than often is the case in more medically oriented microbiology laboratories. Nonetheless, the concentration of knowledge and funding on medical and otherwise economically relevant issues and organisms tends to ground microbial evolution-type studies closer to biomedicine than might otherwise have been the case. By way of example, it can be much simpler to receive funding to study, or otherwise publish in a prestigious location, the evolutionary diversification of a pathogen, e.g., such as of human immunodeficiency virus (HIV), than it can be to fund and publish the same research on viruses affecting a medically and economically irrelevant or otherwise non-charismatic species (e.g., such as hagfish pathogens). These emphases are not necessarily a bad thing, but are worth appreciating especially as a guide towards navigating the microbial population biology literature.

1.3 COMMON FOOD-BORNE MICROORGANISMS 1.3.1 BACTERIA • Acinetobacter The genus Acinetobacter is a group of Gram-negative, non-motile and non-fermentative bacteria belonging to the family Moraxellaceae. They are important soil organisms where they contribute to the mineralisation of, for example, aromatic compounds. Acinetobacter are able to survive on various surfaces (both moist and dry) in the hospital environment, thereby being an important source of infection in debilitated patients. These bacteria are innately resistant to many classes of antibiotics. In addition, Acinetobacter is uniquely suited to exploitation for biotechnological purposes. • Bacillus Bacillus subtilis is one of the best understood prokaryotes in terms of molecular biology and cell biology. Its superb genetic amenability and relatively large size have provided powerful tools to investigate a bacterium in all possible aspects. Recent improvements in technology have provided novel and amazing insights into the dynamic structure of this single cell organism. The organism is a model for differentiation, gene/protein regulation and cell cycle events in bacteria.

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• Clostridium The genus Clostridium comprises a heterogeneous group of anaerobic spore-forming bacteria, including prominent toxin-producing species, such as C. difficile, C. botulinum, C. tetani and C. perfringens, in addition to well-known non-pathogens like solventogenic C. acetobutylicum. Clostridia produce a range of different clostridial toxins including two of the most potent biological toxins known to affect humans. • Corynebacteria Corynebacteria are a diverse group Gram-positive bacteria found in a range of different ecological niches such as soil, vegetables, sewage, skin, and cheese smear. Some are important pathogens while others are of immense industrial importance. • Cyanobacteria Cyanobacteria are a fascinating and versatile group of bacteria of immense biological importance. Thought to be amongst the first organisms to colonize the earth, these bacteria are the photosynthetic ancestors of chloroplasts in eukaryotes such as plants and algae. In addition they can fix nitrogen, survive in very hostile environments (e.g. down to -60°C), are symbiotic, have circadian rhythms, exhibit gliding mobility, and can differentiate into specialized cell types called heterocysts. This makes them ideal model systems for studying fundamental processes such as nitrogen fixation and photosynthesis. • Gram-positive Bacteria Gram-positive bacteria are generally divided into the Actinobacteria and the Firmicutes. The Actinobacteria include some of the most common soil bacteria and some pathogens, such as Mycobacterium, Corynebacterium. • Helicobacter pylori Helicobacter pylori causes peptic ulcers, gastritis and gastric cancer. The bacterium infects up to 50% of the human population. H. pylori has very unique characteristics, such as microaerophily and nitrogen metabolism. • Lactobacillus Lactobacillus is a genus of Gram-positive facultative anaerobic or microaerophilic bacteria. In humans they are symbiotic and are found in the gut flora. Lactobacillus species are used for the production of yogurt, cheese, sauerkraut, pickles, beer, wine, cider, kimchi, chocolate and other fermented foods, as well as animal feeds such as silage. • Legionella Legionella is the genus of bacterium that causes Legionnaires’ Disease also known as Legionellosis. These bacteria are commonly found in aquatic habitats where they can survive and multiply in different protozoa enabling the bacterium to be transmissible and pathogenic to humans. • Mycobacterium Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy. Mycobacteria are aerobic and non-motile bacteria (except for the species Mycobacterium marinum which has been shown to be motile within macrophages) that are

Fundamental Principle of Microbiology

•

•

•

•

•

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characteristically acid-alcohol fast. Mycobacteria do not contain endospores or capsules and are usually considered to be Gram-positive bacteria. Pasteurellaceae The Pasteurellaceae family comprises a large and diverse family of Gram-negative bacteria with members ranging from important pathogens such as Haemophilus influenzae to commensals of the animal and human mucosa. Members of the family Pasteurellaceae cause a wide variety of diseases in humans and animals. Pseudomonas The bacterial genus Pseudomonas includes the opportunistic human pathogen P. aeruginosa, plant pathogenic bacteria, plant beneficial bacteria, ubiquitous soil bacteria with bioremediation capabilities and other species that cause spoilage of milk and dairy products. P. aeruginosa can cause chronic opportunistic infections that have become increasingly apparent in immuno compromised patients and the ageing population of industrialised societies. Staphylococcus Species of Staphylococcus are important pathogens that cause a variety of diseases in humans and animals. In particular, they cause hospital acquired infections and antibiotic resistant strains (MRSA) cause major problems in hospitals. Treponema Treponema pallidum is a gram-negative spirochaete bacterium. There are at least four known subspecies: T. pallidum pallidum, which causes syphilis; T. pallidum pertenue, which causes yaws; T. pallidum carateum, which causes pinta; and T. pallidum endemicum, which causes bejel. Vibrio cholerae Vibrio cholerae is the causative agent of cholera and belongs to a group of organisms whose natural habitats are the aquatic ecosystems. The strains that cause cholera epidemics have evolved from non-pathogenic progenitor strains by acquisition of virulence genes, and V. cholerae represents a paradigm for this evolutionary process.

1.3.2 FUNGI • Candida Candida species are important human pathogens that are best known for causing opportunist infections in immunocompromised hosts (eg transplant patients, AIDS sufferers, cancer patients). Infections are difficult to treat and can be very serious: 30-40% of systemic infections result in death. The sequencing of the genome of C. albicans and those of several other medicallyrelevant Candida species has provided a major impetus for Candida comparative and functional genomic analyses. These have provided a fascinating insight into the molecular and cellular biology of these fungi and these should pave the way for the development of more sensitive diagnostic strategies and novel antifungal therapies. • Pathogenic Fungi Pathogenic fungi are fungi that cause disease in humans or other organisms. The study of pathogenic fungi is referred to as medical

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mycology. Although fungi are eukaryotic organisms many pathogenic fungi are also microorganisms. 1.3.3 VIRUSES • Animal Viruses The study of animal viruses is important from a veterinary viewpoint and many of these viruses causes diseases that are economically devastating. Many animal viruses are also important from a human medical perspective. • Bluetongue Virus Bluetongue virus (BTV), a member of Orbivirus genus within the Reoviridae family causes serious disease in livestock (sheep, goat, cattle). Partly due to this BTV has been in the forefront of molecular studies for last three decades and now represents one of the best understood viruses at the molecular and structural levels. • Coronavirus Coronaviruses are positive-strand, enveloped RNA viruses that are important pathogens of mammals and birds. This group of viruses cause enteric or respiratory tract infections in a variety of animals including humans, livestock and pets. • Bacteriophage The New Phage Biology from genomics to applications. Bacterial viruses, or bacteriophages, are estimated to be the most widely distributed and diverse entities in the biosphere. From initial research defining the nature of viruses, to deciphering the fundamental principles of life, to the development of the science of molecular biology, phages have been ‘model organisms’ for probing the basic chemistry of life. With more recent advances in technology, most notably the ability to elucidate the genome sequences of phages and their bacterial hosts, there has been a resurgence of interest in phages as more information is generated regarding their biology, ecology and diverse nature. Phage research in more recent years has revealed not only their abundance and diversity of form, but also their dramatic impact on the ecology of our planet, their influence on the evolution of microbial populations, and their potential applications. This review focuses on this new post-genomic era of phage biology, from information emerging from genomics and metagenomics approaches through to applications in agriculture, human therapy and biotechnology. • Foot and Mouth Foot-and-mouth disease virus (FMDV) is the prototypic member of the Aphthovirus genus in the Picornaviridae family. This picornavirus is the etiological agent of an acute systemic vesicular disease that affects cattle worldwide. • Cytomegalovirus Cytomegaloviruses are members of the herpesvirus group and can infect humans and other primates. Between 50-80% of adults in developed countries and up to 100% in developing countries are infected with human cytomegalovirus. Infection causes problems in immunocompromised hosts including AIDS victims or patients undergoing organ and stem cell

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transplantation and congenital infection can cause birth defects in the child. Development of an effective vaccine has high priority. • Epstein-Barr Virus Epstein-Barr virus (EBV) is a human gamma herpes virus that remains one of the most successful viral parasites known to man. It is the etiological agent of infectious mononucleosis and is the major biological cofactor contributing to a number of human cancers including B-cell neo-plasms (e.g. Burkitt’s lymphoma, Hodgkin’s disease and immunoblastic lymphomas), certain forms of T-cell lymphoma, and some epithelial tumours (e.g. nasopharyngeal carcinomas and gastric carcinomas). • Papillomavirus Papillomaviruses are oncogenic DNA tumour viruses that infect humans and animals. Human papillomavirus is one of the most common causes of sexually transmitted infection in the world and can also cause cancer. Papillomavirus research has been revolutionised in recent years with the advent of new technologies such as organotypicraft cultures, virus-like particles and transgenic mice. 1.3.4 BACTERIOPHAGE • Phage Bacterial viruses, or bacteriophages, are estimated to be the most widely distributed and diverse entities in the biosphere. From initial research defining the nature of viruses, to deciphering the fundamental principles of life, to the development of the science of molecular biology, phages have been ‘model organisms’ for probing the basic chemistry of life. With more recent advances in technology, most notably the ability to elucidate the genome sequences of phages and their bacterial hosts, there has been a resurgence of interest in phages as more information is generated regarding their biology, ecology and diverse nature. Phage research in more recent years has revealed not only their abundance and diversity of form, but also their dramatic impact on the ecology of our planet, their influence on the evolution of microbial populations, and their potential applications. 1.3.5 GRAM-POSITIVE BACTERIA Gram-positive Bacteria are generally divided into the Actinobacteria and the Firmicutes. The Actinobacteria include some of the most common soil bacteria and some pathogens, including Mycobacterium, Corynebacterium, Bacillus, Staphylococcus. • Bacillus Bacillus subtilis is one of the best understood prokaryotes in terms of molecular biology and cell biology. Its superb genetic amenability and relatively large size have provided powerful tools to investigate a bacterium in all possible aspects. Recent improvements in technology have provided novel and amazing insights into the dynamic structure of this single cell organism. The organism is a model for differentiation, gene/protein regulation and cell cycle events in bacteria.

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• Clostridium The genus Clostridium comprises a heterogeneous group of anaerobic spore-forming bacteria, including prominent toxin-producing species, such as C. difficile, C. botulinum, C. tetani and C. perfringens, in addition to well-known non-pathogens like solventogenic C. acetobutylicum. Clostridia produce a range of different clostridial toxins including two of the most potent biological toxins known to affect humans. • Corynebacteria Corynebacteria are a diverse group of Gram-positive bacteria found in a range of different ecological niches such as soil, vegetables, sewage, skin, and cheese smear. Some are important pathogens while others are of immense industrial importance. • Lactobacillus Lactobacillus is a genus of Gram-positive facultative anaerobic or microaerophilic bacteria. In humans they are symbiotic and are found in the gut flora. Lactobacillus species are used for the production of yogurt, cheese, sauerkraut, pickles, beer, wine, cider, kimchi, chocolate and other fermented foods, as well as animal feeds such as silage. • Mycobacterium Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy. Mycobacteria are aerobic and non-motile bacteria (except for the species Mycobacterium marinum which has been shown to be motile within macrophages) that are characteristically acid-alcohol fast. Mycobacteria do not contain endospores or capsules and are usually considered to be Gram-positive bacteria. • Staphylococcus Species of Staphylococcus are important pathogens that cause a variety of diseases in humans and animals. In particular, they cause hospital acquired infections and antibiotic resistant strains (MRSA) cause major problems in hospitals. 1.3.6 GRAM-NEGATIVE BACTERIA Many species of Gram-negative bacteria are pathogenic. Medically relevant Gramnegative include Acinetobacter, Helicobacter pylori, Legionella, Pasteurellaceae, Pseudomonas, Treponema, Vibrio cholerae. • Acinetobacter The genus Acinetobacter is a group of Gram-negative, non-motile and non-fermentative bacteria belonging to the family Moraxellaceae. They are important soil organisms where they contribute to the mineralisation of, for example, aromatic compounds. Acinetobacter are able to survive on various surfaces (both moist and dry) in the hospital environment, thereby being an important source of infection in debilitated patients. These bacteria are innately resistant to many classes of antibiotics. In addition, Acinetobacter is uniquely suited to exploitation for biotechnological purposes. • Cyanobacteria Cyanobacteria are a fascinating and versatile group of bacteria of immense biological importance. Thought to be amongst the first organisms

Fundamental Principle of Microbiology

•

•

•

•

•

•

•

11

to colonize the earth, these bacteria are the photosynthetic ancestors of chloroplasts in eukaryotes such as plants and algae. In addition they can fix nitrogen, survive in very hostile environments (e.g. down to -60°C), are symbiotic, have circadian rhythms, exhibit gliding mobility, and can differentiate into specialized cell types called heterocysts. This makes them ideal model systems for studying fundamental processes such as nitrogen fixation and photosynthesis. Helicobacter pylori Helicobacter pylori causes peptic ulcers, gastritis and gastric cancer. The bacterium infects up to 50% of the human population. H. pylori has very unique characteristics, such as microaerophily and nitrogen metabolism. Legionella Legionella is the genus of bacterium that causes Legionnaires’ Disease also known as Legionellosis. These bacteria are commonly found in aquatic habitats where they can survive and multiply in different protozoa enabling the bacterium to be transmissible and pathogenic to humans. Pasteurellaceae The Pasteurellaceae family comprises a large and diverse family of Gram-negative bacteria with members ranging from important pathogens such as Haemophilus influenzae to commensals of the animal and human mucosa. Members of the family Pasteurellaceae cause a wide variety of diseases in humans and animals. Pseudomonas The bacterial genus Pseudomonas includes the opportunistic human pathogen P. aeruginosa, plant pathogenic bacteria, plant beneficial bacteria, ubiquitous soil bacteria with bioremediation capabilities and other species that cause spoilage of milk and dairy products. P. aeruginosa can cause chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the ageing population of industrialised societies. Treponema Treponema pallidum is a gram-negative spirochaete bacterium. There are at least four known subspecies: T. pallidum pallidum, which causes syphilis; T. pallidum pertenue, which causes yaws; T. pallidum carateum, which causes pinta; and T. pallidum endemicum, which causes bejel. Vibrio cholerae Vibrio cholerae is the causative agent of cholera and belongs to a group of organisms whose natural habitats are the aquatic ecosystems. The strains that cause cholera epidemics have evolved from non-pathogenic progenitor strains by acquisition of virulence genes, and V. cholerae represents a paradigm for this evolutionary process. Borrelia The genus Borrelia, in the spirochete phylum, is not closely related to any other bacteria and has a highly unusual genome composed of a linear chromosome and multiple circular and linear plasmids that appear to be in a constant state of rearrangement, recombination, and deletion. The determination of the genome sequence of Borrelia strains has facilitated tremendous advances

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in understanding this genus at the molecular and cellular level as well as the pathogenesis of Lyme disease and relapsing fever.

1.4 MICRO-ORGANISMS AND FOOD

The foods that we eat are rarely if ever sterile, they carry microbial associations whose composition depends upon which organisms gain access and how they grow, survive and interact in the food over time. The micro-organisms present will originate from the natural micro-flora of the raw material and those organisms introduced in the course of harvesting/slaughter, processing, storage and distribution. The numerical balance between the various types will be determined by the properties of the food, its storage environment, properties of the organisms themselves and the effects of processing. In most cases this microflora has no discernible effect and the food is consumed without objection and with no adverse consequences. In some instances though, microorganisms manifest their presence in one of several ways: • They can cause spoilage; • They can cause foodborne illness; • They can transform a food’s properties in a beneficial way – food fermentation. 1.4.1 FOOD SPOILAGE/PRESERVATION From the earliest times, storage of stable nuts and grains for winter provision is likely to have been a feature shared with many other animals but, with the advent of agriculture, the safe storage of surplus production assumed greater importance if seasonal growth patterns were to be used most effectively. Food preservation techniques based on sound, if then unknown, microbiological principles were developed empirically to arrest or retard the natural processes of decay. The staple foods for most parts of the world were the seeds – rice, wheat, sorghum, millet, maize, oats and barley – which would keep for one or two seasons if adequately dried, and it seems probable that most early methods of food preservation depended largely on water activity reduction in the form of solar drying, salting, storing in concentrated sugar solutions or smoking over a fire. The industrial revolution which started in Britain in the late 18th century provided a new impetus to the development of food preservation techniques. It produced a massive growth of population in the new industrial centres which had somehow to be fed; a problem which many thought would never be solved satisfactorily. Such views were often based upon the work of the English cleric Thomas Malthus who in his ‘Essay on Population’ observed that the inevitable consequence of the exponential growth in population and the arithmetic growth in agricultural productivity would be overpopulation and mass starvation. This in fact proved not to be the case as the 19th century saw the development of substantial food preservation industries based around the use of chilling, canning and freezing and the first large scale importation of foods from distant producers.

Fundamental Principle of Microbiology

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To this day, we are not free from concerns about over-population. Globally there is sufficient food to feed the world’s current population, estimated to be 6600 million in 2006. World grain production has more than managed to keep pace with the increasing population in recent years and the World Health Organization’s Food and Agriculture Panel consider that current and emerging capabilities for the production and preservation of food should ensure an adequate supply of safe and nutritious food up to and beyond the year 2010 when the world’s population is projected to rise to more than 7 billion. There is however little room for complacency. Despite overall sufficiency, it is recognized that a large proportion of the population is malnourished and that 840 million people suffer chronic hunger. The principal cause of this is not insufficiency however, but poverty which leaves an estimated one-fifth of the world’s population without the means to meet their daily needs. Any long-term solution to this must lie in improving the economic status of those in the poorest countries and this, in its train, is likely to bring a decrease in population growth rate similar to that seen in recent years in more affluent countries. In any event, the world’s food supply will need to increase to keep pace with population growth and this has its own environmental and social costs in terms of the more intensive exploitation of land and sea resources. One way of mitigating this is to reduce the substantial pre- and post-harvest losses which occur, particularly in developing countries where the problems of food supply are often most acute. It has been estimated that the average losses in cereals and legumes exceed 10% whereas with more perishable products such as starchy staples and vegetables the figure is more than 20% – increasing to an estimated 25% for highly perishable products such as fish. In absolute terms, the US National Academy of Sciences has estimated the losses in cereals and legumes in developing countries as 100 million tonnes, enough to feed 300 million people. Clearly reduction in such losses can make an important contribution to feeding the world’s population. While it is unrealistic to claim that food microbiology offers all the answers, the expertise of the food microbiologist can make an important contribution. In part, this will lie in helping to extend the application of current knowledge and techniques but there is also a recognized need for simple, low-cost, effective methods for improving food storage and preservation in developing countries. Problems for the food microbiologist will not however disappear as a result of successful development programmes. Increasing wealth will lead to changes in patterns of food consumption and changing demands on the food industry. Income increases among the poor have been shown to lead to increased demand for the basic food staples while in the betteroff it leads to increased demand for more perishable animal products. To supply an increasingly affluent and expanding urban population will require massive extension of a safe distribution network and will place great demands on the food microbiologist. 1.4.2 FOOD SAFETY In addition to its undoubted value, food has a long association with the transmission of disease. Regulations governing food hygiene can be found in numerous early sources

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such as the Old Testament, and the writings of Confucius, Hinduism and Islam. Such early writers had at best only a vague conception of the true causes of foodborne illness and many of their prescriptions probably had only a slight effect on its incidence. Even today, despite our increased knowledge, ‘Foodborne disease is perhaps the most widespread health problem in the contemporary world and an important cause of reduced economic productivity.’ (WHO 1992.) The available evidence clearly indicates that biological contaminants are the major cause. 1.4.3 FERMENTATION Microbes can however play a positive role in food. They can be consumed as foods in themselves as in the edible fungi, mycoprotein and algae. They can also effect desirable transformations in a food, changing its properties in a way that is beneficial.

1.5 WHAT CAUSES MICROORGANISMS TO GROW

Factors that affect the rate of proliferation of microorganisms are categorized as extrinsic and intrinsic. 1.5.1 EXTRINSIC FACTORS Extrinsic factors relate to the environmental factors that affect the growth rate of microorganisms. 1.5.1.1 Temperature Microbes have an optimum, minimum, and maximum temperature for growth. Therefore, the environmental temperature determines not only the proliferation rate but also the genera of microorganisms that will thrive and the extent of microbial activity that occurs. For example, a change of only a few degrees in temperature may favour the growth of entirely different organisms and result in a different type of food spoilage and foodborne illness. These characteristics have been responsible for the use of temperature as a method of controlling microbial activity. The optimal temperature for the proliferation of most microorganisms is from 14ºC to 40ºC, although some microbes will thrive below 0ºC, and other genera will grow at temperatures up to and exceeding 100ºC. Microbes classified according to temperature of optimal growth include: Thermophiles (high-temperature-loving microorganisms), with growth optima at temperatures above 45ºC. Examples are Bacillus stearother- mophilus, Bacillus coagulans, and Lactobacillus thermophilus. Mesophiles (medium-temperature- loving microorganisms), with growth optima between 20ºC and 45ºC. Exam- ples are most lactobacilli and staphylococci. Psychrotrophs (cold-temperature-tolerant microorganisms), which tolerate and thrive at temperatures below 20ºC. Examples are Pseudomonas and Moraxella Acinetobacter.

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Bacteria, molds, and yeasts each have some genera that thrive in the range characteristic of thermophiles, mesophiles, and psychrotrophs. Molds and yeasts tend to be less thermophilic than do bacteria. As the temperature approaches 0ºC, fewer microorganisms thrive, and their proliferation is slower. Below approximately 5ºC, proliferation of spoilage microorganisms is retarded, and growth of most pathogens ceases. 1.5.1.2 Oxygen Availability As with temperature, availability of oxygen determines which microorganisms will be active. Some microorganisms have an absolute requirement for oxygen. Others grow in the total absence of oxygen, and others grow either with or without available oxy- gen. Microorganisms that require free oxygen are called aerobic microorganisms (Pseudomonas species is an example). Those that thrive in the absence of oxygen are called anaerobic microorganisms (i.e., Clostridium species). Microorganisms that grow with or without the presence of free oxygen are called facultative microorganisms (e.g., Lactobacillus species). 1.5.1.3 Relative Humidity This extrinsic factor affects microbial growth and can be influenced by temperature. All microorganisms have high requirements for water to support their growth and activity. A high relative humidity can cause moisture condensation on food, equipment, walls, and ceilings. Condensation causes moist surfaces, which are conducive to microbial growth and spoilage. Also, microbial growth is inhibited by a low relative humidity. Bacteria require a higher humidity than do yeasts and molds. Optimal relative humidity for bacteria is 92% or higher, whereas yeasts prefer it to be 90% or higher. Molds thrive more if the relative humidity is 85% to 90%. 1.5.2 INTRINSIC FACTORS Intrinsic factors that affect the rate of proliferation relate more to the characteristics of the substrates (foodstuff or debris) that support or affect growth of microorganisms. 1.5.2.1 Water Activity A reduction of water availability will reduce microbial proliferation. The available water for metabolic activity instead of total moisture content determines the extent of microbial growth. The unit of measurement for water requirement of microorganisms is usually expressed as water activity (Aw), defined as the vapor pressure of the subject solution divided by the vapor pressure of the pure solvent: Aw = p ÷ p0, where p is the vapor pressure of the solution and p0 is the vapor pressure of pure water. The approximate optimal Aw for the growth of many microorganisms is 0.99, and most bacteria require an Aw higher than 0.91 for growth. The approximate relationship between fractional equilibrium relative humidity (RH) and Aw is RH = Aw × 100. Therefore, an Aw of 0.95

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is approximately equivalent to an RH of 95% in the atmosphere above the solution. Most natural food products have an Aw of approximately 0.99. Generally, bacteria have the highest water activity requirements of the microorganisms. Molds normally have the lowest Aw requirement, and yeasts are intermediate. Most spoilage bacteria do not grow at an Aw below 0.91, but molds and yeasts can grow at an Aw of 0.80 or lower. Molds and yeasts can grow on partially dehydrated surfaces (including food), whereas bacterial growth is retarded. 1.5.2.2 pH pH is a measurement of log10 of the reciprocal of the hydrogen ion concentration (g/L) and is represented as pH = log10[H+]. The pH for optimal growth of most microorganisms is near neutrality (7.0). Yeasts can grow in an acid environment and thrive best in an inter- mediate acid (4.0 to 4.5) range. Molds tolerate a wider range (2.0 to 8.0), although their growth is generally greater with an acid pH. They can thrive in a medium that is too acid for either bacteria or yeasts. Bacterial growth is usually favoured by nearneutral pH values. However, acidophilic (acid-loving) bacteria grow on food or debris down to a pH of approximately 5.2. Below 5.2, microbial growth is dramatically reduced from that in the normal pH range. 1.5.2.3 Oxidation-Reduction Potential The oxidation-reduction potential is an indication of the oxidizing and reducing power of the substrate. To attain optimal growth, some microorganisms require reduced conditions; others need oxidized conditions. Thus, the importance of the oxidation– reduction potential is apparent. All saprophytic microorganisms that are able to transfer hydrogen as H+ and E– (electrons) to molecular oxygen are called aerobes. Aerobic microorganisms grow more rapidly under a high oxidation-reduction potential (oxidizing reactivity). A low potential (reducing reactivity) favours the growth of anaerobes. Facultative microorganisms are capable of growth under either condition. Microorganisms can alter the oxidation-reduction potential of food to the extent that the activity of other microorganisms is restricted. For example, anaerobes can decrease the oxidation–reduction potential to such a low level that the growth of aerobes can be inhibited. 1.5.2.4 Nutrient Requirements In addition to water and oxygen (except for anaerobes), microorganisms have other nutrient requirements. Most microbes need external sources of nitrogen, energy (carbohydrates, proteins, or lipids), minerals, and vitamins to support their growth. Nitrogen is normally obtained from amino acids and non-protein nitrogen sources. However, some microorganisms utilize peptides and proteins. Molds are the most effective in the utilization of proteins, complex carbohydrates, and lipids because they contain enzymes capable of hydrolyzing these molecules into less complex components.

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Many bacteria have a similar capability, but most yeasts require the simple forms of these compounds. All microorganisms need minerals, but requirements for vitamins vary. Molds and some bacteria can synthesize enough B vitamins for their needs, whereas other microorganisms require a ready-made supply. 1.5.2.5 Inhibitory Substances Microbial proliferation can be affected by the presence or absence of inhibitory substances. Substances or agents that inhibit microbial activity are called bacteriostats. Those that destroy microorganisms are called bactericides. Some bacteriostatic substances, such as nitrites, are added during food processing. Most bactericides are utilized as a method of decontaminating food- stuffs or as a sanitizer for cleaned equipment, utensils, and rooms. 1.5.3 IMPLICIT FACTORS A third set of factors that are important in determining the nature of microbial associations found in foods are described as implicit factors – properties of the organisms themselves, how they respond to their environment and interact with one another. At its simplest, an organism’s specific growth rate can determine its importance in a food’s microflora; those with the highest specific growth rate are likely to dominate over time. This will of course depend upon the conditions prevailing; many moulds can grow perfectly well on fresh foods such as meat, but they grow more slowly than bacteria and are therefore out-competed. In foods where the faster growing bacteria are inhibited by factors such as reduced pH or aw, moulds assume an important role in spoilage. Alternatively, two organisms may have similar maximum specific growth rates but differ in their affinity (Ks) for a growth limiting substrate. If the level of that substrate is sufficiently low that it becomes limiting, then the organism with the lower Ks (higher affinity) will outgrow the other. In Sections 1.5.1 and 1.5.2 we described how microbial growth and survival are influenced by a number of factors and how micro-organisms respond to changes in some of these. This response does however depend on the physiological state of the organism. Exponential phase cells are almost always killed more easily by heat, low pH or antimicrobials than stationary phase cells and often the faster their growth rate the more readily they are killed. This makes sense intuitively; the consequences of a car crash are invariably more serious the faster the car is travelling at the time. At higher growth rates, where cell activity is greater and more finely balanced, the damage caused by a slight jolt to the system will be more severe than the same perturbation in cells growing very slowly or not at all. The precise mechanism leading to cell death is almost certainly very complex. One proposal is that lethal damage is largely a result of an oxidative burst, the production of large numbers of damaging free radicals within the cell in response to the physical or chemical stress applied. This would mean that cell death is in fact a function of the organism’s response to a stress rather than a direct

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effect of the stress itself. A cell’s sensitivity to potentially lethal treatments can also be affected by its previous history. Generally, some form of pre-adaptation will decrease the damaging effect of adverse conditions. Growth or holding organisms such as Salmonella at higher temperatures has been shown to increase their heat resistance. Pre-exposure to moderately low pH can increase an organism’s subsequent resistance to a more severe acid challenge. Growth at progressively lower temperatures can reduce the minimum temperature at which an organism would otherwise grow. Some reaction to stress can be apparent very soon after exposure as existing enzymes and membrane proteins sense and react to the change. Other responses occur more slowly since they involve gene transcription and the production of proteins. The most extensively studied of this type of response is the production of heat shock proteins; proteins produced following exposure to elevated temperatures and which protect the cell from heat damage. Some heat shock proteins, described as chaperones or chaperonins, interact with unfolded or partially unfolded proteins and assist them in reaching their correct conformation. Chaperonins are present in normal cells but obviously far more will be needed during processes such as heating which increase the rate at which cellular proteins denature. Heat shock proteins are encoded by genes which have a specific sigma factor, sigma 32 also known as RpoH, for transcription. Sigma factors are proteins which bind to DNA-dependent RNA polymerase, the enzyme which transcribes DNA into messenger RNA. When bound to the polymerase they confer specificity for certain classes of promoter on the DNA and thus help determine which regions of the genome are transcribed. Another alternative sigma factor RpoS, also known as the stationary phase sigma factor, has been identified in a number of Gram- negative bacteria and a similar regulon sigma B operates in Gram- positive bacteria. RpoS is produced in cells throughout growth but is rapidly degraded in exponential phase cells. As growth slows at the end of exponential phase it accumulates and directs the transcription of a battery of genes associated with the stationary phase, many of which are protective. It is now clear that RpoS is a general stress response regulator and also accumulates in response to environmental stresses such as low pH and osmotic stress. Since the RpoS response confers resistance to a range of stresses, exposure to one factor such as low pH can confer increased resistance to other stresses such as heat. Of equal concern is the observation that RpoS also plays a role in regulating expression of genes associated with virulence in some food borne pathogens and that virulence factors expressed as the cells enter stationary phase can also be induced by stress. The implications of this for food microbiology are considerable, for not only do they suggest that stresses microorganisms encounter during food processing may increase resistance to other stresses, but that they could also increase the virulence of any pathogens present. Until now we have dealt with micro-organisms largely as isolated individuals and have not considered any effects they might have on each other. Cell to cell communication has however been shown to play a part in the induction of stress responses. Molecules

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such as acylhomoserine lactones and proteins secreted by cells in response to a stress have been shown to produce a stress response in others, implying that cells in the vicinity which have not necessarily been directly exposed to the stress may also increase in resistance. Ecologists have identified a number of different ways in which organisms can interact and several of these can be seen in the microbial ecology of food systems. Mutualism, when growth of one organism stimulates the growth of another, is well illustrated by the interaction of the starter cultures in yoghurt fermentation. Similar stimulatory effects can be seen in spoilage associations or in sequences of spoilage organisms seen when growth of one organism paves the way for others. For example, a grain’s water activity may be sufficiently low to prevent the growth of all but a few fungi, once these begin to grow however water produced by their respiration increases the local water activity allowing less xerophilic moulds to take over. Alternatively, one organism might increase the availability of nutrients to others by degrading a food component such as starch or protein into more readily assimilable compounds. Some micro-organisms may remove an inhibitory component and thereby permit the growth of others. This last example has had safety implications in mould-ripened cheeses where mould growth increases the pH allowing less acid tolerant organisms such as Listeria monocytogenes to grow. Alternatively, micro-organisms may be antagonistic towards one another producing inhibitory compounds or sequestering essential nutrients such as iron. 1.5.4 INTERACTION BETWEEN GROWTH FACTORS The effects that factors such as temperature, oxygen, pH, and Aw have on microbial activity may be dependent on each other. Microorganisms generally become more sensitive to oxygen availability, pH, and Aw at temperatures near growth minima or maxima. For example, bacteria may require a higher pH, Aw and minimum temperature for growth under anaerobic conditions than when aerobic conditions prevail. Microorganisms that grow at lower temperatures are usually aerobic and generally have a high Aw requirement. Lowering Aw by adding salt or excluding oxygen from foods (such as meat) that have been held at a refrigerated temperature dramatically reduces the rate of microbial spoilage. Normally, some microbial growth occurs when any one of the factors that controls the growth rate is at a limiting level. If more than one factor becomes limiting, microbial growth is drastically curtailed or completely stopped.

1.6 EFFECTS OF MICROORGANISMS ON SPOILAGE

Food is considered spoiled when it becomes unfit for human consumption. Spoilage is usually equated with the decomposition and putrefaction that results from microorganisms. Davidson (2003) defined spoilage as an undesirable change in the flavour, odour, texture, or colour of food caused by growth of microorganisms and ultimately the action of their enzymes.

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1.6.1 PHYSICAL CHANGES The physical changes caused by microorganisms usually are more apparent than the chemical changes. Microbial spoilage usually results in an obvious change in physical characteristics such as colour, body, thickening, odour, and flavour degradation. Food spoilage is normally classified as being either aerobic or anaerobic, depending on the spoilage conditions, including whether the principal microorganisms causing the spoilage were bacteria, molds, or yeasts. Aerobic spoilage of foods from molds is normally limited to the food surface, where oxygen is available. Molded surfaces of foods such as meats and cheeses can be trimmed off, and the remainder is generally acceptable for consumption. This is especially true for aged meats and cheeses. When these surface molds are trimmed, surfaces underneath usually have limited microbial growth. If extensive bacterial growth occurs on the surface, penetration inside the food surface usually follows, and toxins may be present. Anaerobic spoilage occurs within the interior of food products or in sealed containers, where oxygen is either absent or present in limited quantities. Spoilage is caused by facultative and anaerobic bacteria, and is expressed through souring, putrefaction, or taint. Souring occurs from the accumulation of organic acids during the bacterial enzymatic degradation of complex molecules. Also, proteolysis without putrefaction may contribute to souring. Souring can be accompanied by the production of various gases. Examples of souring are milk, round sour or ham sour, and bone sour in meat. Meat sours, or taints, are caused by anaerobic bacteria that may have been originally present in lymph nodes or bone joints, or that might have gained entrance along the bones during storage and processing. 1.6.2 CHEMICAL CHANGES Through the activity of endogenous hydrolytic enzymes that are present in food- stuffs (and the action of enzymes that microorganisms produce), proteins, lipids, carbohydrates, and other complex molecules are degraded into smaller and simpler compounds. Initially, the endogenous enzymes are responsible for the degradation of complex molecules. As microbial load and activity increase, degradation subsequently occurs. These enzymes hydrolyze the complex molecules into simpler compounds, which are subsequently utilized as nutrient sources for supporting microbial growth and activity. Oxygen availability determines the end products of microbial action. Availability of oxygen permits hydrolysis of proteins into end products such as simple peptides and amino acids. Under anaerobic conditions, proteins may be degraded to a variety of sulfurcontaining compounds, which are odorous and generally obnoxious. The end products of non-protein nitrogenous compounds usually include ammonia. Other chemical changes include action of lipases secreted by microorganisms that hydrolyze triglycerides and phospholipids into glycerol and fatty acids. Phospholipids are hydrolyzed into nitrogenous bases and phosphorus. Lipid oxidation is also accelerated by extensive lipolysis.

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Most microorganisms prefer carbohydrates to other compounds as an energy source since they are more readily utilized for energy. Utilization of carbohydrates by microorganisms results in a variety of end products, such as alcohols and organic acids. In many foods, such as sausage products and cultured dairy products, microbial fermentation of sugar that has been added yields organic acids (such as lactic acid), which con- tribute to their distinct and unique flavours.

1.7 EFFECTS OF MICROORGANISMS ON FOODBORNE ILLNESS

The United States has the safest food sup- ply of all nations. However, the U.S. Center for Disease Control and Prevention (CDC) estimates that there are 76 million foodborne illnesses per year in the United States with approximately 325,000 annual hospitalizations and 5,000 deaths attributable this illness. However, the actual number of confirmed cases documented by the CDC is much lower. The development of gastrointestinal disturbances following the ingestion of food can result from any one of several plausible causes. Although the sanitarian is most interested in those related to microbial origin, other causes are chemical contaminants, toxic plants, animal parasites, allergies, and overeating. Each of these conditions is recognized as a potential source of illness in human. Subsequent discussions will be confined to those illnesses caused by microorganisms. 1.7.1 FOODBORNE DISEASE A foodborne disease is considered to be any illness associated with or in which the causative agent is obtained by the ingestion of food. A foodborne disease outbreak is defined as “two or more persons experiencing a similar illness, usually gastrointestinal, after eating a common food, if analysis identifies the food as the source of illness.” Approximately 66% of all foodborne illness outbreaks are caused by bacterial pathogens. Of the 200 foodborne outbreaks reported each year, approximately 60% are of undetermined etiology. Unidentified causes may be from the Salmonella and Campylobacter species, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, Listeria monocytogenes, Escherichia coli O157, Shigella, Vibrio, and Yersinia enterocolitica, which are transmitted through foods. A wide variety of home-cooked and commercially prepared foods have been implicated in out- breaks, but they are most frequently related to foods of animal origin, such as poultry, eggs, red meat, seafood, and dairy products.

1.8 CONTROLLING MICROBIAL GROWTH

When food processors or consumers want to kill microbes, the least expensive and most effective way is to use heat. By simply cooking food at a relatively high temperature, most problem-causing microorganisms are destroyed. Many of the “ready-to-eat” food

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products that we buy and eat have been precooked to prevent the growth of microbes. Some foods, like fresh ground beef, are not precooked and therefore must be cooked by the consumer before eating. Eating a hamburger that is still pink in the middle means that the inside was not exposed to enough heat to kill microbes that could be present. Couple that with the fact that ground beef has plenty of opportunities for contamination in the production and handling, and you have a recipe for bacterial growth. Blanching, or the exposure of a food to extremely hot water for a few seconds, inactivates enzymes that will deteriorate the food and allow microbes to enter. Blanching is a common process for fruits or vegetables that will be frozen for later use. Pasteurization is a process used in the milk industry to kill microbes by heating milk to a temperature of 145°F (63°C) for 30 minutes. Although pasteurization provides consumers with a safe, quality milk product, the process does not kill all the microorganisms present in milk. It is for this reason that milk must be refrigerated to slow the growth of the surviving microbes. Eventually, even with refrigeration, the microbes will grow and spoil the milk, giving it that sour smell and flavour. Pasteurization is also used with certain juice and egg products. The other possibility for microbial death is the use of extremely cold temperatures. Microbes, like most organisms, have a certain amount of moisture in their cells. When frozen, the moisture expands, causing extensive cell damage and deterioration upon thawing. Although this may seem like a good method of control, the food itself is also damaged when frozen and deteriorates faster upon thawing, providing once again a perfect site for microbial growth. Food producers who wish to provide safe food products that will have longer shelf lives have developed methods of microbial control by changing the environment of the foods themselves. When the environment is changed, the microbes are not eliminated from around the foods, but they have difficulty growing on the food products. One of the easiest ways to make the food environment more hostile for microbes is to reduce the amount of moisture present. Since nearly all microbes require moisture to carry out their life functions, the absence of moisture stops them in their tracks. Another method to change the food environment is to use food additives, or substances designed to retain or improve the desirable characteristics of the food. Food additives, such as sulfur dioxide, potassium sorbate, sodium propionate, and sodium benzoate, are all used to prevent the growth of microbes in food. Other additives can be used to change the pH of food, making it difficult for microbes to thrive. Processing methods can also be used to control microbial growth. Most of these methods can be performed right in our very own kitchens as well as in large-scale processing plants. Some fresh foods are canned before they are shipped and sold to consumers. Canning requires food to be heated to a high temperature while under pressure. The result is the death of microbes because of the heat and the prevention of further contamination because of the vacuum sealing of the container. Vegetables, fruit, and a variety of other products (especially those with high-acid con- tent) that have been properly canned can be stored safely for months and sometimes years.

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As we stated earlier, freezing is another common processing method that kills microbes and prevents their growth. Just think of what our lives would be like with- out frozen pizza! Dehydration is also commonly used by food manufacturers to prevent microbial growth. Dehydration is the removal of water from a food product. When moisture is removed, the microbes cannot function and soon die. Dehydrated foods can be stored for long periods, given that their packaging can keep moisture out. Many of our favourite food items—instant noodles, cocoa mix, snack foods, baking mixes—are dehydrated.

1.9 BRANCHES OF MICROBIOLOGY

The branches of microbiology can be classified into pure and applied sciences. Microbiology can be also classified based on taxonomy, in the cases of bacteriology, mycology, protozoology, and phycology. There is considerable overlap between the specific branches of microbiology with each other and with other disciplines, and certain aspects of these branches can extend beyond the traditional scope of microbiology In general the field of microbiology can be divided in the more fundamental branch (pure microbiology). In this field the organisms are studied as the subject itself on a deeper level. Applied microbiology refers to the fields where the micro-organisms are applied in certain processes. The organisms itself are often not studied as such, but applied to sustain certain processes. 1.9.1 PURE MICROBIOLOGY • Bacteriology: Bacteriology is the branch and specialty of biology that studies the morphology, ecology, genetics and biochemistry of bacteria as well as many other aspects related to them. This subdivision of microbiology involves the identification, classification, and characterization of bacterial species. • Mycology: Mycology is the branch of biology concerned with the study of fungi, including their genetic and biochemical properties, their taxonomy and their use to humans as a source for tinder, medicine, food, and entheogens, as well as their dangers, such as toxicity or infection. – A biologist specializing in mycology is called a mycologist. – Mycology branches into the field of phytopathology, the study of plant diseases, and the two other disciplines that remain closely related because the vast majority of “plant” pathogens are fungi. • Protozoology: Protozoology is the study of protozoa, the “animal-like” (i.e., motile and heterotrophic) protists. • Phycology/algology: Phycology is the scientific study of algae. Also known as algology, phycology is a branch of life science and often is regarded as a subdiscipline of botany. • Parasitology: Parasitology is the study of parasites, their hosts, and the relationship between them. As a biological discipline, the scope of parasitology

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•

•

•

• • • • • •

• • •

is not determined by the organism or environment in question, but by their way of life. This means it forms a synthesis of other disciplines, and draws on techniques from fields such as cell biology, bioinformatics, biochemistry, molecular biology, immunology, genetics, evolution and ecology. Immunology: The study of the immune system. Many components of the immune system are typically cellular in nature and not associated with any specific organ; but rather are embedded or circulating in various tissues located throughout the body. Virology: Virology is the study of viruses – submicroscopic, parasitic particles of genetic material contained in a protein coat – and virus-like agents. It focuses on the following aspects of viruses: their structure, classification and evolution, their ways to infect and exploit host cells for reproduction, their interaction with host organism physiology and immunity, the diseases they cause, the techniques to isolate and culture them, and their use in research and therapy. Virology is considered to be a subfield of microbiology or of medicine. Nematology: Nematology is the scientific discipline concerned with the study of nematodes, or roundworms. Although nematological investigation dates back to the days of Aristotle or even earlier, nematology as an independent discipline has its recognizable beginnings in the mid to late 19th century. Microbial cytology: The study of microscopic and submicroscopic details of microorganisms Microbial physiology: The study of how the microbial cell functions biochemically. Includes the study of microbial growth, microbial metabolism and microbial cell structure Microbial ecology: The relationship between microorganisms and their environment Microbial genetics: The study of how genes are organized and regulated in microbes in relation to their cellular functions Closely related to the field of molecular biology Cellular microbiology: A discipline bridging microbiology and cell biology Evolutionary microbiology: The study of the evolution of microbes. This field can be subdivided into: – Microbial taxonomy: The naming and classification of microorganisms – Microbial systematic: The study of the diversity and genetic relationship of microorganisms Generation microbiology: The study of those microorganisms that have the same characters as their parents Systems microbiology: A discipline bridging systems biology and microbiology Molecular microbiology: The study of the molecular principles of the physiological processes in microorganisms

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1.9.1.1 Other • Astro microbiology: The study of microorganisms in outer space • Biological agent: The study of those microorganisms which are being used in weapon industries. • Nano microbiology: The study of those organisms on nano level. • Predictive microbiology: The quantification of relations between controlling factors in foods and responses of pathogenic and spoilage microorganisms using mathematical modelling 1.9.2 APPLIED MICROBIOLOGY • Medical microbiology: The study of the pathogenic microbes and the role of microbes in human illness. Includes the study of microbial pathogenesis and epidemiology and is related to the study of disease pathology and immunology. This area of microbiology also covers the study of human microbiota, cancer, and the tumor microenvironment. • Pharmaceutical microbiology: The study of microorganisms that are related to the production of antibiotics, enzymes, vitamins, vaccines, and other pharmaceutical products and that cause pharmaceutical contamination and spoil. • Industrial microbiology: The exploitation of microbes for use in industrial processes. Examples include industrial fermentation and wastewater treatment. Closely linked to the biotechnology industry. This field also includes brewing, an important application of microbiology. • Microbial biotechnology: The manipulation of microorganisms at the genetic and molecular level to generate useful products. • Food microbiology: The study of microorganisms causing food spoilage and foodborne illness. Using microorganisms to produce foods, for example by fermentation. • Agricultural microbiology: The study of agriculturally relevant microorganisms. This field can be further classified into the following: – Plant microbiology and Plant pathology: The study of the interactions between microorganisms and plants and plant pathogens. – Soil microbiology: The study of those microorganisms that are found in soil. • Veterinary microbiology: The study of the role of microbes in veterinary medicine or animal taxonomy. • Environmental microbiology: The study of the function and diversity of microbes in their natural environments. This involves the characterization of key bacterial habitats such as the rhizosphere and phyllosphere, soil and groundwater ecosystems, open oceans or extreme environments (extremophiles). This field includes other branches of microbiology such as: – Microbial ecology – Microbially mediated nutrient cycling

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– Geomicrobiology – Microbial diversity – Bioremediation: Use of micro-organisms to clean air, water and soils. • Water microbiology (or aquatic microbiology): The study of those microorganisms that are found in water. • Aeromicrobiology (or air microbiology): The study of airborne microorganisms. • Biotechnology: Telated to recombinant DNA technology or genetic engineering

1.10 APPLICATIONS OF MICROBIOLOGY

While some fear microbes due to the association of some microbes with various human illnesses, many microbes are also responsible for numerous beneficial processes such as industrial fermentation (e.g. the production of alcohol, vinegar and dairy products), antibiotic production and as vehicles for cloning in more complex organisms such as plants. Scientists have also exploited their knowledge of microbes to produce biotechnologically important enzymes such as Taq polymerase, reporter genes for use in other genetic systems and novel molecular biology techniques such as the yeast twohybrid system. Bacteria can be used for the industrial production of amino acids. Corynebacterium glutamicum is one of the most important bacterial species with an annual production of more than two million tons of amino acids, mainly L-glutamate and L-lysine. Since some bacteria have the ability to synthesize antibiotics, they are used for medicinal purposes, such as Streptomyces to make aminoglycoside antibiotics. A variety of biopolymers, such as polysaccharides, polyesters, and polyamides, are produced by microorganisms. Microorganisms are used for the biotechnological production of biopolymers with tailored properties suitable for high-value medical application such as tissue engineering and drug delivery. Microorganisms are used for the biosynthesis of xanthan, alginate, cellulose, cyanophycin, poly(gamma-glutamic acid), levan, hyaluronic acid, organic acids, oligosaccharides and polysaccharide, and polyhydroxyalkanoates. Microorganisms are beneficial for microbial biodegradation or bioremediation of domestic, agricultural and industrial wastes and subsurface pollution in soils, sediments and marine environments. The ability of each microorganism to degrade toxic waste depends on the nature of each contaminant. Since sites typically have multiple pollutant types, the most effective approach to microbial biodegradation is to use a mixture of bacterial and fungal species and strains, each specific to the biodegradation of one or more types of contaminants. Symbiotic microbial communities confer benefits to their human and animal hosts health including aiding digestion, producing beneficial vitamins and amino acids, and suppressing pathogenic microbes. Some benefit may be conferred by eating fermented foods, probiotics (bacteria potentially beneficial to the digestive system) or prebiotics

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(substances consumed to promote the growth of probiotic microorganisms). The ways the microbiome influences human and animal health, as well as methods to influence the microbiome are active areas of research. Research has suggested that microorganisms could be useful in the treatment of cancer. Various strains of non-pathogenic clostridia can infiltrate and replicate within solid tumors. Clostridial vectors can be safely administered and their potential to deliver therapeutic proteins has been demonstrated in a variety of preclinical models.

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2 Food Poisoning and Other Food Borne Hazards

2.1 WHAT IS FOOD POISONING?

Foodborne illness, more commonly referred to as food poisoning, is the result of eating contaminated, spoiled, or toxic food. The most common symptoms of food poisoning include nausea, vomiting, and diarrhea. Food poisoning syndrome results from ingestion of water and wide variety of food contaminated with pathogenic microorganisms (bacteria, viruses, protozoa, fungi), their toxins and chemicals. Food poisoning must be suspected when an acute illness with gastrointestinal or neurological manifestation affect two or more persons, who have shared a meal during the previous 72 hours. The term as generally used encompasses both food-related infection and food-related intoxication. Some microbiologists consider microbial food poisoning to be different from foodborne infections. In microbial food poisoning, the microbes multiply readily in the food prior to consumption, whereas in food-borne infection, food is merely the vector for microbes that do not grow on their transient substrate. Others consider food poisoning as intoxication of food by chemicals or toxins from bacteria or fungi. Consumption of poisonous mushroom leads to mycetism, while consumption of food contaminated with toxin producing fungi leads to mycotoxicosis. Some microorganisms can use our food as a source of nutrients for their own growth. By growing in the food, metabolizing them and producing by-products, they not only

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render the food inedible but also pose health problems upon consumption. Many of our foods will support the growth of pathogenic microorganisms or at least serve as a vector for their transmission. Food can get contaminated from plant surfaces, animals, water, sewage, air, soil, or from food handlers during handling and processing. Food poisoning includes ill effects caused by the ingestion of contaminated food by many ways apart from microbial agents. They may be • Through the addition of proteins • Through eating of inherent poisonous substance such as certain mushrooms, fish and molluscs by mistake • Adulteration of food with poisonous substance such as Argemone mexicana in mustard producing epidemic dropsy The term “food poisoning” is however restricted only to acute gastroenteritis due to bacterial pollution of food or drink. The term “food-borne” disease is defined as “A disease, usually either infectious or toxic in nature, caused by agents that enter the body through the ingestion of food”. Food-borne diseases may be classified as Food-Borne Intoxications Food-borne intoxications are caused 1. Due to naturally occurring toxins in some foods, including – Lathyrism (beta-oxalyl amino-alanine) – Endemic ascitis (Pyrrolizidine alkaloids) 2. Due to toxins produced by certain bacteria, including – Botulism – Staphyloccal toxins 3. Due to toxins produced by some fungi, including – Aflatoxin – Ergot – Fusarium toxins 4. Due to toxins produced by some algae, like – Planktonic dinoflagellates – Diatoms – Cyanobacteria 5. Due to food-borne chemical poisoning 2.1.1 HOW DOES FOOD BECOME CONTAMINATED? Pathogens can be found on almost all of the food that humans eat. However, heat from cooking usually kills pathogens on food before it reaches our plate. Foods eaten raw are common sources of food poisoning because they don’t go through the cooking process. Occasionally, food will come in contact with the organisms in fecal matter. This most commonly happens when a person preparing food doesn’t wash their hands before cooking.

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Meat, eggs, and dairy products are frequently contaminated. Water may also be contaminated with organisms that cause illness. 2.1.2 WHO IS AT RISK FOR FOOD POISONING? Anyone can come down with food poisoning. Statistically speaking, nearly everyone will come down with food poisoning at least once in their lives. There are some populations that are more at risk than others. Anyone with a suppressed immune system or an auto-immune disease may have a greater risk of infection and a greater risk of complications resulting from food poisoning. According to the Mayo Clinic, pregnant women are more at risk because their bodies are coping with changes to their metabolism and circulatory system during pregnancy. Elderly individuals also face a greater risk of contracting food poisoning because their immune systems may not respond quickly to infectious organisms. Children are also considered an at-risk population because their immune systems aren’t as developed as those of adults. Young children are more easily affected by dehydration from vomiting and diarrhea. 2.1.3 HOW IS FOOD POISONING DIAGNOSED? Your doctor may be able to diagnose the type of food poisoning based on your symptoms. In severe cases, blood tests, stool tests, and tests on food that you have eaten may be conducted to determine what is responsible for the food poisoning. Your doctor may also use a urine test to evaluate whether an individual is dehydrated as a result of food poisoning. 2.1.4 HOW IS FOOD POISONING TREATED? Food poisoning can usually be treated at home, and most cases will resolve within three to five days. If you have food poisoning, it’s crucial to remain properly hydrated. Sports drinks high in electrolytes can be helpful with this. Fruit juice and coconut water can restore carbohydrates and help with fatigue. Avoid caffeine, which may irritate the digestive tract. Decaffeinated teas with soothing herbs like chamomile, peppermint, and dandelion may calm an upset stomach. Read about more remedies for an upset stomach. Over-the-counter medications like Imodium and Pepto-Bismol can help control diarrhea and suppress nausea. However, you should check with your doctor before using these medications, as the body uses vomiting and diarrhea to rid the system of the toxin. Also, using these medications could mask the severity of the illness and cause you to delay seeking expert treatment. It’s also important for those with food poisoning to get plenty of rest. In severe cases of food poisoning, individuals may require hydration with intravenous (IV) fluids at a hospital. In the very worst cases of food poisoning, a longer hospitalization may be required while the individual recovers.

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2.2 BACTERIAL FOOD POISONING 2.2.1 FOOD-BORNE DISEASES AND FOOD POISONING Food-borne disease is a disease caused by ingestion of food contaminated by any agent, chemical or biological. Food poisoning is an acute enteritis caused by the ingestion of food, characterized by diarrhea, vomiting, with or without fever and abdominal pains. Food poisoning is normally associated with the small and large intestine. Certain types of food poisoning are described as intoxications and others as infections Intoxications Intoxication involves food poisoning in which the organism grows in food and releases a toxin from the cells. When the toxin is ingested along with the food, it gives rise to the Food Poisoning Syndrome. The presence of organism in the food is irrelevant to disease production. It is the toxin that gives rise to the disease. Bacterial toxins that produce intoxications are the exotoxin types of either enterotoxin (affecting the gut) as in staphylococcal intoxication or neurotoxin (affecting the nervous system) as in botulism. Another category of intoxications are the mycotoxicoses (due to ingestion of mycotoxins) and the diseases caused by algal toxins (shell fish poisoning). Generally, intoxications have short incubation periods. Infections These involve food poisoning caused by the ingestion of live organisms. The organisms grow in the gastrointestinal tract to produce the disease. Most microbial food poisonings fall in this category. For example, salmonellosis caused by Salmonella sp. like Salmonella typhi. Enteritis associated with food poisoning infections is due to the production of exotoxins or endotoxins that act as enterotoxins. In certain other types of food poisoning, as in the case of Clostridium perfringens, live cells need to be ingested for the disease to occur but the organism does not grow and reproduce in the gut. Vegetative cells sporulate after ingestion and enterotoxin released causing the disease symptoms. Since live cells are needed to be ingested to cause the food poisoning, it can be considered as food-borne infection. 2.2.2 FOOD POISONING BACTERIA What are these Microbes? It is estimated that every year more than Eighty one million people are affected by food borne illness every year. Illnesses such as food poisoning are becoming more common as our lifestyles change – for one thing, we eat out more and more food is being prepared in advance. We have no accurate figures on how much food poisoning is the result of

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mishandling by the consumer, but it is thought to be between 12 to 20%. Food safety is all about reducing the risk of becoming sick from eating foods. The principles of food safety are easy to apply in the home, when eating in the outdoors, at your local shop when buying groceries, and even at a restaurant. Most food poisoning incidents are a result of mishandling food – keeping it at the wrong temperature, incorrect re-heating, and cross contamination. To help you handle your food safely, take a look at the following information. Remember that nearly ALL foods need to be handled with care.

2.3 CAUSES OF FOOD POISONING

There are three causes of food poisoning: • Biological - Poisonous Food • Bacterial - Microbes • Chemical

2.3.1 BIOLOGICAL FOOD POISONING Foods containing harmful substances can be poisonous. There are many species of poisonous mushrooms, they may cause illness and in some cases death. These mushrooms are very similar in appearance to the edible variety and may easily be eaten by mistake. Deadly nightshade, which grows throughout Europe and Asia, contains a drug belladonna in all parts of the plant. The drug is used to relieve illness such as asthma, bronchitis and heart disease. However, it may be lethal if this medicine is taken in large doses and there were cases where children have been poisoned by eating the berries of the plant. Potatoes are also members of the nightshade family and green potatoes contain a substance called solanine which causes illness or even death if eaten in large quantities. Therefore, green potatoes should always be discarded. Raw red kidney beans contain a toxic substance called haemagglutinin. A number of cases of poisoning have occurred as a result of cooking the beans in a slow cooker at an insufficient high temperature. Boiling the beans for 10 minutes destroys the toxin. Canned kidney beans are heated sufficiently during processing and are therefore safe. Certain moulds are capable of causing illness by producing toxins in foods known as mycotoxins. The mould Aspergillus flavus produces aflatoxins. This has been found in groundnuts (peanuts), figs, cereals and some other foods. High level of aflatoxin consumption is associated with liver cancer. Some ciguatera fish contains biochemical toxin, such as marine coral reef fish. Consumption of such biochemical toxin may cause numbness in limbs, face, tongue or around the mouth, cold objects perceived as hot and vice versa, dizziness, palpitation and chest pain.

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2.3.2 BACTERIAL FOOD POISONING Bacterial food poisoning is an acute illness caused by the consumption of food contaminated by bacterial pathogens. It is the most common cause of food poisoning and stringent hygiene precautions must be taken in order to prevent outbreaks of this type of illness. Since most food poisoning incidents occur as a result of unhygienic practices, this means that they are preventable. 2.3.2.1 Three main types of bacterial food poisoning include: 1. The infective type which is caused by eating food containing a large number of living bacteria. After being eaten, the bacteria establish themselves in the alimentary canal and when they die they release an endotoxin (e.g. Salmonella poisoning). 2. The toxin type which is caused by eating food containing exotoxin. The toxin is released into food while the bacteria are growing and multiplying in the food. The bacteria themselves may be dead when the food is eaten. (e.g. Staphylococcal poisoning). 3. The third type is also caused by toxin. The toxin is not produced in the food but is released into the alimentary canal after the bacteria have been eaten and while they are growing in the alimentary canal. (e.g. Clostridium perfringen poisoning) Although there are various kinds of bacterial food poisoning, the following are the most prevalent: Pathogenic Bacteria

Growth Temperature Range*

Common Foods Involved

Salmonella spp.

6.5 – 47°C (35 - 37°C)

Staphylococcus aureus

7 – 45°C (37°C)

Vibrio parahaemolyticus

12.8 – 40°C (37°C)

Bacillus cereus

10 – 49°C (30 - 37°C)

Clostridium perfringens

10 – 52°C (43 - 47°C)

Raw or undercooked egg and egg products (e.g. Tiramisu); undercooked meat, poultry and their products (e.g. barbecued and preserved meat, goose intestines, etc.). Foods which have been subject to a large amount of handling with no subsequent cooking or reheating (e.g. lunch boxes, cakes, pastries, sandwiches, etc.). Raw or undercooked seafood, shellfish, marine products and salted food (e.g. jellyfish, cuttlefish, salted vegetables and smoked knuckles, etc.). Leftover cooked rice, fried rice, meat products and vegetables. Cross-contaminated and inadequately cooked meat and meat products (e.g. stew and meat pies, etc.).

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2.3.2.2 Common Contributing Factors to Bacterial Food Poisoning 1. Contamination of Cooked Food Cooked food has been contaminated by food handlers, raw food or pests. 2. Improper Storage of Cooked Food Cooked food has been stored between 4°C and 60°C for a prolonged period. 3. Inadequate Cooking of Food Raw food has not been cooked thoroughly to reduce any pathogen present. 4. Inadequate Reheating of Cooked Food Cooked food has not been reheated to 75°C. 5. Inadequate Thawing of Food Before Cooking Partially thawed food still has a high bacterial count or pathogen content and which needs a longer time to reach the temperature that kills the bacteria and pathogens in cooking, has not been cooked for sufficiently long time. 6. Preparation of Food Too Early In Advance Food has been prepared too early in advance but has not been stored under proper controlled temperature. 7. Infected Food Handlers Food handlers infected with communicable diseases have engaged in handling food. 8. Consumption of Raw Food Raw food that usually has a high bacterial count or pathogen content has been eaten. 9. Use of Unsafe Food Source Food has been purchased from an unauthorised or unreliable source such as hawkers. 10.Use of Leftovers Use of leftover foods (e.g. cooked rice) that have been stored between 4°C to 60°C for a prolonged period. 2.3.3 CHEMICAL FOOD POISONING Chemical food poisoning is caused by the presence of toxic chemicals in food. These substances may be agricultural chemicals, which are used intentionally in crop production. The use of weedkillers and insecticides is essential to ensure food yields. However, some of these substances may be dangerous if used indiscriminately, since they may be toxic if they are consumed in large doses. Weedkillers and insecticides are tested very thoroughly before they are placed on the market and farmers are given detailed instructions as to their proper use. Poisoning may also be caused by the accumulation of certain metals (e.g. lead, mercury and cadmium) in the body. High levels of mercury and cadmium have been found in fish taken from waters polluted by industrial waste. Cases of lead poisoning have arisen as a result of drinking water that has passed through lead pipes.

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In addition, food poisoning may be caused by organic mercury compounds, notably methyl mercury. Between 1953 and 1960, 52 people in the Japanese town of Minamata died and about 100 suffered serious brain damage as a result of eating fish containing high levels of methyl mercury. Investigations showed that the source of mercury was effluent from a local chemical factory. The effluent contained inorganic mercury but this was converted into methyl mercury accumulated in the fish and shellfish living in the bay. Altogether about 900 people showed symptoms of methyl mercury poisoning, as well as many of the seabirds and cats of the area.

2.4 FOODBORNE ILLNESS

Foodborne illness (also foodborne disease and colloquially referred to as food poisoning) is any illness resulting from the food spoilage of contaminated food, pathogenic bacteria, viruses, or parasites that contaminate food, as well as toxins such as poisonous mushrooms and various species of beans that have not been boiled for at least 10 minutes. Symptoms vary depending on the cause, and are described below in this article. A few broad generalizations can be made, e.g.: The incubation period ranges from hours to days, depending on the cause and on how much was consumed. The incubation period tends to cause sufferers to not associate the symptoms with the item consumed, and so to cause sufferers to attribute the symptoms to gastroenteritis for example. Symptoms often include vomiting, fever, and aches, and may include diarrhea. Bouts of vomiting can be repeated with an extended delay in between, because even if infected food was eliminated from the stomach in the first bout, microbes (if applicable) can pass through the stomach into the intestine via cells lining the intestinal walls and begin to multiply. Some types of microbes stay in the intestine, some produce a toxin that is absorbed into the bloodstream, and some can directly invade deeper body tissues.

2.5 CAUSES OF FOODBORNE ILLNESS

Foodborne illness usually arises from improper handling, preparation, or food storage. Good hygiene practices before, during, and after food preparation can reduce the chances of contracting an illness. There is a consensus in the public health community that regular hand-washing is one of the most effective defences against the spread of foodborne illness. The action of monitoring food to ensure that it will not cause foodborne illness is known as food safety. Foodborne disease can also be caused by a large variety of toxins that affect the environment. Furthermore, foodborne illness can be caused by pesticides or medicines in food and natural toxic substances such as poisonous mushrooms or reef fish. 2.5.1 BACTERIA Bacteria are a common cause of foodborne illness. In the United Kingdom during 2000, the individual bacteria involved were the following: Campylobacter jejuni 77.3%,

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Salmonella 20.9%, Escherichia coli O157:H7 1.4%, and all others less than 0.56%. In the past, bacterial infections were thought to be more prevalent because few places had the capability to test for norovirus and no active surveillance was being done for this particular agent. Toxins from bacterial infections are delayed because the bacteria need time to multiply. As a result, symptoms associated with intoxication are usually not seen until 12–72 hours or more after eating contaminated food. However, in some cases, such as Staphylococcal food poisoning, the onset of illness can be as soon as 30 minutes after ingesting contaminated food. Most common bacterial foodborne pathogens are: • Campylobacter jejuni which can lead to secondary Guillain–Barré syndrome and periodontitis • Clostridium perfringens, the “cafeteria germ” • Salmonella spp. – its S. typhimurium infection is caused by consumption of eggs or poultry that are not adequately cooked or by other interactive humananimal pathogens • Escherichia coli O157:H7 enterohemorrhagic (EHEC) which can cause hemolytic-uremic syndrome Other common bacterial foodborne pathogens are: • Bacillus cereus • Escherichia coli, other virulence properties, such as enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroaggregative (EAEC or EAgEC) • Listeria monocytogenes • Shigella spp. • Staphylococcus aureus • Staphylococcal enteritis • Streptococcus • Vibrio cholerae, including O1 and non-O1 • Vibrio parahaemolyticus • Vibrio vulnificus • Yersinia enterocolitica and Yersinia pseudotuberculosis Less common bacterial agents: • Brucella spp. • Corynebacterium ulcerans • Coxiella burnetii or Q fever • Plesiomonas shigelloides 2.5.1.1 ENTEROTOXINS In addition to disease caused by direct bacterial infection, some foodborne illnesses are caused by enterotoxins (exotoxins targeting the intestines). Enterotoxins can produce illness even when the microbes that produced them have been killed. Symptom

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appearance varies with the toxin but may be rapid in onset, as in the case of enterotoxins of Staphylococcus aureus in which symptoms appear in one to six hours. This causes intense vomiting including or not including diarrhea (resulting in staphylococcal enteritis), and staphylococcal enterotoxins (most commonly staphylococcal enterotoxin A but also including staphylococcal enterotoxin B) are the most commonly reported enterotoxins although cases of poisoning are likely underestimated. It occurs mainly in cooked and processed foods due to competition with other biota in raw foods, and humans are the main cause of contamination as a substantial percentage of humans are persistent carriers of S. aureus. The CDC has estimated about 240,000 cases per year in the United States. • Clostridium botulinum • Clostridium perfringens • Bacillus cereus The rare but potentially deadly disease botulism occurs when the anaerobic bacterium Clostridium botulinum grows in improperly canned low-acid foods and produces botulin, a powerful paralytic toxin. Pseudoalteromonas tetraodonis, certain species of Pseudomonas and Vibrio, and some other bacteria, produce the lethal tetrodotoxin, which is present in the tissues of some living animal species rather than being a product of decomposition. 2.5.1.2 EMERGING FOODBORNE PATHOGENS Many foodborne illnesses remain poorly understood. • Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria 2.5.1.3 PREVENTING BACTERIAL FOOD POISONING Prevention is mainly the role of the state, through the definition of strict rules of hygiene and a public services of veterinary surveying of animal products in the food chain, from farming to the transformation industry and delivery (shops and restaurants). This regulation includes: • Traceability: In a final product, it must be possible to know the origin of the ingredients (originating farm, identification of the harvesting or of the animal) and where and when it was processed; the origin of the illness can thus be tracked and solved (and possibly penalized), and the final products can be removed from the sale if a problem is detected; • Enforcement of hygiene procedures such as HACCP and the “cold chain”; • Power of control and of law enforcement of veterinarians. In August 2006, the United States Food and Drug Administration approved Phage therapy which involves spraying meat with viruses that infect bacteria, and thus preventing infection. This has raised concerns, because without mandatory labelling consumers would not be aware that meat and poultry products have been treated with the spray.

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At home, prevention mainly consists of good food safety practices. Many forms of bacterial poisoning can be prevented by cooking it sufficiently, and either eating it quickly or refrigerating it effectively. Many toxins, however, are not destroyed by heat treatment. Techniques that help prevent food borne illness in the kitchen are hand washing, rinsing produce, preventing cross-contamination, proper storage, and maintaining cooking temperatures. In general, freezing or refrigerating prevents virtually all bacteria from growing, and heating food sufficiently kills parasites, viruses, and most bacteria. Bacteria grow most rapidly at the range of temperatures between 40 and 140°F (4 and 60°C), called the “danger zone”. Storing food below or above the “danger zone” can effectively limit the production of toxins. For storing leftovers, the food must be put in shallow containers for quick cooling and must be refrigerated within two hours. When food is reheated, it must reach an internal temperature of 165°F (74°C) or until hot or steaming to kill bacteria. 2.5.2 MYCOTOXINS AND ALIMENTARY MYCOTOXICOSES The term alimentary mycotoxicoses refers to the effect of poisoning by Mycotoxins (The term ‘mycotoxin’ is usually reserved for the toxic chemical products produced by fungi that readily colonize crops) through food consumption. Mycotoxins sometimes have important effects on human and animal health. For example, an outbreak which occurred in the UK in 1960 caused the death of 100,000 turkeys which had consumed aflatoxin-contaminated peanut meal. In the USSR in World War II, 5,000 people died due to Alimentary Toxic Aleukia (ALA). The common foodborne Mycotoxins include: • Aflatoxins – originated from Aspergillus parasiticus and Aspergillus flavus. They are frequently found in tree nuts, peanuts, maize, sorghum and other oilseeds, including corn and cottonseeds. The pronounced forms of Aflatoxins are those of B1, B2, G1, and G2, amongst which Aflatoxin B1 predominantly targets the liver, which will result in necrosis, cirrhosis, and carcinoma. In the US, the acceptable level of total aflatoxins in foods is less than 20 ìg/kg, except for Aflatoxin M1 in milk, which should be less than 0.5 ìg/kg. The official document can be found at FDA’s web site. • Altertoxins – are those of Alternariol (AOH), Alternariol methyl ether (AME), Altenuene (ALT), Altertoxin-1 (ATX-1), Tenuazonic acid (TeA) and Radicinin (RAD), originated from Alternaria spp. Some of the toxins can be present in sorghum, ragi, wheat and tomatoes. Some research has shown that the toxins can be easily cross-contaminated between grain commodities, suggesting that manufacturing and storage of grain commodities is a critical practice. • Citrinin • Citreoviridin • Cyclopiazonic acid • Cytochalasins

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• Ergot alkaloids/ Ergopeptine alkaloids – Ergotamine • Fumonisins – Crop corn can be easily contaminated by the fungi Fusarium moniliforme, and its Fumonisin B1 will cause Leukoencephalomalacia (LEM) in horses, Pulmonary edema syndrome (PES) in pigs, liver cancer in rats and Esophageal cancer in humans. For human and animal health, both the FDA and the EC have regulated the content levels of toxins in food and animal feed. • Fusaric acid • Fusarochromanone • Kojic acid • Lolitrem alkaloids • Moniliformin • 3-Nitropropionic acid • Nivalenol • Ochratoxins – In Australia, The Limit of Reporting (LOR) level for Ochratoxin A (OTA) analyses in 20th Australian Total Diet Survey was 1 µg/kg, whereas the EC restricts the content of OTA to 5 µg/kg in cereal commodities, 3 µg/kg in processed products and 10 µg/kg in dried vine fruits. • Oosporeine • Patulin – Currently, this toxin has been advisably regulated on fruit products. The EC and the FDA have limited it to under 50 µg/kg for fruit juice and fruit nectar, while limits of 25 µg/kg for solid-contained fruit products and 10 µg/kg for baby foods were specified by the EC. • Phomopsins • Sporidesmin A • Sterigmatocystin • Tremorgenic mycotoxins – Five of them have been reported to be associated with molds found in fermented meats. These are Fumitremorgen B, Paxilline, Penitrem A, Verrucosidin, and Verruculogen. • Trichothecenes – sourced from Cephalosporium, Fusarium, Myrothecium, Stachybotrys and Trichoderma. The toxins are usually found in molded maize, wheat, corn, peanuts and rice, or animal feed of hay and straw. Four trichothecenes, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) have been most commonly encountered by humans and animals. The consequences of oral intake of, or dermal exposure to, the toxins will result in Alimentary toxic aleukia, neutropenia, aplastic anemia, thrombocytopenia and/or skin irritation. In 1993, the FDA issued a document for the content limits of DON in food and animal feed at an advisory level. In 2003, US published a patent that is very promising for farmers to produce a trichothecene-resistant crop. • Zearalenone • Zearalenols

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2.5.3 VIRUSES Viral infections make up perhaps one third of cases of food poisoning in developed countries. In the US, more than 50% of cases are viral and noroviruses are the most common foodborne illness, causing 57% of outbreaks in 2004. Foodborne viral infection are usually of intermediate (1–3 days) incubation period, causing illnesses which are self-limited in otherwise healthy individuals; they are similar to the bacterial forms described above. • Enterovirus • Hepatitis A is distinguished from other viral causes by its prolonged (2–6 week) incubation period and its ability to spread beyond the stomach and intestines into the liver. It often results in jaundice, or yellowing of the skin, but rarely leads to chronic liver dysfunction. The virus has been found to cause infection due to the consumption of fresh-cut produce which has fecal contamination. • Hepatitis E • Norovirus • Rotavirus

2.5.4 PARASITES Most foodborne parasites are zoonoses. • Platyhelminthes: – Diphyllobothrium sp. – Nanophyetus sp. – Taenia saginata – Taenia solium – Fasciola hepatica • Nematode: – Anisakis sp. – Ascaris lumbricoides – Eustrongylides sp. – Trichinella spiralis – Trichuris trichiura • Protozoa: – Acanthamoeba and other free-living amoebae – Cryptosporidium parvum – Cyclospora cayetanensis – Entamoeba histolytica – Giardia lamblia – Sarcocystis hominis – Sarcocystis suihominis – Toxoplasma gondii

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2.5.5 NATURAL TOXINS Several foods can naturally contain toxins, many of which are not produced by bacteria. Plants in particular may be toxic; animals which are naturally poisonous to eat are rare. In evolutionary terms, animals can escape being eaten by fleeing; plants can use only passive defences such as poisons and distasteful substances, for example capsaicin in chili peppers and pungent sulfur compounds in garlic and onions. Most animal poisons are not synthesised by the animal, but acquired by eating poisonous plants to which the animal is immune, or by bacterial action. • Alkaloids • Ciguatera poisoning • Grayanotoxin (honey intoxication) • Mushroom toxins • Phytohaemagglutinin (red kidney bean poisoning; destroyed by boiling) • Pyrrolizidine alkaloids • Shellfish toxin, including paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, amnesic shellfish poisoning and ciguatera fish poisoning • Scombrotoxin • Tetrodotoxin (fugu fish poisoning) Some plants contain substances which are toxic in large doses, but have therapeutic properties in appropriate dosages. • Foxglove contains cardiac glycosides. • Poisonous hemlock (conium) has medicinal uses. 2.5.6 OTHER PATHOGENIC AGENTS Prions, resulting in Creutzfeldt–Jakob disease (CJD) and its variant (vCJD) 2.5.7 “PTOMAINE POISONING” In 1883, the Italian, Professor Salmi, of Bologna, introduced the generic name ptomaine (from Greek ptôma, “fall, fallen body, corpse”) for alkaloids found in decaying animal and vegetable matter, especially (as reflected in their names) putrescine and cadaverine. The 1892 Merck’s Bulletin stated, “We name such products of bacterial origin ptomaines; and the special alkaloid produced by the comma bacillus is variously named Cadaverine, Putrescine, etc.” While The Lancet stated, “The chemical ferments produced in the system, the...ptomaines which may exercise so disastrous an influence.” It is now known that the “disastrous...influence” is due to the direct action of bacteria and only slightly to the alkaloids. Thus, the use of the phrase “ptomaine poisoning” is now obsolete. Tainted potato salad sickening hundreds at a Communist political convention in Massillon, Ohio, and aboard a Washington DC cruise boat in separate incidents during a single week in 1932 drew national attention to the dangers of so-called “ptomaine poisoning” in the pages of the American news weekly, Time. Another newspaper article

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from 1944 told of more than 150 persons being hospitalized in Chicago with ptomaine poisoning apparently from rice pudding served by a chain of restaurants.

2.6 FOODBRONE MECHANISM 2.6.1 INCUBATION PERIOD The delay between consumption of a contaminated food and appearance of the first symptoms of illness is called the incubation period. This ranges from hours to days (and rarely months or even years, such as in the case of listeriosis or bovine spongiform encephalopathy), depending on the agent, and on how much was consumed. If symptoms occur within one to six hours after eating the food, it suggests that it is caused by a bacterial toxin or a chemical rather than live bacteria. The long incubation period of many foodborne illnesses tends to cause sufferers to attribute their symptoms to gastroenteritis. During the incubation period, microbes pass through the stomach into the intestine, attach to the cells lining the intestinal walls, and begin to multiply there. Some types of microbes stay in the intestine, some produce a toxin that is absorbed into the bloodstream, and some can directly invade the deeper body tissues. The symptoms produced depend on the type of microbe. 2.6.2 INFECTIOUS DOSE The infectious dose is the amount of agent that must be consumed to give rise to symptoms of foodborne illness, and varies according to the agent and the consumer’s age and overall health. Pathogens vary in minimum infectious dose; for example, Shigella sonnei has a low estimated minimum dose of < 500 colony-forming units (CFU) while Staphylococcus aureus has a relatively high estimate. In the case of Salmonella a relatively large inoculum of 1 million to 1 billion organisms is necessary to produce symptoms in healthy human volunteers, as Salmonellae are very sensitive to acid. An unusually high stomach pH level (low acidity) greatly reduces the number of bacteria required to cause symptoms by a factor of between 10 and 100.

2.7 MICROBIOLOGY OF FOODBORNE ILLNESS

Bacteria are single-celled organisms which multiply by cell division, under appropriate environmental conditions. The conditions that influence bacterial growth are the food itself, acidity, time, temperature, oxygen, and moisture. Most bacteria need nutrients to survive. They obtain these nutrients from food. Bacteria grow best in food that is neutral to slightly acidic (acidity is measured by pH). Microorganisms have different acidity (pH), temperature, and oxygen requirements for optimal growth. Bacteria need time to grow and they grow rapidly between 41°F and 140°F. Bacterial growth is slowed at temperatures below 41°F and limited at temperatures above 140°F. Some bacteria require

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oxygen to grow (aerobic), some grow when there is no oxygen (anaerobic), and some can grow with or without oxygen (facultative). Bacteria will grow when food and water is available. If water is bound or tied up with, for example salts or sugars, it is not available to be used by bacteria. This concept of available water is referred to as water activity (Aw). Some bacteria can be further categorized: • Some bacteria are spore formers. The spore protects the organism during periods of environmental stress. When the conditions become suitable, the organism germinates from the spore and continues the growth cycle. • Some bacteria produce toxins that cause illness. Molds are a multi-cellular fungi that reproduce by fruiting bodies that break and release thousands of microscopic mold spores, each capable of growing under the right conditions. Molds can send “roots” into the food to provide nourishment to the spore. Molds prefer damp, dark environments for optimal growth and they grow readily on almost any food, as well as walls, ceilings, and other areas of high moisture. Some molds produce toxins that can cause illness. Viruses are the smallest known organisms. They cannot multiply in food—they need a human host. Viruses are transmitted to food from infected people. Parasites include worms and protozoa. They cannot multiply in food; they multiply in a host cell.

2.8 PREVENTION OF FOODBORNE ILLNESS

Follow these 4 simple steps to keep food safe: Step1: CLEAN: Wash hands and food contact surfaces and utensils often, between tasks, and if they have become contaminated. Effective cleaning involves removing soil and debris, scrubbing with hot soapy water and rinsing, using potable/drinking water. Sanitizing involves the use of high heat (e.g., a dishwasher) or chemicals (e.g., chlorine bleach) to reduce or eliminate the number of microorganisms to a safe level. • Wash hands with warm water and soap for 20 seconds and dry with a disposable paper towel or clean hand cloth. • Alcohol based hand sanitizers are not a replacement for handwashing. They are not effective if the hands are dirty, they are not effective against Norovirus, and they do not eliminate all types of microorganisms. • Wash cutting boards, dishes, and utensils after preparing each food item and before you use it for the next food. • Use hot, soapy water, rinse with hot water, and air dry or dry with a clean paper towel or clean dish cloth. • Or wash in the dishwasher. • Wash countertops after preparing each food item and before you use it for the next food.

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• Use paper towels or clean dish cloths to wipe kitchen surfaces or spills. • Wash countertops with hot soapy water, rinse with hot water and air dry or dry with a clean paper towel or clean dish cloth. • To sanitize for added protection for bacteria on surfaces, you can use the following: – Dilute mixtures of chlorine bleach and water are a cost-effective method of sanitation. Chlorine bleach is a very effective sanitizer. It comes in several concentrations. – If bleach is 8.25%: measure 1 teaspoon of bleach per 1 gallon of water or 1/ 8 teaspoon of bleach per 1 pint of water. – Apply to the cleaned countertop and allow to sit for 1-2 minutes and air dry or dry with a clean paper towel. – Alternatively, commercial products for sanitizing the home kitchen are available. Follow manufacturer instruction for use. – Wash dish cloths often in a washing machine. – Store sponge in a place so it can dry after use. – To lower the risk of cross-contamination, sanitize the dish sponge often: – Soak in a chlorine bleach solution for 1 min. – Microwave heat a damp sponge for 1 min. – Put sponge in dishwasher cycle. – Replace the dish sponge often. Step 2: SEPARATE to prevent cross contamination. Cross contamination is the transfer of harmful bacteria from uncooked food products (e.g. raw meat, fish, and poultry) or unclean people, countertops, and kitchen equipment to ready-to-eat foods (e.g., fruits, vegetables, deli meats/cheeses, and prepared or cooked foods). • Prevent cross contamination when grocery shopping. – Physically separate raw meat, fish and poultry to prevent their juices from dripping onto other foods. This can be done by: – Segregating raw meat, fish and poultry on one side of the shopping cart. – Placing raw meat, fish and poultry in separate plastic bags (e.g. one bag for chicken, one bag for fish, etc.). • Designate reusable bags for grocery shopping only. Reusable bags for raw meat, fish, or poultry should never be used for ready-to-eat products. – Frequently wash bags. Cloth bags should be washed in a machine and machine dried or air-dried. Plastic-lined bags should be scrubbed using hot water and soap and air-dried. – Separate raw meat, fish and poultry in disposable plastic bags before putting them in a reusable bag – Check that both cloth and plastic-lined reusable bags are completely dry before storing. • Prevent cross contamination when storing food in the refrigerator.

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– In the refrigerator, store raw meats, fish, and poultry below ready-to-eat and cooked foods. – When thawing frozen raw meat, fish and poultry, put the food in a plastic bag or on a plate on the lowest shelf to prevent juices from dripping onto other foods. i. After thawing in the refrigerator, food should remain safe and of good quality for a few days before cooking. Food thawed in the refrigerator can be refrozen without cooking, although quality may be impacted. See Chill section for other methods for thawing. • Prevent cross contamination when handling, preparing, and serving food. – Thoroughly wash your hands before and after handling different foods, after using the bathroom, and anytime they can become contaminated. – Use separate cutting boards for meat and produce. Alternatively, prepare produce first, then meat. – Wash and rinse cutting board, knives, and preparation area after cutting raw meat, fish or poultry. These items can be sanitized after cleaning. – Use a clean serving plate to serve cooked meat. Do not use the plate that held the raw meat, unless it is washed. – Throw away any sauce or dip that has been used to marinade raw meat, fish, or poultry. Do not use this extra sauce as a dip for cooked food unless it is boiled first. Step 3: COOK food thoroughly and use a thermometer to verify the proper temperature was reached. • Cook foods to the safe minimum internal temperature, as indicated in the table below: • To determine that the proper temperature was reached, place a food thermometer in the thickest part of the food and allow the it to equilibrate. – Make sure it’s not touching bone, fat, or gristle. – For whole poultry, insert the thermometer into the innermost part of the thigh and wing and the thickest part of the breast. – For combination dishes, place the thermometer in the center or thickest portion. – Egg dishes and dishes containing ground meat or poultry should be checked in several places. • Clean your food thermometer with hot, soapy water before and after each use! Food Thermometers – Why use them? Not only is it important to monitor the refrigerator temperature (chill foods); but using a thermometer is the only reliable way to ensure that a food is properly cooked. When cooking: • Colour is not a reliable indicator that the food has been cooked to the correct temperature to ensure that foodborne pathogens are destroyed. • Determining “doneness” of hamburger cannot be safely done by looking at the brown colour of the meat or of chicken by looking that the juices run clear.

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• Time alone as an indicator that the food is cooked properly could result in a potential food safety hazard. Recipes may state “x minutes/pound”. However, different thicknesses of a food or ingredients that are used can alter the time needed at a specific temperature to make sure the food has reached the correct temperature to kill all pathogens. • Food thermometers come in several types and styles and range in level of technology and price. There is a lot of good information on how to use a thermometer correctly, proper placement, and how to check to see if it is accurate (see Resources– Thermometers). Pop-up temperature devices are commonly found in turkeys or oven roaster chickens. These devices indicate that the food has come to the correct temperature for safety. However, while these devices are reliable, it is recommended that the temperature be checked in several places with a conventional thermometer to ensure proper cooking. Step 4: CHILL foods promptly. Cold temperatures slow the growth of harmful bacteria. Cold air must circulate to help keep food safe, so do not over fill the refrigerator. Maintain the refrigerator temperature at 41°F or below. Place an appliance thermometer in the rear portion of the refrigerator, and monitor regularly. Maintain the freezer temperature at 0°F or below. • Refrigerate and/or freeze meat, poultry, eggs and other perishables as soon as possible after purchasing. • Consider using a cooler with ice or gel packs to transport perishable food. • Perishable foods, such as cut fresh fruits or vegetables and cooked food should not sit at room temperature more than two hours before putting them in the refrigerator or freezer (one hour when the temperature is above 90°F). • There are three safe ways to thaw food: in the refrigerator (see Separate), in cold water, and in the microwave. Food thawed in cold water or in the microwave should be cooked immediately. • Submerging the food in cold water. It is important to place the food in a bag that will prevent the water from entering. Check the water every 30 minutes to make sure it is cold. Cook food prior to refreezing. • Microwave thawing. Cook food immediately once thawed because some areas of the food may become warm and begin to cook during the thawing process. Cook food prior to refreezing. • Cool leftovers quickly by dividing large amounts into shallow containers for quicker cooling in the refrigerator.

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Food Spoilage

3 Food Spoilage

3.1 FOOD SPOILAGE

Food is considered contaminated when unwanted microorganisms are present. Most of the time the contamination is natural, but sometimes it is artificial. Natural contamination occurs when microorganisms attach themselves to foods while the foods are in their growing stages. For instance, fruits are often contaminated with yeasts because yeasts ferment the carbohydrates in fruits. Artificial contamination occurs when food is handled or processed, such as when fecal bacteria enter food through improper handling procedures. Spoilage is the process in which food deteriorates to the point in which it is not edible to humans or its quality of edibility becomes reduced. Various external forces are responsible for the spoilage of food. Food that is capable of spoiling is referred to as perishable food. Food spoilage is a disagreeable change or departure from the food’s normal state. Such a change can be detected with the senses of smell, taste, touch, or vision. Changes occurring in food depend upon the composition of food and the microorganisms present in it and result from chemical reactions relating to the metabolic activities of microorganisms as they grow in the food. Food spoilage is a metabolic process that causes foods to be undesirable or unacceptable for human consumption due to changes in sensory characteristics. Spoiled foods may be safe to eat, i.e. they may not cause illness because there are no pathogens or a toxin present, but changes in texture, smell, taste, or appearance cause them to be

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rejected. Some ecologists have suggested these noxious smells are produced by microbes to repulse large animals, thereby keeping the food resource for themselves. Food loss, from farm to fork, causes considerable environmental and economic effects. The USDA Economic Research Service estimated that more than ninety-six billion pounds of food in the U.S. were lost by retailers, foodservice and consumers in 1995. Fresh produce and fluid milk each accounted for nearly 20% of this loss while lower percentages were accounted for by grain piroducts (15.2%), caloric sweeteners (12.4%), processed fruits and vegetables (8.6%), meat, poultry and fish (8.5%), and fat and oils (7.1%). Some of this food would have been considered still edible but was discarded because it was perishable, past its sell-by date, or in excess of needs. There are also environmental and resource costs associated with food spoilage and loss. If 20% of a crop is lost, then 20% of the fertilizer and irrigation water used to grow that crop was also lost. Shelf life of a food is the time during which it remains stable and retains its desired qualities. The wide array of available dairy foods challenges the microbiologist, engineer, and technologist to find the best ways to prevent the entry of microorganisms, destroy those that do get in along with their enzymes, and prevent the growth and activities of those that escape processing treatments. Troublesome spoilage microorganisms include aerobic psychrotrophic Gram-negative bacteria, yeasts, molds, heterofermentative lactobacilli, and spore-forming bacteria. Psychrotrophic bacteria can produce large amounts of extracellular hydrolytic enzymes, and the extent of recontamination of pasteurized fluid milk products with these bacteria is a major determinant of their shelf life. Fungal spoilage of dairy foods is manifested by the presence of a wide variety of metabolic byproducts, causing off-odors and flavours, in addition to visible changes in colour or texture. Coliforms, yeasts, heterofermentative lactic acid bacteria, and spore-forming bacteria can all cause gassing defects in cheeses. The rate of spoilage of many dairy foods is slowed by the application of one or more of the following treatments: reducing the pH by fermenting the lactose to lactic acid; adding acids or other approved preservatives; introducing desirable microflora that restricts the growth of undesirable microorganisms; adding sugar or salt to reduce the water activity (aw); removing water; packaging to limit available oxygen; and freezing. The type of spoilage microorganisms differs widely among dairy foods because of the selective effects of practices followed in production, formulation, processing, packaging, storage, distribution, and handling. 3.1.1 SCENARIO OF FOOD SPOILAGE WORLDWIDE The issue of food losses is of high importance in the efforts to combat hunger, raise income and improve food security in the world’s poorest countries. Food losses have an impact on food security for poor people, on food quality and safety, on economic development and on the environment. The exact causes of food losses vary throughout the world and are very much dependent on the specific conditions and local situation in a given country. In broad terms, food losses will be influenced by crop production choices and patterns, internal infrastructure and capacity, marketing chains and channels

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for distribution, and consumer purchasing and food use practices. Irrespective of the level of economic development and maturity of systems in a country, food losses should be kept to a minimum. Food losses represent a waste of resources used in production such as land, water, energy and inputs. Producing food that will not be consumed leads to unnecessary CO2 emissions in addition to loss of economic value of the food produced. Economically avoidable food losses have a direct and negative impact on the income of both farmers and consumers. Given that many smallholders live on the margins of food insecurity, a reduction in food losses could have an immediate and significant impact on their livelihoods. For poor consumers (food insecure or at-risk households), the priority is clearly to have access to food products that are nutritious, safe and affordable. It is important to note that food insecurity is often more a question of access than a supply problem. Improving the efficiency of the food supply chain could help to bring down the cost of food to the consumer and thus increase access. Given the magnitude of food losses, making profitable investments in reducing losses could be one way of reducing the cost of food. But that would, of course, require that financial gains from reduced losses are not outweighed by their costs. How much food is lost and wasted in the world today and how can we prevent food losses? Those are questions impossible to give precise answers to, and there is not much ongoing research in the area. This is quite surprising as forecasts suggest that food production must increase significantly to meet future global demand. Worldwide postharvest fruit and vegetables losses are as high as 30 to 40% and even much higher in some developing countries. Reducing postharvest losses is very important; ensuring that sufficient food, both in quantity and in quality is available to every inhabitant in our planet. The prospects are also that the world population will grow from 5.7 billion inhabitants in 1995 to 8.3 billion in 2025. World production of vegetables amounted to 486 million ton, while that of fruits reached 392 million ton. Reduction of post-harvest losses reduces cost of production, trade and distribution, lowers the price for the consumer and increases the farmer’s income. Fruits and vegetables are very important food commodities not only in India but all over the world. India, which is the second most populated country of the world, is still struggling to achieve self-sufficiency to feed about 800 million people. For this purpose, fruits and vegetables have got their specific importance to provide a balance and healthy diet to the people. India is the second largest producer of vegetables and fourth largest producer of fruits in the world. Though India is producing adequate quantities of fruits and vegetables, yet on account of losses in the field as well as in storage, they become inadequate. Generally, about 30 % fruits and vegetables are rendered unfit for consumption due to spoilage after harvesting. India annually produces fruits and vegetables of the value of about Rs. 7000 crores and wastage may be of the order of Rs. 2100 crores. This is a huge loss of valuable food even when the minimum food requirement of the population is not met. Therefore, it is important not only to grow

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more, but also to save what is grown at high cost. Post harvest loss of fruits and vegetables has been defined as “that weight of wholesome edible product (exclusive of moisture content) that is normally consumed by human and that has been separated from the medium and sites of its immediate growth and production by deliberate human action with the intention of using it for human feeding but which for any reasons fails to be consumed by human.” Not only quantity and quality but even the appearance of fruits and vegetables are affected and their market value is reduced. Most ‘skin deep’ injuries such as ‘fly speck’ in apples do not affect the edible part. Some infections are not harmful as they occur on inedible parts and can be trimmed off before. Fresh fruits and vegetables are perishable and highly prone to these losses because they are composed of living tissues. These tissues must be kept alive and health throughout the process of marketing. These are composed of thousands of living cells which require care and maintenance. Therefore, the reduction of post-harvest loss of fruit and vegetables is a complementary means for increasing production. It may not be necessary to considerably step up the production of fruits and vegetables with the growing demand if the post-harvest loss is reduced to a great extent. The cost of preventing losses after harvest in general is less than preventing a similar additional amount of fruit and vegetable crop of the same quality. Attention to the concept of post-harvest food loss reduction, as a significant means to increase food availability, was drawn by the World Food Conference held in Rome in 1974. The global dairy industry is impressive by large. In 2005, world milk production was estimated at 644 million tons, of which 541 million tons was cows’ milk. The leading producers of milk were the European Union at 142 million tons, India at 88 million tons, the United States at 80 million tons (20.9 billion gallons), and Russia at 31 million tons. Cheese production amounted to 8.6 million tons in Western Europe and 4.8 million tons in the United States. The vast array of products made from milk worldwide leads to an equally impressive array of spoilage microorganisms. A survey of dairy product consumption revealed that 6% of US consumers would eat more dairy products if they stayed fresher longer. Products range from those that are readily spoiled by microorganisms to those that are shelf stable for many months, and the spoilage rate can be influenced by factors such as moisture content, pH, processing parameters, and temperature of storage. 3.1.2 SCENARIO OF FOOD SPOILAGE IN INDIA India is the second major producer of fruits and vegetables and ranks next to Brazil and China respectively, in the world. It contributes 10 percent of world fruit production and 14 per cent of world vegetable production. Fruits and vegetables are more prone to spoilage than cereals due to their nature and composition, and this spoilage occurs at the time of harvesting, handling transportation, storage, marketing and processing resulting in waste. Efficient management of these wastes can help in preserving vital nutrients of our foods and feeds, and bringing down the cost of production of processed

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foods, besides minimizing pollution hazards. According to India Agricultural Research Data Book 2004, the losses in fruits and vegetables are to the tune of 30 per cent. Taking estimated production of fruits and vegetables in India at 150 million tones, the total waste generated comes to 50 million tones per annum. The post-harvest technologies for perishable horticultural produce serve as an effective tool for getting better return to the produce and also help in avoiding wastage both at production site and distribution centers, which will help in regulating the market infrastructure. Like any other food, fruits and vegetables are also prone to microbial spoilage caused by fungi, bacteria, yeast and moulds. A significant portion of losses of fruits and vegetables during post-harvest period is attributed to diseases caused by fungi and bacteria. The succulent nature of fruits and vegetables makes them easily invaded by these organisms. Besides attacking fresh fruits and vegetables, these organisms also cause damage to canned and processed products. Many serious post-harvest diseases occur rapidly and cause extensive break down of the commodity, sometimes spoiling the entire package. It is estimated that 36 % of the vegetable decay is caused by soft rot bacteria. Similarly fruit rot in aonla and other soft fruits caused by fungi is also very destructive. As far as vegetables are concerned, naturally the source of infection is from the field, water used for cleaning the surface, contact with equipment and storage environment. The most common pathogens causing rots in vegetables and fruits are fungi such as Alternaria, Botrytis, Diplodia, Monilinia, Phomopsis, Rhizopus, Pencillium, Fusarium, etc. Among bacteria Ervinia, Pseudomonas, etc. cause extensive damage. High temperature and relative humidity favour the development of post-harvest decay organisms. More acidic tissue is generally attacked by fungi, while fruits and vegetables having pH above 4.5 are more commonly attacked by bacteria, ego bacterial soft rot of potato caused by Ceratocystis, fimbriata, water soft rot of carrot by Sclerotinia sclerotiorum etc. In India, there is a vast scope for growing fruit and vegetable throughout the year in one or other part of the country because the climatic conditions are highly suitable for growing various types of fruits and vegetables. Fruit and vegetable are highly perishable but most important commodity for human diet due to their high nutritional value. They are the cheapest and other source of protective food supplied in fresh or processed or preserved form throughout the year for human consumption. Hence the national picture will improve significantly. Fruit and vegetable are available in surplus only in certain seasons and availability in different regions. In peak season due to improper handling practices, marketing, storage problems around 20-25% fruit and vegetable are spoilt in various stages. Fruit and vegetable are living commodities as they respire. Hence, proper post harvest management handling and processing is required in horticulture crops. A variety of fresh fruit and vegetable in India can be made available in plenty due to favourable agro-climatic situations. Hence there is no dearth for raw material for processing. Product profile being developed in India at present is limited to few fruit and vegetable like

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mango, pineapple, grapes etc. But there is a wider potentiality for processing of papaya, banana, jack, guava, aonla, carambola and other minor fruits. Similarly there is a greater scope for processing cauliflower, carrot, bitter-gourd onion, garlic, watermelon, muskmelon etc. Proper handling, packaging, transportation and storage reduce the post-harvest losses of fruit and vegetables. For every one percent reduction in loss will save 5 million tons of fruit and vegetable per year. Processing and preservation technology helps. There are about 4000 small and large scale processing units in the country which process only about 2.5% of the total fruit and vegetable as against 40-85% in developed countries.

3.2 FOOD SPOILAGE MICROORGANISMS

Chemical reactions that cause offensive sensory changes in foods are mediated by a variety of microbes that use food as a carbon and energy source. These organisms include prokaryotes (bacteria), single-celled organisms lacking defined nuclei and other organelles, and eukaryotes, single-celled (yeasts) and multicellular (molds) organisms with nuclei and other organelles. Some microbes are commonly found in many types of spoiled foods while others are more selective in the foods they consume; multiple species are often identified in a single spoiled food item but there may be one species (a specific spoilage organism, SSO) primarily responsible for production of the compounds causing off-odors and flavours. Within a spoiling food, there is often a succession of different populations that rise and fall as different nutrients become available or are exhausted. Some microbes, such as lactic acid bacteria and molds, secrete compounds that inhibit competitors. Spoilage microbes are often common inhabitants of soil, water, or the intestinal tracts of animals and may be dispersed through the air and water and by the activities of small animals, particularly insects. It should be noted that with the development of new molecular typing methods, the scientific names of some spoilage organisms, particularly the bacteria, have changed in recent years and some older names are no longer in use. Many insects and small mammals also cause deterioration of food but these will not be considered here. 3.2.1 YEASTS Yeasts are a subset of a large group of organisms called fungi that also includes molds and mushrooms. They are generally single-celled organisms that are adapted for life in specialized, usually liquid, environments and, unlike some molds and mushrooms, do not produce toxic secondary metabolites. Yeasts can grow with or without oxygen (facultative) and are well known for their beneficial fermentations that produce bread and alcoholic drinks. They often colonize foods with a high sugar or salt content and contribute to spoilage of maple syrup, pickles, and sauerkraut. Fruits and juices with a low pH are another target, and there are some yeasts that grow on the surfaces of meat and cheese.

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There are four main groups of spoilage yeasts: Zygosaccharomyces and related genera tolerate high sugar and high salt concentrations and are the usual spoilage organisms in foods such as honey, dried fruit, jams and soy sauce. They usually grow slowly, producing off-odors and flavours and carbon dioxide that may cause food containers to swell and burst. Debaryomyces hansenii can grow at salt concentrations as high as 24%, accounting for its frequent isolation from salt brines used for cured meats, cheeses, and olives. This group also includes the most important spoilage organisms in salad dressings. Saccharomyces spp. are best known for their role in production of bread and wine but some strains also spoil wines and other alcoholic beverages by producing gassiness, turbidity and offflavours associated with hydrogen sulfide and acetic acid. Some species grow on fruits, including yogurt containing fruit, and some are resistant to heat processing. Candida and related genera are a heterogeneous group of yeasts, some of which also cause human infections. They are involved in spoilage of fruits, some vegetables and dairy products. Dekkera/Brettanomyces are principally involved in spoilage of fermented foods, including alcoholic beverages and some dairy products. They can produce volatile phenolic compounds responsible for off-flavours. 3.2.2 MOLDS Molds are filamentous fungi that do not produce large fruiting bodies like mushrooms. Molds are very important for recycling dead plant and animal remains in nature but also attack a wide variety of foods and other materials useful to humans. They are well adapted for growth on and through solid substrates, generally produce airborne spores, and require oxygen for their metabolic processes. Most molds grow at a pH range of 3 to 8 and some can grow at very low water activity levels (0.7–0.8) on dried foods. Spores can tolerate harsh environmental conditions but most are sensitive to heat treatment. An exception is Byssochlammys, whose spores have a D value of 1–12 minutes at 90ºC. Different mold species have different optimal growth temperatures, with some able to grow in refrigerators. They have a diverse secondary metabolism producing a number of toxic and carcinogenic mycotoxins. Some spoilage molds are toxigenic while others are not. Spoilage molds can be categorized into four main groups: Zygomycetes are considered relatively primitive fungi but are widespread in nature, growing rapidly on simple carbon sources in soil and plant debris, and their spores are commonly present in indoor air. Generally they require high water activities for growth and are notorious for causing rots in a variety of stored fruits and vegetables, including strawberries and sweet potatoes. Some common bread molds also are zygomycetes. Some zygomycetes are also utilized for production of fermented soy products, enzymes, and organic chemicals. The most common spoilage species are Mucor and Rhizopus. Zygomycetes are not known for producing mycotoxins but there are some reports of toxic compounds produced by a few species.

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Penicillium and related genera are present in soils and plant debris from both tropical and Antarctic conditions but tend to dominate spoilage in temperate regions. They are distinguished by their reproductive structures that produce chains of conidia. Although they can be useful to humans in producing antibiotics and blue cheese, many species are important spoilage organisms, and some produce potent mycotoxins (patulin, ochratoxin, citreoviridin, penitrem). Penicillium spp. cause visible rots on citrus, pear, and apple fruits and cause enormous losses in these crops. They also spoil other fruits and vegetables, including cereals. Some species can attack refrigerated and processed foods such as jams and margarine. A related genus, Byssochlamys, is the most important organism causing spoilage of pasteurized juices because of the high heat resistance of its spores. Aspergillus and related molds generally grow faster and are more resistant to high temperatures and low water activity than Penicillium spp. and tend to dominate spoilage in warmer climates. Many aspergilla produce mycotoxins: aflatoxins, ochratoxin, territrems, cyclopiazonic acid. Aspergilli spoil a wide variety of food and non-food items (paper, leather, etc.) but are probably best known for spoilage of grains, dried beans, peanuts, tree nuts, and some spices. Other molds, belonging to several genera, have been isolated from spoiled food. These generally are not major causes of spoilage but can be a problem for some foods. Fusarium spp. cause plant diseases and produce several important mycotoxins but are not important spoilage organisms. However, their mycotoxins may be present in harvested grains and pose a health risk. 3.2.3 BACTERIA Spore-forming bacteria are usually associated with spoilage of heat-treated foods because their spores can survive high processing temperatures. These Gram-positive bacteria may be strict anaerobes or facultative (capable of growth with or without oxygen). Some spore-formers are thermophilic, preferring growth at high temperatures (as high as 55ºC). Some anaerobic thermophiles produce hydrogen sulphide (Desulfotomaculum) and others produce hydrogen and carbon dioxide (Thermoanaerobacterium) during growth on canned/ hermetically sealed foods kept at high temperatures, for example, soups sold in vending machines. Other thermophiles (Bacillus and Geobacillus spp.) cause a flat sour spoilage of high or low pH canned foods with little or no gas production, and one species causes ropiness in bread held at high ambient temperatures. Mesophilic anaerobes, growing at ambient temperatures, cause several types of spoilage of vegetables (Bacillus spp.); putrefaction of canned products, early blowing of cheeses, and butyric acid production in canned vegetables and fruits (Clostridium spp.); and “medicinal” flavours in canned low-acid foods (Alicyclobacillus) (Chang and Kang, 2003). Psychrotolerant sporeformers produce gas and sickly odors in chilled meats and brine-cured hams (Clostridium spp.) while others produce off-odors and gas in vacuum-packed, chilled foods and milk

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(Bacillus spp.). Lactic acid bacteria (LAB) are a group of Gram-positive bacteria, including species of Lactobacillus, Pediococcus, Leuconostoc and Oenococcus, some of which are useful in producing fermented foods such as yogurt and pickles. However, under low oxygen, low temperature, and acidic conditions, these bacteria become the predominant spoilage organisms on a variety of foods. Undesirable changes caused by LAB include greening of meat and gas formation in cheeses (blowing), pickles (bloater damage), and canned or packaged meat and vegetables. Off-flavours described as mousy, cheesy, malty, acidic, buttery or liver-like may be detected in wine, meats, milk, or juices spoiled by these bacteria. LAB may also produce large amounts of an exopolysaccharide that causes slime on meats and ropy spoilage in some beverages. Pseudomonas and related genera are aerobic, gram-negative soil bacteria, some of which can degrade a wide variety of unusual compounds. They generally require a high water activity for growth (0.95 or higher) and are inhibited by pH values less than 5.4. Some species grow at refrigeration temperatures (psychrophilic) while other are adapted for growth at warmer, ambient temperatures. Four species of Pseudomonas (P. fluorescens, P. fragi, P. lundensis, and P. viridiflava), Shewanella putrefaciens, and Xanthomonas campestris are the main food spoilage organisms in this group. Soft rots of plant-derived foods occur when pectins that hold adjacent plant cells together are degraded by pectic lyase enzymes secreted by X. campestris, P. fluorescens and P. viridiflava. These two species of Pseudomonas comprise up to 40% of the naturally occurring bacteria on the surface of fruits and vegetables and cause nearly half of postharvest rot of fresh produce stored at cold temperatures. P. fluorescens, P. fragi, P. lundensis, and S. putrefaciens cause spoilage of animal-derived foods (meat, fish, milk) by secreting lipases and proteases that cause formation of sulfides and trimethylamine (off-odors) and by forming biofilms (slime) on surfaces (55;73). Some strains are adapted for growth at cold temperatures and spoil these foods in the refrigerator. Enterobacteriaceae are gram-negative, facultatively anaerobic bacteria that include a number of human pathogens (Salmonella, E. coli, Shigella, Yersinia) and also a large number of spoilage organisms. These bacteria are widespread in nature in soil, on plant surfaces and in digestive tracts of animals and are therefore present in many foods. Erwinia carotovora is one of the most important bacteria causing soft rot of vegetables in the field or stored at ambient temperatures. Biogenic amines are produced in meat and fish by several members of this group while others produce off-odors or colours in beer (Obesumbacterium), bacon and other cured meats (Proteus, Serratia), cheeses (several genera), cole slaw (Klebsiella), and shell eggs (Proteus, Enterobacter, Serratia). Temperature, salt concentration, and pH are the most important factors determining which, if any, of these microbes spoil foods. Many Gram-negative bacteria, including pseudomonads and enterobacteriaceae, secrete acyl homoserine lactones (AHLs) to regulate the expression of certain genes, such as virulence factors, as a function of cell density. These AHL quorum-sensing signals may regulate proteolytic enzyme production and iron chelation during spoilage

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of some foods although the role of these signals in other spoilage systems is not clear. Other bacteria are associated with spoilage of chilled, high protein foods such as meat, fish, and dairy products. They may not be the predominant spoilage organisms but contribute to the breakdown of food components and may produce off-odors. Most species are aerobic although some grow at lowoxygen levels and may survive vacuum packaging, and one (Brochothrix) is a facultative anaerobe. Some examples include: Acinetobacter and Psychrobacter, which are predominant bacteria on poultry carcasses on the processing line and have been isolated from a variety of spoiled meat and fish. Acinetobacter grows at a pH as low as 3.3 and has been detected in spoiled soft drinks. These two genera do not produce extracellular lipases, hydrogen sulfide, or trimethylamine (fishy odour) and so are considered to have a low spoilage potential. Alcaligenes is a potential contaminant of dairy products and meat and has been isolated from rancid butter and milk with an off-odour. These bacteria occur naturally in the digestive tract of some animals and also in soil and water. Flavobacterium is found widely in the environment and in chilled foods, particularly dairy products, fish, and meat. It uses both lipases and proteases to produce disagreeable odors in butter, margarine, cheese, cream, and other products with dairy ingredients. Moraxella and Photobacterium are important constituents of the microflora on the surface of fish. Photobacterium can grow and produce trimethylamine in ice-stored, vacuum-packaged fish. Brochothrix has been isolated from meat, fish, dairy products and frozen vegetables. During spoilage, it produces odors described as sour, musty, and sweaty.

3.3 HOW FOOD SPOILS

Food spoilage and deterioration is no accident. It is a naturally occurring process. To understand how to maintain the quality of food and prevent spoilage, we need to know what can cause it. Factors that affect food spoilage include: • Microorganisms • Enzymes • Air • Light • Insects, Rodents, Parasites and Other Creatures • Physical Damage • Temperature • Time 3.3.1 MICROORGANISMS Many types of microorganisms can cause food problems. The microorganisms that can cause food-borne illness are called pathogenic microorganisms. These microorganisms grow best at room temperatures (60-90°F), but most do not grow well at refrigerator or freezer temperatures. Pathogenic microorganisms may grow in

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foods without any noticeable change in odour, appearance or taste. Spoilage microorganisms, including some kinds of bacteria, yeasts and molds, can grow well at temperatures as low as 40°F. When spoilage microorganisms are present, the food usually looks and/or smells awful. Read more about pathogenic microorganisms under Food Poisoning/Foodborne Illness? 3.3.2 ENZYMES Enzymes, substances naturally present in food, are responsible for the ripening process in fruits and vegetables. Enzymes are responsible for texture, colour and flavour changes. For example, as a banana turns from green to yellow to brown, not only does the colour change, but there is also a change in the fruit’ss texture. Unblanched, frozen corn-onthe-cob may taste like the cob over time. This is the result of enzyme action. 3.3.3 AIR Oxidation, a chemical process that produces undesirable changes in colour, flavour and nutrient content, results when air reacts with food components. When fats in foods become rancid, oxidation is responsible. Discolouration of light-coloured fruits can be reduced by using an antioxidant, such as ascorbic acid or citric acid, before freezing. Vapor-proof packaging that keeps air out helps reduce oxidation problems. 3.3.4 LIGHT Light exposure could result in colour and vitamin loss. Light also may be responsible for the oxidation of fats. 3.3.5 INSECTS, RODENTS, PARASITES AND OTHER CREATURES These creatures require food to survive and damage food, making it more vulnerable to further deterioration. 3.3.6 PHYSICAL DAMAGE Bruises and cracks on raw produce leave areas where microorganisms easily may grow. Improperly packaged foods, dented cans and broken packages provide places for microorganisms, air, light and creatures to enter. Gentle handling of food items will help maintain food quality and safety longer. 3.3.7 TEMPERATURE Temperature affects storage time, and food deteriorates faster at higher temperatures. Recommended temperatures for storage areas are: • Cupboard/Pantry 50-70°F • Refrigerator 34-40°F • Freezer 0°F or below

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Microorganisms, both spoilage and pathogenic, grow rapidly at room temperature. To slow microbial growth, the enzymatic and oxidation processes, store foods at lower temperatures. 3.3.8 TIME Microorganisms need time to grow and multiply. Other reactions, such as oxidation and enzyme action, also require time to develop. Purchase reasonable quantities, especially of perishable foods, to help avoid long-term storage.

3.4 TYPES OF FOOD SPOILAGE 3.4.1 MICROBIAL SPOILAGE Microbial spoilage is caused by microorganisms like fungi (moulds, yeasts) and bacteria. They spoil food by growing in it and producing substances that change the colour, texture and odour of the food. Eventually the food will be unfit for human consumption. When food is covered with a furry growth and becomes soft and smells bad, the spoilage is caused by the growth of moulds and yeasts. Microbial spoilage by moulds and yeasts includes souring of milk, growth of mould on bread and rotting of fruit and vegetables. These organisms are rarely harmful to humans, but bacterial contamination is often more dangerous because the food does not always look bad, even if it is severely infected. When microorganisms get access to food, they utilise the nutrients found in it and their numbers rapidly increase. They change the food’s flavour and synthesise new compounds that can be harmful to humans. Food spoilage directly affects the colour, taste, odour and consistency or texture of food, and it may become dangerous to eat. The presence of a bad odour or smell coming from food is an indication that it may be unsafe. But remember that not all unsafe food smells bad. • What is the difference between food contamination and food spoilage? • Food contamination is when food is contaminated with microorganisms or substances and eating it could result in foodborne disease. Food spoilage is any undesired change in the natural colour, taste or texture of food items that makes it unfit for consumption because it has lost its quality and nutritional value. The term contact spoilage is used when microbial spoilage is the result of direct contact or touching between the food and any contaminated or unclean surface such as shelves, food preparation boards or unwashed hands. It also includes food-to-food contact, for example between cooked meat and raw meat or between rotting fruit and sound fruit. 3.4.2 PHYSICAL SPOILAGE Physical spoilage is due to physical damage to food during harvesting, processing or distribution. The damage increases the chance of chemical or microbial spoilage and

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contamination because the protective outer layer of the food is bruised or broken and microorganisms can enter the foodstuff more easily. For example you may have noticed that when an apple skin is damaged, the apple rots more quickly. 3.4.3 CHEMICAL SPOILAGE Chemical reactions in food are responsible for changes in the colour and flavour of foods during processing and storage. Foods are of best quality when they are fresh, but after fruits and vegetables are harvested, or animals are slaughtered, chemical changes begin automatically within the foods and lead to deterioration in quality. Fats break down and become rancid (smell bad), and naturally-occurring enzymes promote major chemical changes in foods as they age. 3.4.3.1 Enzymic Spoilage (Autolysis) Every living organism uses specialised proteins called enzymes to drive the chemical reactions in its cells. After death, enzymes play a role in the decomposition of onceliving tissue, in a process called autolysis (self-destruction) or enzymic spoilage. For example, some enzymes in a tomato help it to ripen, but other enzymes cause it to decay (Figure 8.8). Once enzymic spoilage is under way, it produces damage to the tomato skin, so moulds can begin to can attack it as well, speeding the process of decay. 3.4.3.2 Enzymic Browning When the cells of fruits and vegetables such as apples, potatoes, bananas and avocado are cut and exposed to the air, enzymes present in the cells bring about a chemical reaction in which colourless compounds are converted into brown-coloured compounds. This is called enzymic browning. If the food is cooked very soon after cutting, the enzymes are destroyed by heat and the browning does not occur. For example, apples are prone to discolouration if cut open when raw, but when cooked they do not go brown. 3.4.4 APPEARANCE OF SPOILED FOOD Spoiled food is generally more a problem of appearance than a problem of disease causing. In food spoilage, the changes in appearance or texture of the food, such as rottenness, softness and change in colour, taste or odour are usually obvious, whereas in contaminated food such characteristics may not be noticed. A large majority of the microorganisms responsible for food spoilage are not pathogenic to humans. However, you should advise people in your community that they should not eat food that is spoiled because it is not nutritious and may make them sick (cause vomiting).

3.5 SPOILAGE OF FRUITS AND VEGETABLES

The main sources of microorganisms in vegetables are soil, water, air, and other environmental sources, and can include some plant pathogens. Fresh vegetables are

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fairly rich in carbohydrates (5% or more), low in proteins (about 1 to 2%), and, except for tomatoes, have high pH. Microorganisms grow more rapidly in damaged or cut vegetables. The presence of air, high humidity, and higher temperature during storage increases the chances of spoilage. The common spoilage defects are caused by molds belonging to genera Penicillium, Phytophthora, Alternaria, Botrytis, and Aspergillus. Among the bacterial genera, species from Pseudomonas, Erwinia, Bacillus, and Clostridium are important. Microbial vegetable spoilage is generally described by the common term rot, along with the changes in the appearance, such as black rot, gray rot, pink rot, soft rot, stem-end rot. Vegetables are another tempting source of nutrients for spoilage organisms because of their near neutral pH and high water activity. Although vegetables are exposed to a multitude of soil microbes, not all of these can attack plants and some spoilage microbes are not common in soil, for example, lactic acid bacteria. Most spoilage losses are not due to microorganisms that cause plant diseases but rather to bacteria and molds that take advantage of mechanical and chilling damage to plant surfaces. Some microbes are found in only a few types of vegetables while others are widespread. Erwinia carotovora is the most common spoilage bacterium and has been detected in virtually every kind of vegetable. It can even grow at refrigeration temperatures. Bacterial spoilage first causes softening of tissues as pectins are degraded and the whole vegetable may eventually degenerate into a slimy mass. Starches and sugars are metabolized next and unpleasant odors and flavours develop along with lactic acid and ethanol. Besides E. carotovora, several Pseudomonas spp. and lactic acid bacteria are important spoilage bacteria. Molds belonging to several genera, including Rhizopus, Alternaria and Botrytis, cause a number of vegetable rots described by their colour, texture, or acidic products. The higher moisture content of vegetables as compared to grains allows different fungi to proliferate, but some species of Aspergillus attack onions. Intact, healthy fruits have many microbes on their surfaces but can usually inhibit their growth until after harvest. Ripening weakens cell walls and decreases the amounts of antifungal chemicals in fruits, and physical damage during harvesting causes breaks in outer protective layers of fruits that spoilage organisms can exploit. Molds are tolerant of acidic conditions and low water activity and are involved in spoilage of citrus fruits, apples, pears, and other fruits. Penicillium, Botrytis, and Rhizopus are frequently isolated from spoiled fruits. Yeasts and some bacteria, including Erwinia and Xanthomonas, can also spoil some fruits and these may particularly be a problem for fresh cut packaged fruits. Fruits juices generally have relatively high levels of sugar and a low pH and this favours growth of yeasts, molds and some acid-tolerant bacteria. Spoilage may be manifested as surface pellicles or fibrous mats of molds, cloudiness, and off-flavours. Lack of oxygen in bottled and canned drinks limits mold growth. Saccharomyces and Zygosaccharomyces are resistant to thermal processing and are found in some spoiled

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juices. Alicyclobacillus spp., an acidophilic and thermophilic spore-forming bacteria, has emerged as an important spoilage microbe, causing a smoky taint and other offflavours in pasteurized juices Propionibacterium cyclohexanicum, an acidtolerant nonsporeforming bacterium also survives heating and grows in a variety of fruit juices. Lactic acid bacteria can spoil orange and tomato juices, and some pseudomonads and enterobacteriaceae also spoil juices. These bacteria are not as heat tolerant but may be post-pasteurization contaminants colonization by many, but not all, microbes and are the most important first step in delaying the spoilage process. Microbes require certain conditions for growth, and therefore management of the environment of foods can change these factors and delay spoilage: Many, but not all, microbes grow slowly or not at all at low temperatures, and refrigeration can prolong the lag phase and decrease growth rate of microbes. Many microbes require a high water activity and therefore keeping foods such as grains and cereal products dry will help to preserve them. Some microbes require oxygen, others are killed by oxygen, and still others are facultative. Managing the atmosphere during storage in packaging can retard or prevent the growth of some microbes. Several types of modified atmosphere packaging (MAP) have been developed to retard growth of pathogenic and spoilage organisms. However, microbes are endlessly innovative and eventually seem to circumvent the barriers set against them. Therefore further strategies and multiple hurdles are utilized to extend shelf life. These procedures must be assessed for compatibility with different foods so that there are no significant organoleptic changes in the foods caused by the treatment or preservative. These methods for food preservation will not be covered in depth here.

3.6 SPOILAGE OF DAIRY PRODUCTS

Milk is an excellent medium for growth for a variety of bacteria. Spoilage bacteria may originate on the farm from the environment or milking equipment or in processing plants from equipment, employees, or the air. LAB are usually the predominant microbes in raw milk and proliferate if milk is not cooled adequately. When populations reach about 106 cfu/ml, off-flavours develop in milk due to production of lactic acid and other compounds. Refrigeration suppresses growth of LAB and within one day psychrophilic bacteria (Pseudomonas, Enterobacter, Alcaligenes and some sporeformers) grow and can eventually produce rancid odors through the action of lipases and bitter peptides from protease action. Pasteurization kills the psychrophiles and mesophilic bacteria (LAB), but heat-tolerant species (Alcaligenes, Microbacterium, and the sporeformers Bacillus and Clostridium) survive and may later cause spoilage in milk or other dairy products. Immediately following pasteurization, bacterial counts are usually 10 cm. Brakes and locking devices should comply with the hygienic requirements mentioned above. Preference should be given to single-wheeled castors because dual-wheeled castors are more sensitive to contamination, and are more difficult to inspect and to clean. Castor wheels should be constructed so as to have no concave surfaces facing the horn assembly except that part which joins the hub. The included angles between all vertical and horizontal surfaces shall have a radius of not less than 6mm. Wheels should have solid webs, smoothsided, without ridges or crevices, and their tread-face should be smooth and flat. Rubber wheeled castors should have a tyre, from which tread and shoulder are free of grooves, lugs, voids, sipes, dimples, indentations, carves, etc. wherein foreign matter can penetrate the tyre. If bolted, axle bolt ends should be flush and should not extend more than twoand-a-half exposed threads beyond the retaining nut. Excess threads should be cut off and covered with a “dome” type nut. The use on the axle of cotter pins or castellated nuts to keep the wheel attached to the horn assembly, is not acceptable. Two PTFE washers (combination seals) can be fitted, one either side of the wheel, to prevent direct contact (e.g. metal -to-metal contact) between the wheel and the castor body. Although it is expected that the life of these washers should almost be as long as that of the wheel, these washers can become worn, and then must be replaced immediately. In general, washers (retaining washer under a nut) should not be used between the horn of the castor and the axle retaining nut, because there they are more exposed to impact from the outside. Roller or ball bearings should be used. Roller can carry heavier loads, while ball bearings wheels roll more easily but carry lighter loads. All bearing arrangements must ensure that no crevices or dead areas are present which could adversely affect cleanability and/or functional life. If no self -lubricating bearings (stainless steel with PTFE bushing) are used, they should be lubricated every six months. In corrosive environments, lubrication of bearings should occur once a month. In the food industry where the lubricant is washed away by daily cleaning, lubrication is sometimes required after each washing. Bearings in castors (wheels and swivel horns) should preferably be of the sealed type. These seals used to contain the lubricant oil or the grease in the bearings will wear, ultimately allowing leakage. Their integrity must be regularly checked and they should be replaced at defined maintenance intervals. If “open” ball-race bearings

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are used, they must be cleanable and, when required, capable of being disinfected and re-packed with food grade grease as necessary. 5.6.6 BELT CONVEYOR Conveyor frame should have an open structure (Fig. 5.31) with a minimum of hidden areas/surfaces. But guards are required in places where a drive station, a pulley, rollers or the conveyor belt may cause injury. The guards, however, should be easily to dismount to allow for complete cleaning. Solid cross members as structural members are preferred over hollow section members, although completely sealed hollow section members are still more preferable over open profile angle or channel sections, to minimize horizontal ledges and crevices. Hollow sections should be sealed by welding. Conveying surfaces shall be supported by a minimum amount of carrying surface or bed as required (Fig. 5.32b). The use of solid plate that expands the whole top surface of the conveyor table to provide support to a belt is likely to increase contamination problems, and cause excessive wear of the belt (Fig. 5.32a). Non-removable bearing surfaces for belts cannot be cleaned easily. Rollers shall be used where practical, or line supports that are easily removable for cleaning. The conveyor belt should have minimal debris retention, and running under turned over section of side cladding (overhanging belt edges) is not allowed because the whole surface of the belt cannot be cleaned, and the belt cannot be lifted up to allow cleaning and inspection of internal surfaces and support members. But also pivoted covers can’t be cleaned easily. The use of fixed hinges is not recommended because of the great difficulty of removing debris and microbial slime from between the hinge segments (Fig. 5.32a). Side guides used to contain product should be capable of being removed. But removable guides also may cause problems because of the possibility of the fastening system working loose. The conveyor frame must be designed so that the sides of the belt are turned up to form an integral guide to the belt. Besides this guide cladding can be made removable allowing for effective cleaning (Fig. 5.32b).

Fig. 5.31

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Fig. 5.32

The drive motor of the belt conveyor should not be positioned over the product flow, as this may result in contamination of the product by lubricants discharged from the drive system. Otherwise, an adequately sized drip tray should be fitted. However, motors should be rather located below the line of the product flow because the exposed motor may have a fan that will blow dust and dust-borne microbes around the place. The motor, gears and the chain must be covered to avoid any contamination of food product (e.g., enclosure in a hygienically designed and hermetically sealed housing). However, a chain guard (essential from an occupational safety point of view), when open, may provide a place where product may accumulate, allowing microbes to multiply to large numbers and so posing a contamination risk for the food product on the belt (Fig. 5.33).

Fig. 5.33

Fig. 5.34

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Also notice that drive motors installed below food products are quickly splashed and difficult to keep clean. The motor is also often of a type that cannot be washed with a high pressure hose using water and cleaning agents. In that case, installed below the line of the product flow, the gears and motors of belt drives must be covered. Alternatively, cleanable and sealed motors (wash down or easy clean motors) which do not require ventilation or housings, can be used. Where needed, the motor, gears and the chain should be enclosed in a hygienically designed enclosure or hermetically sealed housing (Fig. 5.34a). IP55/54/67 motors can be easily cleaned and drained of water around the motor, if they are provided with enough air space for cleaning and disinfection, maintenance and repair. Where possible, use drum motors (motorized pulleys) (Fig. 5.34b) that are fully closed, non-ventilated, conveyor belt drives where motor and gearwheels are at the inside, submerged in an bath of food grade lubricant, providing at the same time lubrication and cooling. Drum motors make gears and chains redundant. The design of rollers, pulleys and sprockets shall be free of end recesses and shall be closed if hollow. A welded construction should be preferred to a sealed design (Fig. 5.35).

Fig. 5.35

Embedded reinforcements, as well as fabric backing materials in conveyor belts, must be covered to avoid contact with the product. Cut edges of belts which incorporate reinforcing materials must be sealed to prevent penetration by wicking (capillary action) of liquids into the interior (Fig. 5.36).

Fig. 5.36

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5.6.7 COVERS AND GUARDS It is difficult to obtain motors, gearboxes, etc. that meet the recommendation of EN 1672-2. Protecting any of these items by means of covers or guards is recommended. These guards must also protect the food product from contact with drive parts such as lubricated chains, sprocket wheels, etc. The requirement of guarding machinery to ensure safety in operation may easily conflict with hygiene requirements unless considerable care is taken in its design, construction, installation and maintenance. However, these housings or guards should be removable to provide access for cleaning. From a hygienic and safety point of view, totally removable covers, guards or cladding rather should be avoided. They may not be put back, creating a hazard for the operators in the environment of the process equipment and exposing the food product at risk. Covers and guards also may become damaged during removal. Bars, perforated/punched sheet and weld mesh (Fig. 5.37) stainless steel guards with an open area of 40-50% give good protection from moving equipment parts, and permit access for cleaning and disinfection by spray nozzles or hosing down procedures. For good drainability, covers should always have an angle and should be free of panel joints.

Fig. 5.37 Example of a hygienically designed guard (courtesy of P.T. Group)

Where possible, hinged covers and guards that pivot outboard should be used. But use as few hinges as possible, and use hinges with the least number of parts. In view of cleaning and disinfection, continuous and piano hinges are not allowed. Block or pin hinges are a possible option, but should have removable hinge pins or be lift-off. Finally, the exterior of enclosures is more easily to clean if internal hinges are used.

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Fig. 5.38

5.6.8 MAINTENANCE ENCLOSURES Maintenance enclosures (e.g. electric control panels, junction boxes, pneumatic/ hydraulic enclosures) must be designed, constructed, and maintainable to ensure that the product, water, or product liquid does not penetrate into, or accumulate in or on the enclosure. The cabinet and operator panel are mounted where they will be least exposed to splashes. Electrical control cabinets mounted on the exterior of the equipment shall be watertight and sealed to the supporting member with food standard silicon seal, or spaced sufficiently away from the member to permit cleaning of all surfaces. A minimum of 20 mm between the control and supporting member shall be provided. Electrical enclosures can also be sealed to a wall (with food standard silicone seal), or shall be spaced away at least 30 mm or at a distance equal to 1/5 of the shortest dimension of the electrical enclosure parallel to that wall. The distance between the cabinet base and the floor should be no less than 0.3 m. Horizontal surfaces should be minimised or avoided, by installing a top roof with a minimum 30° inclination towards the front to allow water to run off and prevent that tools are placed on the top. The front edge of the inclining cabinet top should reach beyond the front door and the seal (Fig. 5.38). To prevent condensate dripping from the field box into the product, field boxes should not be placed in or above the contact area. Furthermore, field boxes should be located such that easy access for maintenance and cleaning is practicable. All connections (e.g. cable ladders or wire trays, trunking, conduit, cable, etc.) to cabinets or field boxes should be made via the bottom side of the cabinet. Connections of cables and wires to housings must be sealed.

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The control and indicator devices must be constructed of durable and mechanical stable (unbreakable, resistant to steam, moisture and the actions of cleaning and sanitizing agents, abrasion and corrosion resistant) material. Commonly used food grade plastics for the construction of control devices and indicator lights are Polyamide (PA), Polycarbonate (PC), Polyoxymethylene (POM), Silicone and Acrylonitrile butadiene Styrene (ABS). Control devices and indicator lights in contact with food should be shaped such as to avoid the accumulation of dirt and bacteria, and to facilitate cleaning (Fig. 5.39). The device heads must have smooth and crevice free surfaces that are easy to clean. Device head to front panel transitions must be smooth, without corners and edges. Push buttons, when touched, should not penetrate deeply in the front panel far beyond an (protruding) frame edge surrounding the button. Connections must be conceived in such a way, that protruding parts, strips and concealed corners are restricted to a minimum. The connections of inside surfaces must be made with curves of sufficient diameter. Seals should fill the gaps between the fixed and moving device parts, to avoid the ingress of product residues, lubricants and organic materials. A perfect, hermetic seal is also required to prevent the ingress of moisture, dust and dirt within the control panel. An IP67 or IP67K ingress protection rating for control panel enclosures is highly recommended. The preferred installation positions for control and indicator devices are declining and vertical surfaces, such that fluids (splashed food and cleaning solutions) are able to flow from the control panel, at least in cleaning position. Adequate space should be provided between control and indicator devices for easy cleaning. More hygienic alternatives to control panels with push buttons and selection switches are membrane panels with a 2% inclination or touch screen displays.

Fig. 5.39 Control panel with hygienic control and indicator devices.

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5.7 HYGIENIC DESIGN CLOSED EQUIPMENT FOR PROCESSING OF LIQUID FOOD 5.7.1 PROCESS AND UTILITY LINES 5.7.1.1 Hygienic Design of Process and Utility Lines To avoid the formation of standing “pools” of liquid that can support the growth of microorganisms, process and utility piping runs should be sloped to at least 3% in the direction of flow and should be properly supported to prevent sagging (Fig. 5.40 and Fig. 5.41).

Fig. 5.40

Fig. 5.41 Non-drainable pipe (courtesy of Knuth Lorenzen, personal communication)

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Blanked-off tees should be avoided where possible as they constitute a potential hazard. A dead space, being an area outside the product flow, where liquid or gas can become stagnant and where water is not exchanged during flushing, is formed. An air pocket may be present if the branch of a blanked-off tee is pointing vertically upwards (Fig. 5.42a). Hence it will prevent liquids (cleaning solutions, disinfectant solutions or hot water) from reaching all surfaces to be treated, with as result that cleaning-in-place and decontamination processes will be unsatisfactory. Drain points pointing downwards act as a dead leg (Fig. 5.42b) are not acceptable because they provide an area of entrapment which may not be reached by cleaning or sterilizing procedures, and hence they lead to contamination of the product. During a hot water treatment, the hot water also will stagnate in the downwards pointing pocket, so that the temperature of the surfaces in the dead area may be lower than required as the consequence of heat loss. A downwards pointing dead area also will collect condensate during steam sterilisation (Fig. 5.42c), with as result that again the temperature of the surfaces in the dead area may be lower than required.

Fig. 5.42

The direction of the flow of food product has a significant influence on the residence time in the dead leg. When the food product flows in the direction as indicated in Fig. 5.43a, b &c, part of the product will stand still in the dead leg, especially if the length or depth of the T-section is too long. If the length of the T-section is equivalent to the diameter of the main pipe, a flow velocity of 2 m/s in the main pipe already results in a reduced velocity of 0.3 m/s in the T-section. This decrease in flow velocity provides a relatively stable pocket or ‘dead leg’ in which product residues can accumulate and microorganisms begin to multiply. Long T-sections outside of the main flow of cleaning solutions are also very difficult to clean. During cleaning there is much less transfer of thermal (heat), chemical (detergent and disinfectant chemicals) and mechanical energy (action of turbulent flow) to the food residues in the zones and T -sections which are outside the main flow of cleaning liquids than to the soil in the main flow. Notice that flow away from the ‘dead’ leg (Fig. 5.43a & c) gives rise to more contamination problems and worse cleaning, as velocities in these dead legs are even much lower. A properly designed food processing line should not have unnecessary dead legs, and where they can’t be excluded, they should be in the correct position for the selected cleaning and decontamination process and should be as short as possible. For pipe

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diameters of 25 mm or larger, T-sections should have a depth/length of preferably under 28 mm, while for smaller pipe diameters this length should be smaller than the diameter. Blanked-off tees should be positioned such that they are a few degrees above the horizontal. The ‘dead-leg’ will then be drainable but not necessarily cleanable even if made as short as possible. If a sensor must be installed in a process line, it should be installed in a bend on a shortened tee in a position that the flow of cleaning fluid should be directed into the tee (Fig. 5.43e and f). Where an angle valve is installed in the process piping circuit, this valve also must be mounted in a shortened tee so that no or a minimum of annular space above the side branch is formed. Again the flow of cleaning solution must be directed into the tee. For most liquids, the dead leg should be positioned as shown in Fig. 5.43e, d, f. Especially the configuration in Fig. 5.43f is quite acceptable, because the flow directed into the short “dead leg” provides sufficiently high velocities for proper cleaning. If the dead leg is very short, configuration Fig. 5.43d is acceptable, although flow across a ‘dead leg’ results in much lower velocities within it and thus only provides moderate cleaning. Configuration Fig. 5.43e may not be suitable, if products contain any particulate matter, which may accumulate in the dead leg. In all cases, the cleaning procedure must take the presence of the dead leg into account.

Fig. 5.43

Flow diversion should not be done in a way that would cause part of the product to stand still in a dead leg. The two-valve system for flow diversion (Fig. 5.44a) creates a dead leg towards the closed valve. The correct type of valve is shown in Fig. 5.44b.

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Fig. 5.44

For horizontal piping, eccentric reducers should be used instead of concentric reducers, because the latter provides a dead spot where condensate and dirt may collect (Fig. 5.45).

Fig. 5.45

5.7.1.2 Hygienic Integration of Process and Utility Piping in Food Factories Welding of attachments on food processing support piping is not recommended. They can cause stress on the pipe and the part of the supporting anchoring structure. All hangers and supports have to be designed in such a way that they either move together with the pipe (roll or slide) or that they can swing without exposing any stress either on the pipe or on the part of the supporting anchoring structure. All process and utility piping should be grouped together in pipe trains whenever possible. All these process and utility piping should preferably be positioned in a way that all exterior surfaces are readily accessible, to allow cleaning from all sides. The points of use should also be grouped, in an attempt to minimize individual ceiling

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drops. Vertical entrance of piping into the equipment is more hygienic than horizontal piping runs. Running of process and utility piping over open equipment in food preparation areas cannot be accepted, and nesting of ductwork should be avoided. 5.7.2 HOSES The use of hoses is less recommended, because failure of hoses can occur due to overstretching, kinking, rough handling, mechanical impact, ageing, fatigue, abrasion, corrosive atmospheres, etc., and because the chance that leakage of liquid occurs is much higher than when fixed piping is used. Therefore, hoses need regularly inspection for damage, deterioration and cleanliness. They should be cleaned and maintained in good mechanical condition. Braided (woven wire or fabric) covers on hoses should not be used.

Fig. 5.46 Incorrect and correct installation of hoses on fixed pipes

Hoses out of service shall be pendant without touching the floor, and may never hang over open process equipment. Notice that hoses attached to stainless steel pipes should be clamped at the very end of the pipe to minimize the amount of dead space between the clamped portion and the end of the pipe (Fig. 5.46). Hoses should not exceed 3 meters in length. When not in use, the ends of the hoses should be covered or capped to maintain proper hygienic conditions. 5.7.3. PIPE JOINTS 5.7.3.1 Welded Pipe Joints It is strongly recommended that the number of joints, whether welded or detachable, is minimised. Cold bending of pipes is highly preferable to the use of prefabricated bends which have to be installed using joints. Although more hygienic, this is still true for welded joints (Fig. 5.47) as they also remain the weaker places in a process system. Welding is the preferred method of joining, provided that it is done correctly. Stainless steel sanitary tubing joints should be made by automatically orbital welding (Fig. 5.47) where possible and hand welding in those places that are difficult to access. However, those welds that are difficult to access should wherever possible, be completed in the workshop prior to installation on the plant. The applied materials should be easily

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weldable, and higher alloyed filler metal in comparison to the welded material should be used to improve the corrosion resistance. Piping with the correct interior diameters should be applied because any mismatch in diameters or thickness may result in misalignment introducing a step in the wall or bore. If the diameters of the pipes to be joined are not the same, then the smaller pipe should be expanded to match the larger. Misalignment also can be due to incorrect fitting up (missed coincidence between the axes of the two coupled components) prior to welding. Alignment and clamping tools are available to ensure accurate alignment. Misalignment tolerance must be limited to less than 20% of the wall thickness.

Fig. 5.47 Stainless steel sanitary tubing joints should be made by automatically orbital welding where possible

For proper welding, the parts to be welded should be adequately prepared. Cutting should be done with a mechanical mill or saw to ensure that the cut face is exactly at right angles to the longitudinal axis of the pipe. Any burrs must be removed with either a file or emery paper. Care must be taken not to remove the corner edges of the pipe, as this can give rise to problems with fusion of the root of the weld. The pipe surface 25 mm either side of the weld should be roughened up with a stainless steel wire brush, or emery paper. Then both pipe ends and roughened surface area should be degreased with a solvent and cleaned from contaminants. Any organic substances remaining on the metal surface are vaporised during the welding process and form bubbles (porosity) in the weld metal, that may trap product. After two deburred pipe ends are aligned and butted together to a gap of less than 0.25 mm between both pipe faces, a butt weld joint is made by fusing together the two stainless steel edges with the aid of filler material. If the gap during the joint preparation is too wide, a crack running along the weld metal itself may be the result (centre line cracking). Full penetration welds, should be used whenever possible to avoid pockets

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where volumes of gas or contaminants can be trapped. Single pass welds should be utilized instead of multi-pass welds to avoid trapped volumes. The weld metal should exactly fill the joint and remain flush with the surface. Under-penetration leaves a crevice at the joint, while excessive over-penetration can give rise to hold up of product in pipework once taken into service. The weld metal in the joint must be fully fused to the parent, otherwise a crevice will form at the interface between weld and plate. Weld zones should be continuous, smooth, and flush with the parent metal. Welding always should occur with sufficient weld seam protection, because insufficient inert gas shielding or no internal purge will result in roughened welds of lower corrosion resistance, that are prone to increased adhesion of soiling and that are difficult to clean. Typically, where inert gas shielding was inadequate, significant discolouration or carbonisation in the heat affected zone is observed. Weld slag and debris generated within the pipe must be removed from the inside and outside of the weld by proper maintenance and cleaning practice with an alkaline detergent solution prior to the start of the production process. This is followed by rinsing with water of good microbiological quality, usually chlorinated water to 2 ppm available chlorine maximum. After draining the access points should be covered and sealed. In some circumstances there is an additional requirement to passivate the weld area on the product contact side. The welds may be mechanically polished (outside) or electropolished (inside and outside), but air leakage should be monitored after the polishing procedure. Weld seams finally should be visually inspected on any discolouration and surface breaking defects, usually by endoscopy and aided by dye penetrant tests that highlight these defects. Inspection personnel should be trained and act with caution to avoid internal surface damages while handling endoscopic tools. 5.7.3.2 Detachable Pipe Joints Pipe work may be designed for rapid regular dismantling to permit cleaning, or the plant may be designed for cleaning-in-place (CIP) or sterilizing-in-place (SIP) without dismantling the plant. In such equipment it is important to avoid crevices and gaps where product residues can accumulate and potentially begin to decompose. Therefore, from a hygienic point of view, the use of threaded piping is not recommended, because they provide crevices and areas where bacteria can adhere and proliferate. To make detachable joints, also the use of conventional O-ring grooves is not recommended, because these groove designs leave a considerable free space in the groove. Other hygienic requirements for detachable joints include coaxial alignment of the two mating bores, axial stop for controlled compression of the seal, room for thermal expansion of the seal, and avoidance of sharp edges such that seals are not damaged. Where there are depressions and steps of more than 0.2 mm in the pipe work, the flow of cleaning fluid may not thoroughly wash the surface and proper drainability of the piping will be hampered. Hence, when making bolted flange fittings, a lot of care should be taken to

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avoid offsets, gaps, penetrations, and voids. A further aspect to be considered is that the seal material must be compatible with both the system product and also the cleaning fluids which may be at a much higher temperature. A number of specific pipe couplings and also seal arrangements have been developed for hygienic applications. Some types are covered by national, international or internal company standards, but many of these have been in use for some considerable time and are not considered to be compatible with current requirements in some areas of the food and drink industry. 5.7.4 HYGIENIC DESIGN OF PUMPS 5.7.4.1 Hygienic Design of Centrifugal Pumps Whilst it is often convenient for the arrangement of pipework to orientate the casing of a centrifugal pump so that the outlet port is pointing vertically up, this will result in the pump casing retaining liquid up to the level of the inlet port. The pump casing is drainable through the outlet port if the pump’s outlet is arranged to point horizontally at the bottom, or the pump casing can be made drainable through its suction port if installed in vertical execution (Fig. 5.48).

Fig. 5.48 The centrifugal pump is installed in vertical execution, and hence fully drainable through its suction port

5.7.4.2 Hygienic Design of Rotary Lobe Pumps Rotary lobe pumps having unhygienic design features can only be cleaned effectively after dismantling. To avoid any introduction of contaminants into food product and to allow for CIP without dismantling, rotary lobe pumps should be hygienically designed.

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Metal-to-metal joints should be eliminated by hygienic application of O-rings; O-ring groove design should be improved and O-rings should be positioned more appropriately, or alternatively gaskets having controlled compression should be used; sharp corners must be rounded to a minimum radius of 3 mm; the length of the annular space within the mechanical seals should be reduced by changing the design of these mechanical seals (e.g., the elements of the mechanical seal should be reversed and the radial distance increased); any exposed threads (e.g., threads of the rotor shafts, Fig. 5.49a) should be covered by crevice-free domed retainer nuts; or even better, the rotors and shafts should be designed as an integral construction so that rotor retaining nuts and associated metalto-metal joints can be eliminated, and so that the inside of the front cover can be made completely flat and free of space holes for rotor retainers. Some types of rotary lobe pumps are traditionally positioned in such a way that draining is impossible without dismantling but the same type of pumps can also be designed for installation in a drainable position. In example given, the inlet and outlet ports of rotary lobe pumps have been arranged traditionally in the horizontal position as this has again been convenient for connecting the pipework. This results in the retention of liquid in the casing up to the level of the inlet and outlet ports. Now, lobe pumps are available with the ports arranged in the vertical plane (Fig. 5.49b) so that it is possible to drain the casing.

Fig. 5.49

5.7.5 SENSORS AND INSTRUMENTATION Incorrect mounting of sensors in process lines will result in large dead areas which are unacceptable (Fig. 5.50). Instrument branches, that could become a dead leg when not properly installed, should be installed vertically upwards to keep condensates, debris, suspended solid particles, flakes, etc. from collecting in the sensor or from falling into the sensor and the measurement system. However, the length of the dead area must be as short as possible and its cleanability must be demonstrated. For all pipe diameters the length of the upstand should be smaller than its diameter (l d” d).

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Fig. 5.50

It is possible to avoid such dead areas by mounting e.g. the pressure transmitter on a swept tee (Fig. 5.51). However, swept tees must be used with caution, as a swept tee in a horizontal pipeline could hamper draining. Swept tees should be mounted in a vertical pipeline. Dimension ‘l’ must be as short as possible relative to dimension ‘d’, maximum l = d. Alternatively, pressure transmitters with tubular membranes, with the same inner diameter as the adjacent pipelines can be installed in standard spherical valve bodies welded into the piping by means of clamp fittings. The stainless steel diaphragms are sealed by O-rings fitted into grooves such that there is no metal-to-metal joint on the product side (Fig. 5.52). This way of mounting of pressure transmitters provides a deadspace free, flush transition from the process line to the pressure transmitters.

Fig. 5.51

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Fig. 5.52

Temperature measurement is usually based on electronic detection of a change in resistance. The actual temperature sensor elements used integrate either Platinum thin film resistors (Pt100 etc.), or employ other sensing elements with a varying electrical resistance against temperature (NTC or PTC resistors). Also semiconductor devices are common. The temperature sensor element itself is covered by a protective sleeve, a highly-polished, closed tube typically made of stainless steel. Only one surface of the thermowell has fluid contact, the sensor being installed inside. For these temperature sensors, a close thermal and mechanical contact to the liquid to be measured is needed. Therefore, often a paste with high thermal conductivity is used inside thermowells.

Fig. 5.53

Temperature sensors may not be mounted on a too long tee branch because an unacceptable large dead area is then created. Thermowells with flanged process connection (Fig. 425.53) can be integrated into the process, installed by means of clamp fittings in standard spherical valve bodies welded into the piping. The sheath of the probe is welded into one of two blanks which are sealed to the spherical valve body by

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O-rings fitted into grooves such that there is no metal-to-metal joint on the product side. This way of mounting of a temperature sensor provides a dead-space free, flush transition from the process line to the blank containing the thermowell. A surface probe with the inner diameter of its pipe the same as that of the adjacent piping is, from a hygienic point of view, an excellent choice. However, the thermowell can also be directly fitted via an orbital welded pocket (Fig. 5.54). Attention should be given to the quality of the weld, which must be smooth and continuous. Furthermore, to avoid shadow areas, the direction of the flow must be as indicated.

Fig. 5.54

For temperature measurement in tanks and larger vessels, the thermowells can be continuously welded to the tanks with welding balls or welding collars, after which the inner welding seam is polished and passivated after welding. Sensors also can be installed via a hygienic process connection sandwiched (detachable seal joints such as O-rings) into the pipeline (Fig. 5.55). The dimensions of the O-ring and the design of the groove to be used for mounting sensors are critical to achieving controlled compression of the seal. The O-ring needs periodic maintenance with an inspection of the O-ring upon dismantling. Used O-rings should not be re-installed.

Fig. 5.55

5.7.6 HYGIENIC DESIGN OF VALVES Valves are used to change the direction of the flow of product or cleaning solutions (selection of the product routing), to regulate the flow and pressure, to protect a process system against overpressure. The cleanabilty of a valve is largely determined by its

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internal geometry, the way in which the inlet and outlet connections are made, and the seal between the fluid and the external environment. The seals may be under a static load or dynamic with linear or rotary motion. Valves must meet the following hygienic requirements: • Fully drainable, without the need to dismantle; • Resistant to wear and easy to maintain; • Minimum number of seals, positively retained and flush with adjacent surfaces; • Dynamic seals on valve shafts in contact with product must provide an absolute barrier between the product and the environment to prevent microbial recontamination; • Where unavoidable, springs in contact with product should have minimum surface contact area; • Allow rapid visual detection of internal leakage. Hygienic requirements for different types of valves: • Diaphragm valves used as back pressure valve need visual detection of leakage (usually there are leakage holes in the valve bonnet), because damage to the diaphragm can result in product leaking through into the non-product side. Such an event may give rise to contamination, and cleaning and disinfection will become nearly impossible. To avoid premature rupture, it should be replaced at regular intervals depending upon the operating conditions. Diaphragm valves must be installed for full drainability. • Butterfly valves comprise a disc, and further a rubber seal clamped between the halves of the body providing both a seat for the disc to close on and a seal for the disc spindles. If properly designed, they are hygienic low-cost valves, with as main properties: low resistance to flow, and their appropriateness to be automated and cleaned in-place. Butterfly valves with a stream-lined disk free of external ribs are hygienic. However, product containing fibrous material may build up on the leading edge of the disc, and butterfly valves are suitable as long as the seals are not worn. Seals can wear and break down after a period of time due to the frequent opening and closing of the butterfly valve. Product can also migrate along the shafts due to product pressures in the system. Therefore, butterfly valves should preferably disassembled for manual cleaning. If butterfly valves are in use, appropriate cleaning and maintenance schedules must be implemented. • Traditional ball valves are considered as unsuitable for process installations that are cleaned in-place. Due to the presence of crevices in their internal construction, the area between ball, housing and seal face is uncleanable. Food product is transferred in the annular dead space when the valve is operated from its open to its closed position. When the ball valve is then rotated back from its closed to its open position to allow CIP, the food product trapped in the annular space between the sphere and the housing will not be removed by

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cleaning-in-place. Moreover, ball valves may retain condensate in their internal cavities. Often the design incorporates cavity fillers or encapsulating seals to prevent product flow around the exterior of the ball but product still may find its way under the seat surface and become an area for bacterial growth. Ball valves in existing installations must be disassembled completely for manual cleaning. However, the design and construction of a ball valve are such that it is not easily dismantled for cleaning. Certain ball valves with improved design allow for cleaning-in-place, especially in a half open position. For some applications, connections have been made to the housing so that the annular space may be continuously purged with steam throughout production. Plug valves are unsuitable for CIP, because product is carried around the clearance between the plug and the body during the rotation of the plug. Threeway plug cock valves allow 90° changes in flow direction of both food product and cleaning solutions. They have the disadvantage that they neither can be automated or cleaned in-place. However, plug valves can be easily manually cleaned after dismantling, which - due to their simple design – can be done very easily. Pressure relief valves are valves where the valve head is lifted off its seat when the product pressure exceeds that at which the valve has been set. Product then may be discharged to drain through the discharge port. To flush the inside of the valve body and the discharge port during cleaning-in-place, the valve must be opened by moving the lever through 90°. The valve body must be installed in a position so that it is fully drainable to the outlet side, and should be mounted on a short tee to avoid a large ‘dead-leg’ in which product will be retained throughout the production. Check valves with springs, hinges and flappers should be avoided as they quickly become contaminated and could give rise to cleaning problems. When spring loaded check valve are used, the coil springs having product contact surfaces shall have at least 2 mm openings between coils, including the ends when the spring is in a free position. Spring-loaded check valves must be fully disassembled for manual cleaning. The use of ball-type check valves is the preferred practice. Springless floating ball check valves have a streamlined internal design which may reduce the potential for material to clog or hang up. Check-valves must be installed in a position that allows full drainage of the check valve. Tank outlet valves should be installed as close as possible to the product vessel to reduce the dead-leg formed by the stub pipe that connects the bottom valve with the vessel. They may be manually or mechanically operated and cleaned depending upon their design features. Mixproof valves are an essential part of automated processing, not only separating two different products but also preventing product contamination

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from cleaning fluids during mechanical cleaning. The valve uses double seats that can be operated independently, separated by a self-draining opening to the atmosphere between the valve seats. The vent space must also be cleanable and avoid a pressure build-up in case of a leak from a seal. The outlet from the vent line must be visible so leakage can be easily detected. A steam or sterile barrier may also be applied in the atmospheric opening (vent) to prevent ingress of microorganisms. • Linear plug and stem valves may incorporate a lip seal to limit microbial contamination via the reciprocating shaft. This seal is easily cleanable but will not prevent the ingress of microorganisms. A hole is required to detect product leakage when the lip seal wear becomes excessive. Arrangements incorporating an O ring seal are less hygienic because product can enter the clearance around the stem and become trapped in the O-ring groove from which it cannot be removed by in-place cleaning. For aseptic processing applications where ingress of micro-organisms must be prevented, the shaft may be sealed by means of a diaphragm and bellows. In case of the diaphragm type, the diaphragm must be replaced at regular intervals and a leakage hole must be provided that indicates failure of the diaphragm. With respect to the bellows sealed linear plug and stem valve, the bellows will rupture after a period of service and need to be. • replaced at regular intervals. Moreover, if the product contains particulates, there may be a cleaning problem because particulate material may become trapped in the convolutions of the bellows. A steam barrier between the atmospheric and product sides of the valve stem is another method of preventing ingress of microorganisms.

5.8 INSTALLATION OF THE FOOD PROCESSING EQUIPMENT IN THE FOOD FACTORY 5.8.1 CLEARANCE WITH RESPECT TO THE FLOOR, WALLS AND ADJACENT EQUIPMENT There should be enough clearance under the machine to allow for adequate cleaning and inspection to be carried out effectively. With that purpose, the process equipment should be installed as high off the ground as possible. The minimum height should be a function of the depth of the bottom surface above the floor (indicative: 150-300 mm). For large sized equipment, greater distances apply (at least 0.5 m from walls), as it is necessary to be able to walk around such equipment and at least with enough room to facilitate cleaning. If the equipment is sealed against the mounting surface, care must be taken to avoid gaps, cracks or crevices where insects or micro-organisms can remain/ survive after cleaning.

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Installation of large equipment (e.g., freezing equipment, meat curing chambers, etc.) on feet is technically not always possible. An alternative is sealing the equipment onto the factory floor. Proper sealing the perimeter between the equipment and the subfloor must prevent water from accidentally getting into this space. But sealing, especially with silicone, has not always proven to be successful in excluding wet and unhygienic conditions. Equipment must not be mounted beneath tanks or vessels so that maintenance and cleaning are impeded but must be easily accessible. Increased elevation of tanks and vessels facilitates cleaning and maintenance operations beneath them but water and condensation running down their sides may allow microbial growth and certainly must not fall onto exposed product.

Fig. 5.56

5.8.2 RAISED WALKWAYS AND STAIRS Raised walkways or stairs (Fig. 5.56) over exposed product should be avoided because dirt may be transferred from clothing or footwear onto product lines beneath. The use of covers and hygienically designed walkways should be both considered. The decking of platforms and steps (cross-overs on conveyor-systems) should be constructed from solid plates containing a raised anti-slip material as deck. The steps can be given a small inclination for improved drainability. Mesh must be avoided to prevent soil from being transferred into the product. Further fully-welded continuous kick plates should be in place, designed as a onepiece construction. Platforms and stairs should have generous radii in the corners of kick plates, etc., to allow cleaning and disinfection. Handrails should not overhang the walkway and must be attached to the inside of the walkway. Risers of staircases must be enclosed and the steps should be constructed of the same anti-slip material as the deck.

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5.9 HYGIENE PRACTICES DURING MAINTENANCE OPERATIONS IN THE FOOD INDUSTRY 5.9.1 MAINTENANCE AND REPAIR, A NECESSARY EVIL Physical equipment of any field or of any plant and industry are susceptible to failure through breakdown, deterioration in performance owing to wear and tear with time and to obsolescence due to improvement in technologies. Therefore, machinery should be regularly checked with respect to its performance. Equipment maintenance checks should include an assessment of the equipment’s overall condition and integrity (e.g., is it working properly), the sources of physical contaminants (e.g. damaged, lost or worn parts, rust, loose/flaking paint, broken parts such as needles and blades, loose parts on equipment prone to vibration, polymeric deposits, friction, fatigue, chemical reaction, etc.), the microorganism harbourage sites (e.g., worn or frayed hoses, gaskets or belts, porous welds, product contact surfaces). Increase in noise, lubricant consumption, temperature rise or increased leakage is usually the consequence of failure of equipment and its components. Worn parts should be replaced as soon as practical, not only to ensure that production is maintained but also to prevent that debris from worn or broken parts enters the product or contaminates the production line. The operator also must ensure equipment used for critical measurements is calibrated, and uniquely identifiable. It must be used within its design and capacity (e.g. accuracy, calibration range, conditions of use). Items requiring calibration could include thermometers, temperature recorders, scales, test weights, metal detectors, gas analyzers, pressure or heat sensors, chemical assessment equipment, flow meters, etc. 5.9.2 SCHEDULED PREVENTIVE MAINTENANCE Scheduled preventive maintenance should be preferred over inefficient “breakdown” maintenance and repetitive repair. No longer does the maintenance department have the luxury of extended periods of available equipment downtown in order to carry out maintenance. Instead the maintenance function is moving towards a more predictive approach. If the failure characteristics of the equipment are known, predictive maintenance can detect the failure well in advance and appropriate actions can be taken in a planned and organized manner. Predictive maintenance makes use of a group of emerging scientific technologies that can be employed to detect potential failures: vibration analysis, thermal imaging, ultrasonic measurement and oil analysis. The maintenance technicians should be skilled to use these diagnostic tools, and they must have detailed knowledge of the operating characteristics of the equipment to make the correct failure diagnosis. By means of a risk analysis, the manufacturer may define which parts of the system are critical, allowing to define the necessary treatment (to which interval, to which time point, and with which measures). That maintenance

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schedule should be frequently reviewed during the initial operating period of an installation to establish the optimum maintenance frequency. 5.9.3 PROPER A PRIORI DESIGN, INSTALLATION AND WORKING PRACTICES THAT MAY REDUCE THE OCCUR-RENCE OF UNHYGIENIC CONDITIONS DURING MAINTENANCE AND REPAIRS Proper design and installation of the processing equipment and utility services, and common-sense measures create the appropriate conditions to keep up a sanitary process environment during maintenance and repairs: • Equipment should be of such a design that the need for physical entry into the system is minimized. Enough space and clearance should be provided so that all equipment parts and components are readily and easily accessible for inspection, maintenance and troubleshooting. • Mechanical, electrical, pneumatic, hydraulic and electronic components, together with distribution conduits, valves, pumps, pressure reducers, gas cylinders, vacuum sources, compressors, etc. should be relocated to a technical room or technical corridor adjacent to the production room, so that maintenance personnel can access the technical area without special gowning or disruption of the cleanliness of the high hygiene space below. • Use lamps with high light output so that the factory staff can perform inspections of the food processing equipment and the process environment more easily and profoundly, enhancing the detection of grease, leaking oil, failures, maintenance residues, etc. Torches to light dark places with process equipment should be resistant against breakage. • Maintenance managers and supervisors should implement “Maintenance Best Practice”, eliminating the sources of breakdown and contamination that cause downtime, quality holds and lost profits. • Correct maintenance attitudes must help to ensure that the production area and products are kept free from contamination by undesirable microorganisms, filth, debris, or machine parts. Regularly audits should be done to verify if the maintenance staff or contractors have adopted the correct hygienic practices during maintenance operations. 5.9.4 MAINTENANCE AND REPAIR OPERATIONS ACCORDING TO THE PRINCIPLES OF HYGIENIC DESIGN Maintenance and repairs should occur according to the principles of proper hygienic design to ensure that safe food is produced once production is resumed. The following recommendations should be followed: • The construction materials used during maintenance and repair must be compatible with the food product or process aid they contain, and may not introduce contaminants that would present a risk to food safety. Piping and

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components should be constructed out of the same materials to prevent contact corrosion between dissimilar metals. Work in black steel and stainless steel must always be kept separated. Spare parts should be pre-packed in plastic, stored segregated from other non-stainless steel products. The inlet and outlet connections of the product should be fitted with protective caps before being sealed with shrink wrap in order to prevent the ingress of impurities, insects and small animals in pipes and fittings. Prior to use, process equipment and components should be examined for debris, oil, or grease; and if necessary should be cleaned. The body and internal parts must be handled carefully to ensure that the machined surfaces are not damaged. Use as much as possible piping with the same internal and external diameter over the whole factory, in particular to avoid misalignment (missed coincidence between the axes of two coupled pipe components) prior to welding. Re-assemble piping and equipment components using a new seal, and check for leaks and re-tighten as necessary. All fastening devices should be secured firmly. If old insulation containing asbestos has to be removed, all precautions should be taken to avoid the spreading of asbestos fibres in the food processing environment. For insulation work, preference should be given to rigid foam rather than fibrous materials that have already proven to be an excellent harbourage of dust, insects and rodents. Afterwards, the insulation should be covered with properly sealed cladding of appropriate thickness, that resists tear and abrasion. When a new cable has to be installed, it should not be supported from a previously installed cable because a hygienically unacceptable entangled cable bundle may be formed. The cables should be fastened individually in a distance no less than 25 mm from each other to allow for proper cleaning. The use of temporary devices, such as tape, wire, string, etc. should be avoided. If strips are the only option, they should preferably be of a stainless steel type that can be detected by means of a metal detector. Alternatively, a plastic strip of a colour that is not omnipresent in the food product and food factory could be used. Temporary fixes should be replaced in a timely manner by permanent repairs. Always determine the correct installation situation and direction of fluid flow. Install for maximum cleanability and drainability. Calibrated equipment that is non-conforming (i.e. broken, expired calibration period) must be identified as non-conforming, and further recalibrated, repaired or replaced.

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5.9.5 PERSONAL HYGIENE PRACTICES DURING MAINTENANCE OPERATIONS IN THE FOOD INDUSTRY Before the onset of maintenance and repair operations, all maintenance workers shall comply with the requirements for personal hygiene appropriate to the area where maintenance and repairs will be executed: • Both the food manufacturer’s own maintenance staff and contractors should follow the food manufacturer’s guidance with respect to personal safety and hygiene. • It is recommended to force the maintenance staff or contractors to fill out a health questionnaire before allowing that maintenance staff or contractor to enter the food production area. The food manufacturer must restrict access of any person with obvious health problems such as the flu, colds, skin lesions, uncovered sores or wounds, etc. All personnel is in fact responsible for reporting any such condition to their supervisor before beginning or continuing work. • The use of cosmetics, medical substances (ointments, plaster or band-aid for wound healing, safety pins) or other chemicals (suntan products, etc.) on the skin are not allowed. • Eating, drinking, chewing (gum, toothpicks, straws, etc.) and smoking, are not allowed during maintenance operations. • Maintenance staff or contractors are not allowed to enter the food production area with their casual clothes. They should be stored away from the production area. Protective clothing shall be worn, not only to safeguard the person’s casual clothes during the work but also to protect the food product. In order to avoid contamination of work surfaces, maintenance personnel should wear clean coveralls. • Maintenance workers that worked in a less clean area that has high microbiological activities (raw materials) must change their garments prior to entering a high clean area where sensitive food products (e.g., finished products) are produced. Hair nets, headbands, caps, bump hat, hard hats, beard nets or other devices must be worn to control hair lost into the food, onto food surfaces and into packaging. • All piercings, jewelry, watches should be removed. • Hands should be washed thoroughly, including in between fingers, before entering a food processing area and after eating, drinking, smoking or using the restroom. The use of gloves may be advisable. Gloves are to be maintained in a clean, sanitary and intact condition. Gloves used in less hygienic (raw material) side of the plant must not be used in high hygienic risk areas. • Foot wear should be clean. If it is necessary to stand on or over machinery, the process equipment shall be covered to prevent foot wear dirt and debris from contaminating the surface. It is also recommended to cover footwear with overshoes just prior to walking on the process equipment.

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• Maintenance staff or contractors must remove all unsecured objects which could fall into the product, such as pens, pocket notebooks, small screw drivers, pencils behind the ear, non-attached ear plugs, nuts and bolts in shirt pocket, etc. They must be stored in the tool box or the carrier used to bring parts to the work site. 5.9.6 HYGIENE PRACTICES DURING MAINTENANCE OPERATIONS IN THE FOOD INDUSTRY 5.9.6.1 Recommended Hygiene Practices to be taken before the Onset of Maintenance and Repair Operations The following measures and actions will create the appropriate hygienic conditions to execute maintenance and repair without compromising the safety of the food produced with that equipment when production resumes: • Some work such as drilling or welding will inevitably produce debris and dust. Where possible, production operators should remove food processing equipment from the processing room before repairs are made. Coverings such as tarps or plastic sheeting (polyethylene or equivalent film) can be draped over equipment to reduce contamination. • Maintenance could be done in a separate room outside the food processing area. • If entry in process equipment is required, a plastic cover film must be laid down on the bottom of the process equipment. • Where practical, maintenance tools should be dedicated for use in specific areas of their operation to avoid cross contamination. • Tools used for repairs and maintenance must not come in contact with, or compromise the hygienic status of any product or packaging material. The maintenance tools must be free of rust, peeling paint, niches and threads; and without wooden handles or knurling soft rubber grips. They should be noncorrosive, easy to clean and inspect, with smooth finish and hard plastic grips, and with fitted heads for equipment longevity. They must be designed in a way that they can’t damage the process equipment. • The maintenance tools must be clean and used with care so that they cannot be left in the production equipment. • Maintenance equipment and tools may not transfer microorganisms in a hygienic room from its prior use in a less hygienic area. • Ordinary steel wool or steel brushes should never be used on stainless steel surfaces as particles of steel may get embedded in stainless steel surfaces and rust. • Debris from engineering workshops (such as swarf and other unwanted materials) must be prevented from entering processing or support areas. This is especially

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important where engineering workshops have access ways (e.g. doorways) that lead into processing or support areas. This may be achieved by keeping doors closed, the use of swarf mats, boot washes, etc. 5.9.6.2 Recommended Hygiene Practices during Maintenance and Repair The following hygiene practices should be followed during maintenance and repair: • During maintenance operations, light sources used to provide the necessary light for proper maintenance and repair should not be placed above open process equipment, or the lamp should be housed in a shatter-resistant fixture to avoid that shattering of glass may lead to broken fragments falling into that open processing equipment during its maintenance. By using a protective PTFE coating, one may also maintain the integrity of the lamp in the event of breakage. Light sources used during maintenance operations should not contain mercury. • Opening the distribution system will expose the system to particles from the outside environment. The contamination risk can be minimized by using strict specifications on how to conduct activities, such as cutting pipe work, and handling pipes and components before the actual installation. Precautions should be taken to prevent the distribution of any contamination residues or mechanical damage residues in the surroundings. Vacuum cleaners should be applied to extract maintenance debris at the place where the maintenance takes place, drip pans should be used to collect oil, etc. Equipment openings must be protected to maintain the interior of the process equipment and components free from any external contamination. • Equipment components subjected to maintenance, spare parts and tools should not be placed on the ground or walking surface (e.g., deck), but on a plastic pallet, in a receptacle, a box, a carrier or a trolley provided with a plastic cover. In the food processing area, no wooden pallets should be used to store new or replaced equipment components. • Whenever parts and tools are stored in the production area, they should preferably be kept in rooms or lockers reserved for that use. • Equipment components in service should be clearly indicated, and/or placed in quarantaine. • Take care not to lose nuts, bolts, etc. when removing them from machinery. Because small parts easily can be misplaced, loose bolts, nuts, screws, rivets, washers, etc. should be stored in maintenance receptacles. • Bolts, nuts, screws, etc. of a lower alloy composition may not be left behind on stainless steel, because they may induce corrosion. • Maintenance personnel should not walk on the cladding of insulated piping to prevent that it becomes damaged. • Food grade maintenance chemicals (lubricants, heat transfer liquids, etc.) that do not provoke corrosion should be used.

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• Personnel must be trained and suitably skilled in the correct access, handling and use of approved maintenance compounds, or have access to documented directions. • Maintenance products (oils, greases, lubricants, ammonia, glues, chemical products, etc.) should not be left in the food processing environment when maintenance operations are ceased (e.g., during the night, during weekends, during collective holidays, etc.). They shall be stored separately from food products in clearly labeled (identifying the maintenance compound) and closed containers (e.g., bulk supply) in dedicated secure storage facilities. • Maintenance compounds that are “in-use” or for “immediate-use” may be stored in processing and support areas, but only in quantities necessary for immediate use. When transferred from their original container (e.g., bulk supply) to a new container (e.g., “in-use” or for “immediate-use”), the latter must be labeled with the name of the maintenance compound. • Empty maintenance compound containers must not be re-used in a way that food product could get contaminated. All containers/implements should be labeled ‘for chemical use only’. • Excessive lubricant and grease should be removed to prevent them from coming into contact with the product or food contact areas. • Avoid placing dirty, greasy, oily hands on any surface with which the product comes into contact. 5.9.6.3 Recommended Hygiene Practices after Maintenance and Repair After maintenance and repair operations, the following practices should be followed: • Maintenance tools or machinery must be removed or returned to storage without delay once maintenance or repair work is completed. Therefore, maintenance technicians must verify if all maintenance tools and components are removed after maintenance and repair to ensure nothing is left where it may enter the product or damage equipment. An inventory can be made of all tools prior to maintenance • Any maintenance waste and other refuse (e.g., packaging materials, broken components, failed parts, dirt, dust, spilled oil) must be regularly removed to a suitable storage area and without delay. • Equipment that could be a source of contamination must be physically isolated from processing lines and product, or removed from processing areas. Damaged or decommissioned equipment that remains in processing areas must be clearly identified as such, to ensure that it is not used. Decommissioned equipment may be stored outdoors, but should be placed on a hard standing (e.g. concrete, sealed or paved area) and covered. • If emergency repairs were required during production, any product that may have been left sitting for long periods of time or become contaminated during repairs should be disposed of.

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• The operator must have a procedure to ensure that equipment returned to use (e.g., after repairs and maintenance, re-commissioning or having previously been idle) is not a source of contamination to product, because of bad maintenance or repair, because repair is not conform the rules of appropriate hygienic design, or because maintenance dirt is left. • Maintenance debris (e.g., abraded particles, swarf) must be flushed from the system after maintenance and repairs. • When it was necessary to “break in” to the system for maintenance or inspection, equipment should be thoroughly cleaned any time maintenance or repairs of any type are performed in a food processing facility. The equipment and area should be cleaned with solutions of detergents and disinfectants in the right concentration, then rinsed, and finally dried prior to resuming production. 5.9.7 EVALUATION OF THE QUALITY OF MAINTENANCE WORK DONE AND RECORD KEEPING Before production resumes, the food manufacturer must evaluate if finished maintenance operations and repairs meet the expectations with respect to the quality of the maintenance and repairs. In this perspective, the following practices should be followed: • Equipment must be subjected to a pre-operational check before processing recommences. Are all technical problems solved? Are maintenance and repairs done in a way that the process equipment allows to produce safe food products once production resumes? • Equipment operating under validated conditions must be revalidated if the repairs and maintenance activity may affect its validated status (e.g. replacing temperature probes/sensors in ovens/freezers). • Maintenance records or job sheets (including when and how the defect/ breakdown was repaired, who conducted the work, who has signed-off that it was completed and that appropriate equipment return to use procedures followed). Comprehensive maintenance records will assist the operator to verify that the repairs and maintenance programme is working correctly.

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6 Basic of Toxicology Related to Food

6.1 INTRODUCTION

Toxicology is a discipline, overlapping with biology, chemistry, pharmacology, medicine, and nursing, that involves the study of the adverse effects of chemical substances on living organisms and the practice of diagnosing and treating exposures to toxins and toxicants. The relationship between dose and its effects on the exposed organism is of high significance in toxicology. Factors that influence chemical toxicity include the dosage (and whether it is acute or chronic), route of exposure, species, age, sex, and environment. Toxicologists are experts on poisons and poisoning. Toxicology is defined as the study of the adverse effects of chemicals on living organisms. Its origins may be traced to the time when our prehistoric ancestors first attempted to introduce substances into their diets that they had not encountered previously in their environments. By observing which substances could satisfy hunger without producing illness or death, ancient people developed dietary habits that improved survival and proliferation of the species in their traditional environment and allowed them to adapt to new environments. In its modern context, toxicology draws heavily on knowledge in chemical and biological fields and seeks a detailed understanding of toxic effects and means to prevent or reduce toxicity. In many instances, the original discoveries of toxins that caused devastating human illness and suffering have led to the development of the toxin as a probe of biological function that is used today to study basic mechanisms and to develop cures for human maladies as diverse as postpartum hemorrhage, psychosis, and cancer.

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A brief history of documented uses of toxic agents serves to illustrate the importance of these substances since ancient cultures. The Ebers papyrus of about 1500 BCE, one of the oldest preserved medical documents, describes uses of many poisons such as hemlock, aconite arrow poison, opium, lead, and copper. By 399 BCE, death by hemlock poisoning was a wellestablished means of capital punishment in Greece, most notably in the forced suicide of Socrates. Around this same time, Hippocrates discussed bioavailability and overdosage of toxic agents, and intended poisonings— used mostly by aristocratic women as a means of dispatching unwanted husbands—were of common occurrence in Rome. By about 350 BCE, Theophrastus, a student of Aristotle, made many references to poisonous plants in his first De Historia Plantarum. In about 75 BCE, King Mithridates VI of Pontus (in modern Turkey) was obsessed with poisons and, from a young age, took small amounts of as many as 50 poisons in the hopes of developing resistance to each of them. This practice apparently induced a considerable resistance to poisons, since according to legend, to avoid enemy capture, the standard poisonous mixture was not effective in a suicide attempt by the vanquished king and he had to fall on his sword instead. The term “mithridatic” refers to an antidotal or protective mixture of low but significant doses of toxins and has a firm scientific basis. However, the claim that vanishingly small doses of toxic agents also produce protective effects, which is the claimed basis for homeopathy, does not have scientific support. In 82 BCE, Lex Cornelia (Law of Cornelius) was the first law to be enacted in Rome that included provisions against human poisonings. In approximately 60 CE, Dioscorides, a physician in the Roman armies of Emperors Nero, Caligula, and Claudius, authored a six to eight volume treatise that classified poisons on the basis of origin (plant, animal, mineral) and biological activity, while avoiding the common practice of classification based on fanciful theories of action that were considered important at the time, such as the theory of humors, which posed that body function is regulated by the proper balance of fluids called black bile, yellow bile, phlegm, and blood. This treatise often suggested effective therapies for poisonings such as the use of emetics, and was the standard source of such information for the next 1500 years. Paracelsus (1493–1541) is considered to be the founder of toxicology as an objective science. Paracelsus, who changed his name from Phillip von Hohenheim, was an energetic, irascible, and iconoclastic thinker. He was trained in Switzerland as a physician and traveled widely in Europe and the Middle East to learn alchemy and medicine in other traditions of the day. Although astrology remained an important part of his philosophy, he eschewed magic in his medical practice. His introduction of the practice of keeping wounds clean and allowing them to drain to allow them to heal won him considerable acclaim in Europe. Most notably for toxicology, Paracelsus was the first person who attributed adverse effects of certain substances to the substance itself and not to its association with an evil or angered spirit or god. Paracelsus is accredited with conceiving the basic concept of toxicology, which often is stated as follows:

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All substances are poisons; there is none that is not a poison. The right dose differentiates the poison from a remedy. Although this and other concepts developed by Paracelsus were groundbreaking and major advances in thinking about disease for the time, they put him at odds with the major medical practitioners. As a result, he was forced to leave his home medical practice and spent several of his final years traveling. He was 48 when he died, and there are suspicions that his enemies caught up with him and ended his very fruitful life. How ironic it would be if the father of toxicology were murdered by poisoning! It is useful to evaluate the significance of the Paracelsus axiom in our daily lives by considering examples of wellknown substances with low and high toxicity. Water might be considered one of the least toxic of the substances that we commonly encounter. Can it be toxic? Indeed, there are many reports of water toxicity in the scientific literature. For example, in 2002 a student at California State University at Chico was undergoing a fraternity initiation ordeal in which he was required to drink up to five gallons of water while engaged in rigorous calisthenics and being splashed with ice cold water. Consumption of this amount of water in a short span of time resulted in the dilution of the electrolytes in his blood to the point that normal neurological function was lost and tragically the young man died.

6.2 BASIC PRINCIPLES

The goal of toxicity assessment is to identify adverse effects of a substance. Adverse effects depend on two main factors: i) routes of exposure (oral, inhalation, or dermal) and ii) dose (duration and concentration of exposure). To explore dose, substances are tested in both acute and chronic models. Generally, different sets of experiments are conducted to determine whether a substance causes cancer and to examine other forms of toxicity. Factors that influence chemical toxicity: • Dosage – Both large single exposures (acute) and continuous small exposures (chronic) are studied. • Route of exposure – Ingestion, inhalation or skin absorption • Other factors – Species – Age – Sex – Health – Environment – Individual characteristics

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6.3 BRANCHES OF TOXICOLOGY

The science of toxicology has flourished from its early origins in myth and superstition, and is of increasing importance to many aspects of modern life. Modern toxicology employs cutting-edge knowledge in chemistry, physiology, biochemistry and molecular biology, often aided by computational technology, to deal with problems of toxic agents in several fields of specialization. The major traditional specialties of toxicology address several specific societal needs. Each specialty has its unique educational requirements, and employment in some areas may require professional certification. Clinical toxicology deals with the prevention, diagnosis, and management of poisoning, usually in a hospital or clinical environment. Forensic toxicology is the application of established techniques for the analysis of biological samples for the presence of drugs and other potentially toxic substances, and usually is practiced in association with law enforcement. Occupational toxicology seeks to identify the agents of concern in the workplace, define the conditions for their safe use, and prevent absorption of harmful amounts. Environmental toxicology deals with the potentially deleterious impact of man-made and natural environmental chemicals on living organisms, including wildlife and humans. Regulatory toxicology encompasses the collection, processing, and evaluation of epidemiological and experimental toxicology data to permit scientifically based decisions directed towards the protection of humans from the harmful effects of chemical substances. Furthermore, this area of toxicology supports the development of standard protocols and new testing methods to continuously improve the scientific basis for decision-making processes. Ecotoxicology is concerned with the environmental distribution and toxic effects of chemical and physical agents on populations and communities of living organisms within defined ecosystems. Whereas traditional environmental toxicology is concerned with toxic effects on individual organisms, ecotoxicology is concerned with the impact on populations of living organisms or on ecosystems. Food toxicology focuses on the analysis and toxic effects of bioactive substances as they occur in foods. Food toxicology is a distinct field that evaluates the effects of components of the complex chemical matrix of the diet on the activities of toxic agents that may be natural endogenous products or may be introduced from contaminating organisms, or from food production, processing, and preparation.

6.4 TYPES OF TOXICOLOGY 6.4.1 MEDICAL TOXICOLOGY Medical toxicology is the discipline that requires physician status (MD or DO degree plus specialty education and experience). Medical toxicology is a subspecialty of medicine focusing on toxicology and providing the diagnosis, management, and

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prevention of poisoning and other adverse effects due to medications, occupational and environmental toxicants, and biological agents. Medical toxicologists are involved in the assessment and treatment of a wide variety of problems including acute or chronic poisoning, adverse drug reactions (ADRs), drug overdoses, envenomations, substance abuse, industrial accidents, and other chemical exposures. Medical toxicology is officially recognized as a medical subspecialty by the American Board of Medical Specialties. Its practitioners are physicians, whose primary specialization is generally in emergency medicine, occupational medicine, or pediatrics. Medical toxicology is closely related to clinical toxicology, with the latter discipline encompassing non-physicians as well (generally pharmacists or scientists). 6.4.1.1 Professional Services and Venues • In emergency departments, intensive care units, and other inpatient units, medical toxicoloists provide direct treatment and bedside consultation of acutely poisoned adults and children. • In outpatient clinics, offices, and job sites, medical toxicologists evaluate the health impact from acute and chronic exposure to toxic substances in the workplace, home and general environment. • In regional poison control centers, medical toxicologists provide advice. • In medical schools, universities, and clinical training sites, medical toxicologists teach, research, and provide advanced evidence-based patient care. • In industry and commerce, medical toxicologists contribute to pharmaceutical research and drug safety. • In government agencies, such as the Centers for Disease Control and Prevention and the Food and Drug Administration, medical toxicologists help with health policy. In some of these settings, medical toxicologists are employed to help other physicians to prepare for dealing with the aftermath of crimes such as bioterrorism and war crimes such as chemical warfare and biological warfare. • In clinical laboratories and forensic laboratories, medical toxicologists analyze and interpret diagnostic tests and forensic studies. 6.4.2 CLINICAL TOXICOLOGY Clinical toxicology is the discipline that can be practiced not only by physicians but also other health professionals with a master’s degree in clinical toxicology: physician extenders (physician assistants, nurse practitioners), nurses, pharmacists, and allied health professionals. 6.4.3 COMPUTATIONAL TOXICOLOGY Computational toxicology is a discipline that develops mathematical and computerbased models to better understand and predict adverse health effects caused by chemicals, such as environmental pollutants and pharmaceuticals. Within the Toxicology in the

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21st Century project, the best predictive models were identified to be Deep Neural Networks, Random Forest, and Support Vector Machines, which can reach the performance of in vitro experiments.

6.5 TESTING METHODS

Toxicity experiments may be conducted in vivo (using the whole animal) or in vitro (testing on isolated cells or tissues), or in silico (in a computer simulation). 6.5.1 NON-HUMAN ANIMALS The classic experimental tool of toxicology is testing on non-human animals.[8] As of 2014, such animal testing provides information that is not available by other means about how substances function in a living organism. 6.5.2 ALTERNATIVE TESTING METHODS While testing in animal models remains as a method of estimating human effects, there are both ethical and technical concerns with animal testing. Since the late 1950s, the field of toxicology has sought to reduce or eliminate animal testing under the rubric of “Three Rs” - reduce the number of experiments with animals to the minimum necessary; refine experiments to cause less suffering, and replace in vivo experiments with other types, or use more simple forms of life when possible. Computer modeling is an example of alternative testing methods; using computer models of chemicals and proteins, structure-activity relationships can be determined, and chemical structures that are likely to bind to, and interfere with, proteins with essential functions, can be identified. This work requires expert knowledge in molecular modeling and statistics together with expert judgement in chemistry, biology and toxicology. In 2007 the National Academy of Sciences published a report called “Toxicity Testing in the 21st Century: A Vision and a Strategy” which opened with a statement: “Change often involves a pivotal event that builds on previous history and opens the door to a new era. Pivotal events in science include the discovery of penicillin, the elucidation of the DNA double helix, and the development of computers.... Toxicity testing is approaching such a scientific pivot point. It is poised to take advantage of the revolutions in biology and biotechnology. Advances in toxicogenomics, bioinformatics, systems biology, epigenetics, and computational toxicology could transform toxicity testing from a system based on whole-animal testing to one founded primarily on in vitro methods that evaluate changes in biologic processes using cells, cell lines, or cellular components, preferably of human origin.” As of 2010 that vision was still unrealized. As of 2014 that vision was still unrealized. In some cases shifts away from animal studies has been mandated by law or regulation; the European Union (EU) prohibited use of animal testing for cosmetics in 2013.

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6.6 DOSE-RESPONSE

Since there are both toxic and non-toxic doses for any substance, we may also enquire about the effects of intermediate doses. In fact, the intensity of a biological response is proportional to the concentration of the substance in the body fluids of the exposed organism. The concentration of the substance in the body fluids, in turn, is usually proportional to the dose of the substance to which the organism is subjected. As the dose of a substance is increased, the severity of the toxic response will increase until at a high enough dose the substance will be lethal. This so-called individual dose-response can be represented as a plot of degree of severity of any quantifiable response, such as an enzyme activity, blood pressure, or respiratory rate, as a function of dose. The resulting plot of response against the log10 of concentration will provide a sigmoidal curve (as illustrated in Figure 6.1) that will be nearly linear within a mid-concentration range and will be asymptotic to the zero response and maximum response levels. This response behaviour is called a graded dose response since the severity of the response increases over a range of concentrations of the test substance. Toxicity evaluations with individual test organisms are not used often, however, because individual organisms, even inbred rodent species used in the laboratory, may vary from one another in their sensitivities to toxic agents. Indeed, in studies of groups of test organisms, as the dose is increased, there is not a dose at which all the organisms in the group will suddenly develop the same response. Instead, there will be a range of doses over which the organisms respond in the same way to the test substance. In contrast to the graded individual dose-response, this type of evaluation of toxicity depends on whether or not the test subjects develop a specified response, and is called an all-ornone or quantal population response. To specify this group behaviour, a plot of percent of individuals that respond in a specified manner against the log of the dose is generated.

Fig. 6.1 Dose-reponse

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Let us consider, for example, the generation of a dose-response curve for a hypothetical hypertensive agent. The test substance would be administered in increasing doses to groups of 10 subjects or test organisms. The percentage of individuals in each group that respond in a specific way to the substance (e.g., with blood pressure 140/100) then is determined. The data then are plotted as percent response in each group versus the log of the dose given to each group. Over a range of low doses, there will be no test subjects that develop the specified blood pressure. As the dose increases, there will be increased percentages of individuals in the groups that develop the required blood pressure, until a dose is reached for which a maximum number of individuals in the group respond with the specified blood pressure. This dose, determined statistically, is the mean dose for eliciting the defined response for the population. As the dose is further increased, the percentages of individuals that respond with the specified blood pressure will decrease, since the individuals that responded to the lower doses are now exhibiting blood pressures in excess of the specified level. Eventually, a dose will be reached at which all the test subjects develop blood pressures in excess of the specified level. When the response has been properly defined, information from quantal dose-response experiments can be presented in several ways. A frequency response plot (Figure 6.2) is generated by plotting the percentage of responding individuals in each dose group as a function of the dose. The curve that is generated by these data has the form of the normal Gaussian distribution and, therefore, the data are subject to the statistical laws for such distributions. In this model, the numbers of individuals on either side of the mean are equal and the area under the curve represents the total population. The area under the curve bounded by the inflection points includes the number of individuals responding to the mean dose, plus or minus one standard deviation (SD) from the mean dose, or 95.5% of the population. This mean value is useful in specifying the dose range over which most individuals respond in the same way.

Fig. 6.2 Comparison of shapes of the dose-response curves between Normal Frequency Distribution and Quantal Dose- Response

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Frequency-response curves may be generated from any set of toxicological data where a quantifiable response is measured simply by recording the percentage of subjects that respond at each dose minus the percentage that respond at the lower dose. Generally, the frequency-response curve obtained by experiment only approaches the shape of the true normal distribution. Such curves illustrate clearly, however, that there is a mean dose at which the greatest percentage of individuals will respond in a specific way. There will always be individuals who require either greater (hyposensitive) or smaller (hypersensitive) doses than the mean to elicit the same response. Although the frequency-response distribution curves often are used for certain kinds of statistical analyses of dose-response data, the cumulative- response data presentation is employed more commonly, especially for representing lethal response data. The cumulative-response curve may be generated for non-lethal frequency-response data by plotting log dose versus percentage of individuals responding with at least a specified response. As illustrated in Figure 6.2, if the blood pressure responses used in the previous example are plotted as the percentage of individuals in each dosing group that respond with at least a level of 140/100, the resulting curve will be sigmoidal. Several important values used to characterize toxicity are obtained from this type of curve. The NOAEL (no observed adverse effect level) is the highest dose at which none of the specified toxicity was seen. The LOAEL (lowest observed adverse effect level) is the lowest dose at which toxicity was produced. The TD50 is the statistically determined dose that produced toxicity in 50% of the test organisms. If the toxic response of interest is lethality, then LD50 is the proper notation. At a high enough dose, 100% of the individuals will respond in the specified manner. Since the LD and TD values are determined statistically and based on results of multiple experiments with multiple test organisms, the values should be accompanied by some means of estimating the variability of the value. The probability range (or p value), which is commonly used, generally is accepted to be less than 0.05. This value indicates that the same LD or TD value would be obtained in 95 out of a hypothetical 100 repetitions of the experiment. The cumulative-response curves can facilitate comparisons of toxic potencies between compounds or between different test populations. For example, for two substances with non- overlapping cumulative dose-response curves, the substance with the curve that covers the lower dose range is clearly the more toxic of the two. If prior treatment of a test population with substance A results in a shift of the dose-response curve to the right for toxin B, then substance A exerts a protective effect against substance B. In the case where the dose-response curves for different toxins overlap, the comparison becomes a bit more complex. This can occur when the slopes of the dose-response curves are not the same. These hypothetical compounds have the same LD80, and are said to be equally toxic at this dose. Below this dose, however, compound A produced the higher percentage of toxicity than compound B and, therefore, compound A is more toxic. At doses above the LD80, compound B produces the higher percentage of lethality and, thus, is the more toxic substance. Based on the LD50 values only, compound A is more toxic than

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compound B. Thus, in comparing the toxicities of two substances, the toxic response must be specified, the dose range of toxicity must be stated, and if the toxicities are similar, the slopes of the linear portions of the dose-response curves must be indicated.

6.7 POTENCY

Although all substances exhibit toxic and lethal dose-response behaviour, there is a wide range of LD50 values for toxic substances. By convention, the toxic potencies fall into several categories. A list of LD50 values for several fairly common substances, along with a categorization of the toxicities from extreme to slight, are provided in Table 6.1. Substances with LD50 values greater than about 2 g/kg body wt. generally are considered to be of slight toxicity and that relatively large amounts, in the range of at least one cup, are required to produce a lethal effect in an adult human and are easily avoided under most circumstances. However, exposure to substances in the extreme category with LD50 < 1 mg/kg requires only a few drops or less to be lethal and may be a considerable hazard. Table 6.1 Potency of Common Toxins Agent

LD50 (mg/kg)

Ethyl alcohol Sodium chloride BHA/BHT (antioxidants) Morphine sulfate Caffeine Nicotine Curare Shellfish toxin Dioxin Botulinum toxin

9,000 4,000 2,000 900 200 1 0.5 0.01 0.001 0.00001

6.8 HORMESIS

Toxicity

Slight Moderate High

Extreme

Hormesis is a dose-response phenomenon characterized by a low dose beneficial effect and a high dose toxic effect, resulting in either a J-shaped or an inverted U-shaped dose-response curve. A hormetic substance, therefore, instead of having no effect at low doses, as is the case for most toxins, produces a positive effect compared to the untreated subjects. A representative dose-response curve of such activity is presented in Figure 6.3. Substances required for normal physiological function and survival exhibit hormetic dose-response behaviour. At very low doses, there is an adverse effect (deficiency), and with increasing dose beneficial effects are pro- duced (homeostasis). At very high doses, an adverse response appears from toxicity. For example, high doses of vitamin A can

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cause liver toxicity and birth defects while vitamin A deficiency contributes to blindness and increases the risk of disease and death from severe infections. Non-nutritional substances may also impart beneficial or stimulatory effects at low doses but produce toxicity at higher doses. Thus, chronic alcohol consumption at high doses causes esophageal and liver cancer, whereas low doses can reduce coronary heart disease. Another example is radiation, which at low levels induces beneficial adaptive responses and at high levels causes tissue destruction and cancer.

Fig. 6.3 Hormesis dose-response curve

6.9 MARGIN OF SAFETY

Safety is defined as freedom from danger, injury, or damage. Absolute safety of a substance cannot be proven since proof of safety is based on negative evidence, or the lack of harm or damage caused by the substance. A large number of experiments can be run that may build confidence that the substance will not cause an adverse effect, but these experiments will not prove the safety of the substance. There is always the chance that the next experiment might show that the substance produces an adverse effect in standard or new testing protocols. In addition, our concept of safety continues to evolve and we are now aware that even minute changes, for example, in the activity of an important enzyme, could portend a highly negative effect in the future. Indeed, our concept of safety in regard to toxic exposure continues to develop as our knowledge of biochemical and molecular effects of toxins, and our ability to measure them, grow. Since absolute safety cannot be proven, we must evaluate relative safety, which requires a comparison of toxic effects between different substances or of the same substance under different conditions. When the experimental conditions for toxicity testing in a species have been carefully defined, and the slopes of the dose-response curves are nearly the same, the toxicities of two substances can often be calculated simply by determining the ratio of the TD50s or LD50s. Often, however, a more useful concept is the comparison of doses of a substance that elicit desired and undesired effects. The margin of safety of a substance is the range of doses between the toxic and

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beneficial effects; to allow for possible differences in the slopes of the effective and toxic dose-response curves, it is computed as follows: Margin of Safety (MS) = LD1/ ED99 LD1 is the 1% lethal dose level and ED99 is the 99% effective dose level. A less desirable measure of the relative safety of a substance is the Therapeutic Index, which is defined as follows: Therapeutic Index (T1) = LD50/ ED50 TI may provide a misleading indication of the degree of safety of a sub- stance because this computation does not take into account differences in the slopes of the LD and ED response curves. Nevertheless, this method has been used traditionally for estimations of relative safety. The dose- response data presented in Figure 6.4 serves to illustrate how the use of TI can provide misleading comparisons of the relative toxicities of substances.

Fig. 6.4 The dose-response data serves to illustrate how the use of TI can provide misleading comparisons of the relative toxicities of substances

In this example, drug A and drug B have the same LD50 = 100 mg/kg and ED50 = 2 mg/kg. The comparison of toxicities, therefore, provides the same TI = 100/2 = 50. Therapeutic index does not take into account the slope of the dose-response curves. Margin of safety, however, can overcome this deficiency by using ED99 for the desired effect and LD1 for the undesired effect. Thus, MS = LD1 = ED99 = 10/10 = 1 for drug A; and for drug B = 0:002 = 10 = 0:0002 Therefore, according to the MS comparison, drug B is much less safe than drug A. For substances without a relevant beneficial biological response, the concepts of MS and TI have little meaning. Many substances as diverse as environmental contaminants and food additives fall into this category. For these substances, safety of exposures is estimated based on the NOAEL adjusted by a series of population susceptibility factors to provide a value for the Acceptable Daily Intake (ADI). The ADI is an estimate of the level of daily exposure to an agent that is projected to be without adverse health impact on the human population. For pesticides and food

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additives, it is the daily intake of a chemical, which during an entire lifetime appears to be without appreciable risk on the basis of all known facts at the time, with the inclusion of additional safety factors. The ADI is computed as follows: ADI = NOAEL/ (UF × MF) where UF is the uncertainty factor and MF is the modifying factor. UF and MF provide adjustments to ADI that are presumed to ensure safety by accounting for uncertainty in dose extrapolation, uncertainty in duration extrapolation, differential sensitivities between humans and animals, and differential sensitivities among humans (e.g., the presumed increased sensitivity for children compared to adults). The common default value for each uncertainty factor is 10, but the degree of safety provided by factors of 10 has not been quantified satisfactorily and is the subject of continuing experimentation and debate. Thus, for a substance that triggers all four of the uncertainty factors indicated previously, the calculation would be ADI = NOAEL/10,000. In some cases, for example, if the metabolism of the substance is known to provide greater sensitivity in the test organism compared to humans, an MF of less than 1 may be applied in the ADI calculation.

6.10 BIOLOGIC FACTORS THAT INFLUENCE TOXICITY

It is clear from the foregoing discussion that all substances can exhibit toxicity at sufficiently high doses and there will be a range of sensitivities among individuals to the toxic effects. We will now consider physiologic and anatomical factors that can influence this sensitivity. The scheme presented in Figure 6.5 summarizes biological processes that can modulate responses, both beneficial and adverse, to an administered chemical.

Fig. 6.5 Biological processes that can modulate responses, both beneficial and adverse, to an administered chemical

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Tissue absorption generally is required for most substances to exhibit their toxic effects. This absorption, for example, can result in a limited distribution of the substance at or near the point of contact, or it can lead to the entry of the substance into the blood or lymph circulation and distribution into the entire body. When a substance enters the biologic fluid, it can exist in a free form or a form in which it is bound, most often to proteins in the blood. Also, while in the body fluid, the substance can be translocated in bound or free form to distant sites in the body. Storage sites are body compartments in which the compound is bound with sufficiently high affinity to reduce its free concentration in the general circulation. Bone, lipid tissue, and liver are common sites of storage of xenobiotics. The residence time for the substance in the storage site can be as long as decades and depends on the binding affinity for the site and the concentration of the substance in the circulating fluid. As the concentration of the substance in the body fluid drops due to a cessation in exposure, the substance will be released to the circulation at a rate that depends on its binding affinity for a component of the binding tissue. Biotransformation sites are locations in cells of certain organs that mediate the metabolism of xenobiotics. The most active tissues for this biotransformation are the portals of entry in the liver and small intestine. In most cases, biotransformation converts the substance to an oxidized and conjugated form that is water soluble and more readily excreted via the urine or the bile. In some cases, however, intermediates in the biotransformation process are responsible for the toxic effects of the administered substance. Finally, the xenobiotic or its activated metabolite will encounter its site of action and toxicity. The molecular target is a component of a metabolic or signaling pathway that is important to the normal function or development of the organ. Although a toxic agent may adversely affect the functions of many tissue macromolecules and the cells that contain them, these effects may not be important to the well being of the organ and the organism, and are not considered to be central sites of toxic action. Each of these factors that influence toxicity will be discussed in the following sections.

6.11 ABSORPTION

For a substance to gain access to a specific effector site within an organelle of a complex organism, the substance must often pass through a series of membranes. Although the membranes in various cells of the organism— such as the skin keratinocytes, intestinal enterocytes, vascular endothelial cells, liver hepatocytes, and the nuclear membrane— have certain characteristics that distinguish them from one another, the basic compositions of the membranes are very similar. An accepted general membrane model is illustrated in Figure 6.6. In this model, the membrane is represented as a phospholipid bilayer with hydrophilic outer portions and a hydrophobic interior. Proteins are dispersed throughout the membrane with some proteins traversing the entire width and projecting beyond the

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surfaces of the membrane. The basic cell membrane is approximately 7.5 to 10 nanometers thick and is elastic. It is composed almost entirely of phospholipids and proteins with small quantities of carbohydrates on the surface. A closer look at the chemical structure of the phospholipid component of the membrane provides insight into the effect of its composition on the function of the membrane. As represented in Figure 6.7, the polar head of the phospholipid is composed of a phosphate moiety bound to other small molecules such as choline, serine, ethanolamine, and inositol that can increase the polarity of the phospholipid or serve as sites for further modifications that control cell function, for example, by addition of carbohydrate or phosphate groups.

Fig. 6.6 General membrane model of animal cell

The composition of the lipid components of the phospholipid contributes to the fluidity of the membrane and, thereby, can affect cell function. For example, adequate fluidity of the membrane is maintained, in part, by incorporation of cisunsaturated fatty acids. The cisdouble bonds decrease the strength of interactions between adjacent lipid chains compared to lipids that are saturated or that contain trans-double bonds. Modification in fluidity can affect many cellular functions, including carrier-mediated transport, properties of certain membrane-bound enzymes and receptors, membrane transporters, immunological and chemotherapeutic cytotoxicity, and cell growth. Another important feature of the cell membranes is the presence of aqueous channels or pores. Although water can diffuse passively at a low rate through the continuous phospholipid bilayer of the membrane, some cell types exhibit much higher rates of water transport than others due to the presence of pores in the membrane. The

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transmembrane proteins that form these pores comprise a family of about 12 members called either aquaporins, if they allow passage of only water, or aquaglyceroporins, if they allow passage of glycerol and other small neutral solutes. The conformation of the most studied aquaporins, aquaporin 1 (AQP1) from red blood cells, is indicated in Figure 6.8 As visualized from the extracellular surface, AQP1 forms an elegant and highly symmetrical tetramer in the pore. Water passes through channels in each of the AQP1 molecules in the pore. The rates of passage of water and solutes through these pores depend on the size of the pore, which can be tissue specific and may be hormonally regulated. For example, channels in most cell types are less than 4 nm in diameter and allow passage of molecules with a molecular weight of only a few hundred Daltons. In contrast, the pores in the kidney glomerulus are much larger at approximately 70 nm, and allow passage of some small proteins (< 60,000 Da).

Fig. 6.7 Chemical structure of the phospholipid component of the membrane

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Fig. 6.8 Aquaporins, aquaporin 1 (AQP1) from red blood cells

6.12 BIOLOGICAL DETERMINATION OF TOXICANTS 6.12.1 ACUTE TOXICITY The assay for detecting a poison is usually by observing the toxic effect itself. Since it is rarely desirable to use humans, a “model system,” usually rats or mice, must be selected to use in the identification process. The first toxicity test is generally an acute toxicity test using experimental animals most commonly in a single dose. The toxic effect that occurs within 24 hours of exposure is recorded. The primary purpose of an acute toxicity test is to determine the level of the substance that induces mortality in experimental animals. This is the point at which the median lethal dose (LD50) is determined. Information obtained from these acute toxicity tests generally is used as the basis for establishing dose and route of exposure for subsequent prolonged toxicity tests. Except in the rare instance, if the sub- stance is found to be too acutely toxic for food use, it will be tested for genetic toxicity, metabolism, and pharmacokinetics. 6.12.2 GENETIC TOXICITY The primary objective of genetic toxicity testing is to determine the tendency of the substance to induce mutations in the test organism. A mutation is an inheritable change in the genetic information of a cell. Approximately 10% of all human diseases may have a genetic component and thus may arise from a mutation of one form or another.

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It is well known, for example, that Down’s syndrome, Kleinfelter’s syndrome, sicklecell anemia, and cystic fibrosis arise from specific genetic changes. Most if not all cancers are thought to have their origin in one or more mutations. With the exception of hormones, most substances, approximately 85 to 90%, are carcinogenic in animal species and have been shown to be mutagenic by one assay or another. Although much more information is required before a converse correlation can be established, the fact that a sub- stance is shown to be mutagenic by appropriate tests places it under considerable suspicion as a possible carcinogen. The decision-tree approach proposes a battery of genetic tests early in the testing scheme. It is suggested that there is a high degree of correlation between mutagenicity and carcinogenicity. Therefore, a substance can be banned from use in food because of its carcinogenic probability based on the results of mutagen tests alone. If the substance does not show high carcinogenic probability based on mutagenicity tests, it must be tested further, including long-term carcinogenicity tests. Although the details of an appropriate array of mutagenicity tests are the subject of continuing controversy, the general outline seems to be fairly well established. Assays for which there seem to be general support include analyses of point mutations (localized changes in DNA) in micro-organisms and in mammalian cells, investigation of chromosomal changes (major recombination of genetic material) in cultured mammalian cells and in whole animals, and investigation of cell transformation (tumors produced by implantation in animals) using cultured human or other mammalian cells. 6.12.3 BIOASSAY Bioassay is a commonly used shorthand term for biological assays. Bioassays typically are conducted to measure the effects of a substance on living organisms, including microorganisms and experimental animals. Generally, bioassay is a simple and convenient method to detect toxicants but it may not be useful for quantitative analysis for low levels of substances compared with advanced instrumental methods. Bioassay has been used for various purposes including measurement of the pharmacological activity of new or chemically undefined substances; investigation of the function of endogenous mediator; determination of the side-effect profile, including the degree of drug toxicity; assessing the amount of pollutants being released by a particular source, such as waste- water or urban run-off; and assessing the mutagenicity of chemicals found particularly in foods. 6.12.3.1 Bacterial Reverse Mutation Assay Among many bioassays used in various fields, the bacterial reverse mutation assay is one of the most widely and commonly used methods in food toxicology research. The purpose of the bacterial reverse mutation assay is to evaluate a chemical’s genotoxicity by measuring its ability to induce reverse mutations at selected loci in several bacterial strains. This assay, commonly called the Ames assay (test), was developed originally

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by Dr. Bruce Ames (Professor at University of California, Berkeley) in the early 1970s. It is sensitive to a wide range of mutagenic chemicals. 6.12.3.2 The Host-Mediated Assay Another test that uses microbial organisms to determine the mutagenic potential of a substance is known as the host-mediated assay. In this test, a bacterial organism is injected into the peritoneal cavity of a mammal, usually a rat, and the animal is treated with the test substance. The test substance and its metabolites enter the circulation of the animal, including the peritoneal cavity. After an appropriate period, the test organism is removed from the peritoneal cavity and examined for induction of mutations. 6.12.3.3 The Dominant-Lethal Test A third mutagenesis assay, known as the dominant-lethal test, determines genetic changes in mammals. In this test males are treated with the test substance and mated with untreated females. The dominant-lethal mutation will arise in the sperm and may kill the zygote at any time during development. Females are dissected near the end of gestation and the number of fetal deaths and various other reproductive abnormalities are noted. Assays for point mutations also have been developed using mammalian cell lines. One cell line that has been used extensively is the Chinese hamster ovary cell for which resistances to various substances such as 8- azaguanine is used as markers. In contrast to the Salmonella system, the Chinese hamster ovary cell lines mainly detect forward mutations. How- ever, one problem often encountered with such assays is the variability in metabolic capability of the cell line. Thus, in some cases, tissue homogenates such as those used in the Ames Assay or test cells are incorporated in the host-mediated assay, as is most commonly done with bacterial cells. Mutation of the more general type (those that are not point mutations) may be determined by scoring induced chromatid and chromosomal aberrations. Structural changes in chromosomes may be caused by breaks in the chromosomal unit. If the two ends of the break remain separated, chromosomal materials are lost, resulting in visible breaks in the chromosome. 6.12.3.4 The Cell Transformation Assay The cell transformation assay, in which mammalian cells are used, is an important aspect of any array of short-term genetic toxicity tests. Many cell lines have been developed for the measurement of malignant transformation following exposure to a test substance. A commonly used cell line is embryo fibroblasts from rats, hamsters, and mice. After a period of normal growth, cells are suspended in an appropriate buffer, treated with a test sub- stance, and then portions of cells are tested for survival

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rates. The remaining material is plated out on an appropriate medium, and the transformed cells are observed at the colony stage. Malignancy of cells can be confirmed by the production of tumors following transplantation of transformed cells into the appropriate host. If the genetic toxicology studies lead to the finding of mutagenesis, with the implication of possible carcinogenicity, a risk assessment is applied. If the substance is mutagenic in several assays that are correlated with human carcinogenesis, and the intended use of the substance results in appreciably high human exposures, then with no further testing, the substance may be banned from further use. If the substance is determined to be of low mutagenic risk because, for example, it is mutagenic in several assays but only at very high doses, or the mutagenic activity is observed in only one of the assays, then further studies must be conducted. 6.12.4 METABOLISM Generally, following mutagenicity tests, metabolic studies would be conducted. The objective of this phase of testing is to gain both a general and quantitative understanding of the absorption, biotransformation, disposition (storage), and elimination characteristics of an ingested substance after single and repeated doses. If the biological effects of metabolites are known, the decision to accept or reject the substance can be made on this basis. For example, if all the metabolites can be accounted for and they are all known to be innocuous substances, then the test substance is considered safe. However, if certain metabolites are toxic or if most of the parent substance is retained within certain tissues, then further testing may be indicated. Further evidence of the potential hazard of a substance may also be derived from the knowledge that if the substance has appreciable toxicity in the metabolism of a test species, it may have a similar effect in human metabolism. Thus, knowledge of the metabolism and pharmacokinetics of a sub- stance is essential for establishing the relevance of results from animal testing to projecting likely hazards in humans. 6.12.5 SUBCHRONIC TOXICITY Based on the results of these initial investigations, subchronic toxicity studies may be designed. The objective of the subchronic studies is to determine possible cumulative effects on tissues or metabolic systems. Generally, sub- chronic tests are performed for several months’ duration and may extend to one year. Conventional subchronic studies designed to evaluate the safety of food components usually are limited to dietary exposure for 90 days in two laboratory species, one of which is a rodent. Subchronic tests include daily inspection of physical appearance and behaviour of the test animal. Weekly records of body weight, food consumption, and characteristics of excreta are maintained. Periodic hematological and eye examinations are performed in addition to biochemical tests of blood and urine. Under certain circumstances, tests are run for hepatic, renal, and gastrointestinal functions along with measurements of blood pressure and body

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temperature. All animals are autopsied at the termination of the experiment and examined for gross pathologic changes, including changes in the weights of the major organs and glands. 6.12.6 TERATOGENESIS Teratogenesis testing is an important aspect of subchronic testing. Teratogenesis may be defined as the initiation of developmental abnormalities at any time between zygote formation and postnatal maturation. Relatively little is known about the mechanisms of teratogenesis since it may be caused by radiation, a wide range of chemicals, dietary changes, infection, temperature extremes, or physical trauma. Furthermore, we cannot predict whether a specific substance will be teratogenic based on chemical structures. Since our knowledge of mechanisms of teratogenesis is relatively primitive, teratogenesis assays rely primarily on prolonged testing periods in animals. Administration of substances to bird embryos has been used with some success. However, since the embryos develop with no metabolic interchange with the outside environment, in contrast to the placenta- mediated interchange for mammalian embryos, teratogenesis testing in mammals is much preferred. The phase of embryonic development most susceptible to adverse influences is organogenesis. As illustrated in Figure 6.9, the human fetus is most susceptible to anatomical defects at around 30 days of gestation. That is, exposure to a teratogenic influence around this period is most likely to pro- duce anatomical defects in the developing fetus. One of the major problems in teratogenesis testing is that organisms may be susceptible to teratogenesis for only a few days during the growth of the fetus. If the test substances are not administered precisely at this time, the teratogenic effect will go undetected. Exposure to a teratogen prior to organogenesis may produce no effect or may lead to fetal death and no teratogenic response will be seen. Exposure to a teratogen following the period of organogenesis may lead to functional problems that may be relatively difficult to observe and may not be detected as teratogenic effects. Factors that determine the effective dose of the substance to which the fetus is exposed are (1) the efficiency of the maternal homeostatic processes and (2) the rate of passage of a teratogen across the placenta. The maternal homeostatic processes depend on several factors, including the efficiency of liver metabolism and possible excretion of the substance into the bile, possible metabolism and urinary excretion by the kidney, and tissue storage and protein binding. These processes work together in the maternal system to reduce the overall concentration of the substance to which the developing fetus is exposed. The placenta can also serve as an effective barrier in the passage of certain water-soluble substances of large molecular weight into the fetal circulatory system. However, in the case of certain more lipid- soluble compounds (e.g., methyl mercury) the placenta does little to retard passage into the fetal system.

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Fig. 6.9 Degree of sensitivity of human fetuses to anatomical defects at various times during gestation

Teratogenesis testing protocols should include both short-term (1– 2 days) treatments of pregnant females during organogenesis and continuous treatments during gestation. Teratogenesis tests that include short- term dosing avoid effects of maternal adaptive systems such as induction of metabolic pathways in the liver. This testing protocol also avoids preimplantation damage, increases the likelihood that the embryos will survive to the period of organogenesis, and ensures that critical periods of organ development are covered. Furthermore, this continuous dosing protocol monitors cumulative effects both in the maternal and fetal systems. For example, changes in concentrations and composition of metabolites to which the fetus is exposed during gestation vis-a‘-vis the diminished metabolic activity of the maternal liver are closely monitored, and the level of saturation of maternal storage sites in relation to a rise in the concentration of the test substance in the fetal system may be screened. Since adverse effects on the reproductive system may arise from many causes, tests of reproductive toxicity may include treatment of males prior to mating, short-term dosage of females starting prior to mating and continuing on to lactation, short- and long-term dosing of females during the period of organogenesis and in other periods, and pre- and postnatal evaluation of the offspring. These tests can involve large numbers of animals and periods of time comparable to what would be required for carcinogenesis tests. As a result, measurement of reproductive toxicity can be a very time consuming and expensive procedure. Both mechanistic understanding and testing efficiency are sorely needed in this important area of toxicology. Toxic effects observed in this battery of acute and subchronic tests are evaluated to determine if the tests are relevant to actual conditions of exposure. Many substances at this point in the testing procedure can be rejected from use if their toxicity is sufficiently high. On the other hand, a final decision to accept a relatively non-toxic substance cannot be made if the substance did not satisfy various additional requirements including

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not being consumed at a substantial level, not possessing a chemical structure leading to suspicion of carcinogenicity, no effects on subchronic toxicity testing that would suggest the possibility that long-term exposure would lead to increased toxicity, or no positive results in tests of genetic toxicity. 6.12.7 CHRONIC TOXICITY The objective of chronic toxicity testing is to assess toxicity resulting from long-term, relatively low-level exposure, which would not be evident in sub- chronic testing. Testing protocols require administration of the test substance by an appropriate route and in appropriate dosages for the major portion of the test animal’s life. Chronic toxicity tests are designed so that each treated and control group will include sufficient numbers of animals of both sexes of the chosen species and strain to have an adequate number of survivors at the end of the study for histopathological evaluation of tissues and for statistical treatment of the data. Selecting the proper size of the test group is a major problem in chronic toxicity tests. Table 6.2 indicates the required group sizes as determined by statistical theory. Large numbers of animals must be used if low percentage effects are to be detected. To reduce the numbers of animals required in theory to detect small percentage effects, protocols involving large doses generally are used. However, this practice is coming under increasing scrutiny since the test organism is likely to respond quite differently to high doses of the test material than to low doses. For example, the rates of enzymatic processes such as absorption, excretion, metabolism, and DNA repair are highly sensitive to substrate concentration and are saturable. Thus, high doses of a substance may produce toxic effects by over- whelming a system that readily disposes of low doses. Table 6.2 Theoretical Sizes of Test Groups Required to Determine Toxicity at Indicated Frequencies and Level of Significance True Frequency of Toxic Effect Level of significance Least number of animals for each dose

0.05 58

1 in 20 0.001 134

1 in 100 0.05 295

0.001 670

Despite prudent food-additive laws in the United States, it is still viewed that there is no safe dose of a carcinogen. However, research continues into the existence of a threshold dose below which exposure to a carcinogen may be safe. In most cancer tests, 50 animals of each sex are used for each dose level. Body weights are recorded periodically throughout the testing period, and the level of food consumption is monitored. Animals are examined for obvious tumors, and at the end of the experiment the animals are autopsied and subjected to detailed pathological examination. Rats and mice are widely used in chronic testing because of their relatively low cost and the large volume of knowledge available concerning these animals. The strain of animals used for the test depends on the site of toxicity of the test substance and the

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general susceptibility of the strain to various toxic agents. Generally, strains with some known sensitivity to a range of carcinogens are used. It is likely that a carcinogenic effect will be shown in these animals if the substance is indeed carcinogenic. Variations in diet can also considerably complicate interpretation of the results from chronic toxicity testing. Administration of semisynthetic diets can result in increased tumor yield with several types of carcinogens com- pared to experiments using unrefined diets. Diets that provide insufficient calories result in decreased tumor incidence whereas protein deficiency retards tumor growth. For example, dimethylaminoazobenzeneinduced carcinogenesis is enhanced with riboflavin deficiency in rats. The influences of various dietary components on carcinogenesis are often complex and the mechanism of action is often specific to the carcinogen in question. Many dietary components, such as certain indoles, flavonoids, and certain pesticides that induce xenobiotic metabolizing systems in the liver and other tissues, will decrease the carcinogenic potency of many substances. Even when the various aspects of chronic toxicity tests mentioned in the previous discussion are considered, several other more or less incidental factors can influence the outcome. For example, the temperature and humidity of the room in which the animals are housed must be carefully controlled, as must the type of bedding used in the cages. Cedar wood used as bedding has influenced the outcome of cancer testing, perhaps due to induction of xenobiotic metabolizing enzymes by volatiles from the cedar. Furthermore, cancer tests that are said to differ only with respect to the time of year during which they were performed have produced different results. Thus, it is necessary for even the most well-designed chronic toxicity tests that reproducibility of experimental results be determined. The chronic toxicity test provides the final piece of biological information on whether to accept or reject a substance suggested for food use. If no carcinogenic effects are found, this information, along with all previous data and the estimations of exposure, will be used in the overall risk assessment of a substance. If a substance is determined to be a carcinogen, then in most instances current US law prohibits its use as a food additive. Further testing is needed only if some tests are considered faulty or if unexpected findings make the test design retrospectively inadequate to answer the questions raised.

6.13 TOXICOLOGY AS A PROFESSION

A toxicologist is a scientist or medical personnel who specializes in the study of symptoms, mechanisms, treatments and detection of venoms and toxins; especially the poisoning of people. To work as a toxicologist one should obtain a degree in toxicology or a related degree like biology, chemistry, pharmacology or biochemistry. Toxicologists perform many different duties including research in the academic, non-profit and industrial fields, product safety evaluation, consulting, public service and legal regulation.

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REQUIREMENTS To work as a toxicologist one should obtain a degree in toxicology or a related degree like biology, chemistry or biochemistry. Bachelor’s degree programmes in toxicology cover the chemical makeup of toxins and their effects on biochemistry, physiology and ecology. After introductory life science courses are complete, students typically enroll in labs and apply toxicology principles to research and other studies. Advanced students delve into specific sectors, like the pharmaceutical industry or law enforcement, which apply methods of toxicology in their work. The Society of Toxicology (SOT) recommends that undergraduates in postsecondary schools that don’t offer a bachelor’s degree in toxicology consider attaining a degree in biology or chemistry. Additionally, the SOT advises aspiring toxicologists to take statistics and mathematics courses, as well as gain laboratory experience through lab courses, student research projects and internships. Duties Toxicologists perform many more duties including research in the academic, non-profit and industrial fields, product safety evaluation, consulting, public service and legal regulation. In order to research and assess the effects of chemicals, toxicologists perform carefully designed studies and experiments. These experiments help identify the specific amount of a chemical that may cause harm and potential risks of being near or using products that contain certain chemicals. Research projects may range from assessing the effects of toxic pollutants on the environment to evaluating how the human immune system responds to chemical compounds within pharmaceutical drugs. While the basic duties of toxicologists are to determine the effects of chemicals on organisms and their surroundings, specific job duties may vary based on industry and employment. For example, forensic toxicologists may look for toxic substances in a crime scene, whereas aquatic toxicologists may analyze the toxicity level of wastewater. Compensation The salary for jobs in toxicology is dependent on several factors, including level of schooling, specialization, experience. The U.S. Bureau of Labour Statistics (BLS) notes that jobs for biological scientists, which generally include toxicologists, were expected to increase by 21% between 2008 and 2018. The BLS notes that this increase could be due to research and development growth in biotechnology, as well as budget increases for basic and medical research in biological science.

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7 Food Additives

7.1 INTRODUCTION

A food additive is a substance or mixture of substances, other than basic food components, added to food in a scientifically controlled amount. They are added to food to preserve flavour or improve its taste and appearance. Some additives have been used for centuries; for example, preserving food by pickling—with vinegar or 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 twentieth century, many more additives have been introduced, of both natural and artificial origin. These additions can be made during production, processing, storage, and packaging. It is natural for people to desire better foods, not only from the perspective of health but also for taste, colour, or texture. Hence, a tremendous number of substances have been used since the beginning of the twentieth century to enhance food acceptance. There are two categories of food additives. The first category, intentional additives, are purposely added to perform specific functions. They include preservatives, antibacterial agents, bleaching agents, antioxidants, sweeteners, colouring agents, flavouring agents, and nutrient supplements. The second type of additives are incidental, and may be present in finished food in trace quantities as a result of some phase of production, processing, storage, or packaging. An incidental additive could be a substance present in food due to migration or transfer from the package or processing equipment. Since most food additives are intentionally added substances, only intentional additives are discussed here.

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Substances intentionally added to foods vary from preservatives to flavouring materials. Table 7.1 indicates the approximate number of substances used for each purpose to date. Approximately 300 substances are recognized as food additives, and 60 to 70 food additives are ingested daily by every person in the United States. Table 7.2 lists the most common food additives used for various purposes. Table 7.1 Approximate Number of Different Types of Food Additives. Purpose of Additive Preservatives Antioxidants Sequestrants Surfactants Stabilizers Bleaching, maturing agents Buffers, acids, alkalies Colouring agents Special sweeteners Nutrient supplements Flavouring agents Natural flavouring materials

Number of Different Additives 30 28 44 85 31 24 60 35 9 >100 >700 >350

Table 7.2 Most Common Chemicals Developed as Food Additives. Purposes

Chemicals

Preservatives Antioxidants Sweeteners Colouring agents Flavouring agents Bleaching agents Nutrient supplements

Benzoic acid, sorbic acid, p-oxybenzoic acid, hydrogen peroxide, AF-2 Ascorbic acid, DL-a-tocopherol, BHA, propyl gallate Saccharine, dulcin, sodium cyclamate Food Red No. 2, Food Yellow No. 4, Scarlet Red, Indigo carmine Safrole, methyl anthranilate, maltol, carbon CaOCl2, NaOCl, NaClO2, SO2 Vitamins

As defined by the FDA, the five main reasons to use additives in foods are as follows. • To maintain product consistency. Emulsifiers give products a consistent texture and prevent them from separating. Stabilizers and thickeners give smooth uniform texture. Anticaking agents help substances such as salt to flow freely. • To improve or maintain nutritional value. Vitamins and minerals are added to many common foods such as milk, flour, cereal, and margarine to make up for those likely to be lacking in a person’s diet or lost in processing. Such fortification and enrichment has helped reduce malnutrition among the US population. All products containing added nutrients must be appropriately labeled. • To maintain palatability and wholesomeness. Preservatives retard product spoilage caused by mold, air, bacteria, fungi, or yeast. Bacterial contamination

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can cause food-borne illness, including life-threatening botulism. Antioxidants are preservatives that prevent fats and oils in baked goods and other foods from becoming rancid or developing an off-flavour. They also prevent cut fresh fruits such as apples from turning brown when exposed to air. • To provide leavening or control acidity/alkalinity. Leavening agents that release acids when heated can react with baking soda to help cakes, biscuits, and other baked goods to rise during baking. Other additives help modify the acidity and alkalinity of foods for proper flavour, taste, and colour. • To enhance flavour or impart desired colour. Many spices and natural and synthetic flavours enhance the taste of foods. Colours, likewise, enhance the appearance of certain foods to meet consumer expectations. Examples of substances that perform each of these functions are provided in Table 7.2. Despite the fact that food additives undergo extensive laboratory testing before they are put into commercial food products, the use of additives in foods has engendered great controversy and widespread public concern. There are two basic positions concerning the use of food additives. One is that all additives are potential health threats and should not, on that basis, be used. The other is that unless an additive is proved to be hazardous, using it to protect food from spoilage or to increase its nutritional completeness, palatability, texture, or appearance is well justified. The former opinion has been voiced by some consumers. Their concern is that basic food materials already are contaminated by many toxic substances such as pesticides and microorganisms. Nonetheless, once additives are approved for use in a food product, people will be ingesting them continuously. Therefore, even when an acceptable daily intake (ADI) has been officially established and each product remains within those limits, total ingestion of certain additives from various sources may exceed the ADI. This position holds that the chronic toxicities, such as carcinogenicity and teratogenicity, of food additives have not yet been sufficiently studied. In fact, most food additives are used without any information being made available to consumers about their chronic toxicities. Due to the high costs of testing and other factors, progress in research on the chronic toxicities of food additives is very slow. The second basic position regarding food additives points out their many benefits: Were it not for food additives, baked goods would go stale or mold overnight, salad oils and dressings would separate and turn rancid, table salt would turn hard and lumpy, canned fruits and vegetables would become discoloured or mushy, vitamin potencies would deteriorate, beverages and frozen desserts would lack flavour, and wrappings would stick to the contents. Within the current structure of the food processing industry in the United States, it would be virtually impossible to abandon food additives entirely. However, in order to provide the maximum protection to the consumers, it is wise to study any potential toxicity of the food additives in current use.

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7.2 REGULATIONS

The quality of the food supply in the United States is regulated by numerous state and federal laws. Before the turn of the last century, most states had laws that protected consumers from hazardous or improperly processed foods, but there was no corresponding federal regulatory framework. Public sentiment became focused on the wholesomeness of the food supply following the publication of Upton Sinclair’s novel, The Jungle, in which the deplorable conditions in slaughter houses were described. In addition Dr. Harvey W. Wiley, who was a chemist in the US Department of Agriculture from 1883 to 1930, began conducting chemical and biological analyses of substances in food and discovered instances of misbranded and adulterated food. Since colonies of experimental animals had not yet been developed, Wiley conducted biological testing on himself and a group of young men, who became known as the Poison Squad. Because of the work of Wiley and his group, reliable information on the adulteration, toxicity, and misbranding of food was obtained. Thus, the Pure Food and Drug Act finally passed in 1906 after years of effort to enact such a law. However, it has been suggested that it passed only because it was presented along with the Meat Inspection Act, a response to the public outcry generated by Sinclair’s novel. The 1906 Pure Food and Drug Act forbade the production of misbranded or adulterated food products in the District of Columbia, and prohibited interstate distribution of fraudulent or unhealthy products. It banished such chemical preservatives as boric acid, salicylic acid, and formaldehyde; additionally, it defined food adulteration as the addition of poisons or deleterious materials, the extraction of valuable constituents and the concealment of the resulting inferiority, substitution of other constituents, and the mixture of substances that would adversely affect health. The next major piece of legislation was the Federal Food, Drug, and Cosmetic Act of 1938 (referred to as the 1938 Act), which added further provisions to the 1906 legislation. It defined food as: • Substances used as food or drink by people or animals • Chewing gum • Substances used for components of any such food materials The law also established standards of product identity and fill levels, prohibited adulteration, mandated truthful labeling, and restricted the use of chemicals to those required in the manufacturing of the food, with specific tolerance levels set for chemicals with appreciable toxicity. The Food Additive Amendment that was added to the law in 1958 had a great impact on the food manufacturing industry. Although this amendment to the 1938 Act gave official recognition of the US government’s tolerance regarding the use of food additives and acceptance of the necessity of additives for a wholesome and abundant food supply, it also took the government out of the business of toxicity testing. The amendment stipulated that any food additive was to be proven safe by the manufacturer, who must also prove toxicity. Thus, the additive manufacturer was requested to bear the burden

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of lengthy delays in production and the cost of the millions of dollars required for such testing. The amendment, however, did not apply to additives in use prior to 1958. The potential hazards of these substances initially were assessed based on the opinions of specialists working in the general field of toxicology. Those substances that were considered unsafe for general use in foods were prohibited from further use or strict limits were placed on the levels that could be added to foods. Those substances for which no concerns were expressed were considered “generally recognized as safe” (GRAS). Substances on the GRAS list can be used by manufacturers within the general tenets of good manufacturing practices. Also included in the Food Additive Amendment of 1958 was the Delaney Clause, which was proposed in response to the increasing concern over the possible role of food additives in human cancer. It states that no substance shall be added to food if it is found by proper testing to cause cancer in people or animals. Although the Delaney Clause seems to be a very straightforward and simple way of handling a potentially dangerous class of substances, it provides little room for scientific interpretation of data and remains controversial. The Delaney Clause gives no consideration to the specific dose of a substance likely to be encountered in foods and there is no provision for interpretation of animal results in terms of the site of carcinogenesis, known sensitivity of the experimental model, or the specific dose of carcinogens required to produce the cancer. Although several substances have been banned from food use because of apparent carcinogenicity, in no case has the Delaney Clause been invoked. All substances banned from use have been banned under the general safety provisions of the 1938 Act. The Colour Additive Amendments of 1960 took special notice of colour additives and their potential for misuse and toxicity. The amendments differentiated between natural and synthetic colour additives and required: • Listing of all colour additives on labels • Certification of batches of listed colours where this was deemed necessary to protect public health • Retesting for safety of previously certified colours using modern techniques and procedures where any questions of safety had arisen since the original listing • Testing of all colour additives for food in long-term dietary studies that included carcinogenesis and teratogenesis studies in two or more animal species There are currently two classifications considered suitable for use in food colours, GRAS colours and certified colours. GRAS colours are generally pigments that occur naturally in plants and animals or that are simple organic or inorganic compounds. GRAS colours are exempt from the certification procedure required for most of the synthetic colours. In order to be certified, a sample of each batch of a colour must be submitted to the FDA for analyses. The batch receives approval for use if the sample meets previously established standards of quality. Each batch must be analyzed to ensure that the chemical compositions in the new batches are the same as the composition of the batch that was subjected to biological testing.

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The practice of periodic review is now also used for all food additives. Since 1970, the FDA has been reviewing GRAS substances and ingredients with prior sanctions according to current standards of safety. Under the cur-rent regulatory framework, no substance that appears directly or indirectly in the food may be added to or used on or near food, unless: • The substance is GRAS • The substance has been granted prior sanction or approval • A food additive regulation has been issued, establishing the conditions for its safe use All non-flavour substances on the GRAS list are under review individually in accordance with present-day safety standards while all of the roughly 1000 flavouring agents used in food are reevaluated under a separate programme that considers these substances by chemical class. After a GRAS review is completed, a substance with previous GRAS status will be: • Reaffirmed as GRAS • Classified as a food additive, and conditions of use, levels of use, and any other limitations will be specified • Placed under an interim food additive regulation, which indicates that further toxicological information must be obtained for the substance • Prohibited from use Substances in the interim food additive category can be used in foods while the testing is going on if there is no undue risk to the public. A substance with GRAS status has no specific quantitative limitations on its use; however, its use is restricted by general definitions of adulterated food as specified in the 1938 Act and by what is designated good manufacturing practice (GMP). GMP includes the following conditions for substances added to food: • The quantity of a substance added to food does not exceed the amount reasonably required to accomplish its intended physical, nutritional, or other technical effect in food. • The quantity of a substance that becomes a component of food as a result of its use in the manufacturing, processing, or packaging of food in which it is not intended to accomplish any physical or other technical effect in the food itself shall be reduced to the extent reasonably possible. GMP also specified that the substance is of appropriate food grade and is prepared and handled as a food ingredient. Generally, food additives are regulated in amendments under Section 409 of the 1938 Act. The law requires not only that food additives be safe at the levels used, but also effective in accomplishing their intended effect. Ineffective additives cannot legally be used regardless of their safety. How-ever, this law specifically excludes certain classes of compounds. Pesticide residues in raw agricultural products are not legally considered to be food additives in the United States, although they are subject to regulation by the

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EPA and the individual states. Colourants are regulated separately under the Colour Additive Amendments in the 1938 Act passed in 1960. Prior to the 1958 food additive amendments in the 1938 Act, most food additives were not regulated directly, although certain individual substances were banned by the FDA. Additives that were in common and widespread use in 1958 were exempted from requirements of safety testing on the basis of long-term experience with these compounds. They were termed GRAS under conditions of their intended use, usually at the lowest practical level and in accordance with good manufacturing practices. Although the FDA has specifically listed hundreds of additives as GRAS, the published list is not inclusive. Food additives also have been targeted by the Food Quality Protection Act (FQPA), which took effect in 1996, even though this law was intended mainly to apply to pesticide residues in food. The major impact of FQPA on food additives is elimination of the Delaney Clause. The elimination of the Delaney Clause was part of the 1996 FQPA that substituted a more manageable “risk cup” approach to assessing total human exposure and risk to many chemicals including food additives.

7.3 PRESERVATIVES

One of the most important functions of food additives is to preserve food products from spoilage. Preservatives prevent the spoilage of foods caused by the action of microorganisms or oxidation. The development of methods of food preservation was essential to the prehistoric transition of humans from nomadic hunter-gatherer tribes, to settled agricultural communities. Since the dawn of history, people have struggled to preserve enough food to survive from one growing season to the next. Smoke was probably the first preservative agent to be discovered. Common salt also was used in pre-historic times. Ancient Egyptians made use of vinegar, oil, and honey, substances that still find application today. In the thirteenth century, Wilhelm Beukels discovered “pickling,” a process of preserving food by anaerobic fermentation in brine, a solution of salt in water. In Ancient Assyria, Greece, and China, sulfur dioxide, which normally was described as a fumigant, was also utilized as a preservative. By the late Middle Ages, it was widely used throughout Europe for preserving wine and possibly other applications as well. However, during the late fifteenth century, in what may have been the first legal actions directed against chemical food additives, a number of decrees were promulgated to regulate the use of sulfur in wine production. No other chemical preservatives were introduced until the late eighteenth century when Hofer suggested that borax (hydrated sodium borate) be used. Yet, even at the present time, it is estimated that up to one-third of the agricultural production of the United States is lost after harvest. Synthetic chemicals began to be used as food preservatives at the beginning of the twentieth century, and the widespread use of these chemicals made a broad variety of

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food available to more people for longer periods of time. As mentioned earlier, there are many criticisms of the use of food preservatives. However, the considerable time between the production and the consumption of food today makes some use of preservative necessary in order to prevent spoilage and undesirable alterations in colour and flavour. Many microorganisms, including yeasts, molds, and bacteria, can produce undesirable effects in the appearance, taste, or nutritional value of foods. A number of these organisms produce toxins that pose high risks to human health. Atmospheric oxygen can also adversely affect foods, as for example in the development of rancidity in fats. The word preservative gradually has been given broader connotation, covering not only compounds that suppress microbes but also compounds that prevent chemical and biochemical deterioration. The action of preservatives is not to kill bacteria (bactericidal) but rather to delay their action by suppressing their activity (bacteriostatic). Before being considered for use in food, a chemical preservative must ful-fill certain conditions: • It must be non-toxic and suitable for application. • It must not impart off-flavours when used at levels effective in controlling microbial growth. • It must be readily soluble. • It must exhibit antimicrobial properties over the pH range of each particular food. • It should be economical and practical to use. 7.3.1 BENZOIC ACID Benzoic acid, which usually is used in the form of its sodium salt, sodium benzoate (Figure 7.1), long has been used as an antimicrobial additive in foods. It is used in carbonated and still beverages, syrups, fruit salads, icings, jams, jellies, preserves, salted margarine, mincemeat, pickles and relishes, pie, pastry fillings, prepared salads, fruit cocktail, soy sauce, and caviar. The use level ranges from 0.05 to 0.1%.

Fig. 7.1 Structures of benzoic acid and sodium benzoate

Benzoic acid in the acid form is quite toxic but its sodium salt is much less toxic (Table 7.3). The sodium salt is preferred because of the low aqueous solubility of the free acid. In vivo, the salt is converted to acid, which is the more toxic form. Subacute toxicity studies of benzoic acid in mice indicated that ingestion of benzoic acid or its

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sodium salt caused weight loss, diarrhea, irritation of internal membranes, internal bleeding, enlargement of liver and kidney, hypersensitivity, and paralysis followed by death. When benzoic acid (80 mg/kg body weight) and sodium bisulfate (160 mg/kg body weight) or their mixture (benzoic and/sodium bisulfate ¼ 80 mg/160 mg) were fed to mice for 10 weeks, the death rate was 66% from the mixture and 32% from benzoic acid alone. Four-generation reproductive and developmental toxicities of benzoic acid were examined using diets containing 0, 0.5, and 1% of benzoic acid fed to male and female rats housed together for eight weeks. The second generation was observed through its entire life cycle and the third and fourth generations were examined by autopsy. No changes in normal patterns of growth, reproduction, or lactation during life were recorded and no morphological abnormalities were observed from the autopsies. Table 7.3 Acute Toxicity of Sodium Benzoate, Animal

Method

LD50 (mg/kg)

Rat Rat Rabbit Rabbit Dog

Oral Intravenous injection Oral Subcutaneous injection Oral

2,700 1714 124 2,000 2,000 2,000

Degradation pathways for benzoic acid also have been studied in detail and the results have supported the harmlessness of this substance. The metabolic breakdown pathways of benzoic acid are shown in Figure 7.2.

Fig. 7.2 The metabolic breakdown pathways of benzoic acid.

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Fig. 7.3 Structures of sorbic acid

The total dose of benzoic acid is excreted within 10 to 14 hours and 75 to 80% is excreted within 6 hours. After conjugation with glycine, 90% of benzoic acid appears in the urine as hippuric acid. The rest forms a glucuronide, 1-benzoylglucuronic acid. The lower aliphatic esters of benzoic acid are first hydrolyzed by esterase, which abounds in the intestinal wall and liver. The resulting benzoic acid subsequently is degraded in the usual manner. 7.3.2 SORBIC ACID AND POTASSIUM SORBATE Sorbic acid and its salts have broad-spectrum activity against yeast and molds, but are less active against bacteria. The antimicrobial action of sorbic acid was discovered independently in the United States and Germany in 1939, and since the mid-1950s sorbates have been increasingly used as preservatives. The structures of these compounds are shown in Figure 7.3. Sorbates generally have been found superior to benzoate for preservation of margarine, fish, cheese, bread, and cake. Sorbic acid and its potassium salts are used in low concentrations to control mold and yeast growth in cheese products, some fish and meat products, fresh fruits, vegetables, fruit beverages, baked foods, pickles, and wines. Sorbic acid is practically non-toxic. Table 7.4 shows acute toxicity of sorbic acid and its potassium salt. Animal studies have not shown obvious problems in tests performed with large doses for longer time periods. When sorbic acid (40 mg/kg/day) was injected directly into the stomach of male and female mice for 20 months, no differences were observed in survival rates, growth rates, or appetite between the injected mice and the control. When the dose was increased to 80 mg/kg/day for three additional months, however, some growth inhibition was observed. When potassium sorbate (1 and 2% in feed) was fed to dogs for three months, no pathological abnormalities were observed. This evidence indicates that the subacute toxicity of sorbic acid is negligible. Two-generation reproduction and developmental toxicities of sorbic acid using various animals have shown that neither sorbic acid nor its potassium salt induces malignant growths in animals. For example, rats fed with 5% sorbic acid in feed for two generations (1000 days) showed no changes in growth rates, rates of reproduction, or any other behaviours.

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Food Hygiene and Toxicology Table 7.4 Acute Toxicity of Sorbic Acid and Its Potassium Salt.

Animal

Compound

Method

LD50 (g/kg)

Rat Rat Mouse Mouse Mouse Mouse

Sorbic acid Potassium sorbate Sorbic acid Potassium sorbate Sorbic acid Potassium sorbate

Oral Oral Oral Oral Intraperitoneal Intraperitoneal

10.5 4.2 >8 4.2 2.8 1.3

As a relatively new food additive, sorbate has been subject to stringent toxicitytesting requirements. It may well be the most intensively studied of all chemical food preservatives. In 90-day feeding studies in rats and dogs and a lifetime feeding study in rats, a 5% dietary level of sorbates procured no observable adverse effects. However, at a 10% dietary level in a 120-day feeding study, rats showed increased growth and increased liver weight. This has been attributed to the caloric value of sorbate at these high dietary levels since it can act as a substrate for normal catabolic metabolism in mammals. Sorbates are not mutagenic or tumorigenic, and as noted previously, no reproductive toxicity has been observed. 7.3.3 HYDROGEN PEROXIDE Hydrogen peroxide is used as an agent to reduce the number of bacteria in dairy products or other foodstuffs. In the dairy industry, hydrogen peroxide also has been used as a substitute for heat pasteurization in the treatment of milk and as a direct preservative in keeping the quality of the milk. In Japan, it has been used as a preservative for fishpaste products. Hydrogen peroxide also has a bleaching effect. The use of highly pure hydrogen peroxide in manufactured cheese has been approved by the United States Food and Drug Administration (industrial grade hydrogen peroxide is usually a 3–35% aqueous solution; a commercial home product is a 3% aqueous solution). Acute toxicities (LD50) of hydrogen peroxide for rats are 700 mg/kg/b.w. and 21 mg/kg/b.w. by subcutaneous injection and intravenous injection, respectively. When large amounts of hydrogen peroxide were injected directly into the stomachs of rats, weight and blood protein concentrations were changed slightly. When hydrogen peroxide was mixed with feed, however, no abnormalities were observed. The use of bactericides has been limited due to their toxicity to humans, and only hydrogen peroxide currently is recognized for use. 7.3.4 AF-2 [2-(-FURYL)-3-(5-NITRO-2-FURYL)ACRYLAMIDE] The antibacterial activity of nitrofuran derivatives was first recognized in 1944. The disclosed antibacterial properties of these compounds had created a new group of antimicrobial agents. Such activity is dependent on the presence of a nitro group in the 5-position of the furan ring. Numerous 5-nitrofuran derivatives have been synthesized

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and certain compounds have been used widely in clinical and veterinary medicine and as antiseptics for animal foods. AF-2 (Figure 7.4) was legally approved for use in Japan in 1965 and was added to soybean curd (tofu), ham, sausage, fish ham, fish sausage, and fish paste (kamaboko). The safety-testing data on which the compound was approved were those obtained for acute and chronic toxicity for two years and reproductive potential for four generations using mice and rats. At the time, no attention was paid to mutagenicity. In 1973, AF-2 was proved to be mutagenic in various microbial test systems. The mutagenicity of this food additive strongly suggested its carcinogenicity and the risk of its use as a food additive. Within a year or two after the discovery of its mutagenicity, the actual carcinogenicity of this chemical was demonstrated in animal studies. Since that time, more emphasis has been placed on finding causative agents of cancer. Short-term bioassays had received much attention because they were able to screen suspected carcinogens. The mechanism of carcinogenesis is not yet clear, but a close relation between carcinogens and mutagens has been demonstrated. AF-2 was the first example of compound that was shown to be a carcinogen. The fact that AF-2 was discovered first to be mutagenic proved the value of mutagenicity testing as a screening method for carcinogens.

Fig. 7.4 Structure of 2-(2-furyl)- 3-(5-nitro-2-furyl)acrylamide (AF-2)

7.4 ANTIOXIDANTS

One of the most common types of food deterioration is an undesirable change in colour or flavour caused by oxygen in air (oxidative deterioration). Oxidation causes changes not only in colour or flavour but also decreases the nutritional value of food and sometimes produces toxic materials. Since most foods consist mainly of carbohydrates, fats, proteins, and water, micro-biological spoilage is one of the most important factors to be considered in preserving the carbohydrate and protein portions of food products. However, oxidation, particularly atmospheric oxidation, is the chief factor in the degradation of fats and fatty portions of foods. Oxidative deterioration of fat results not only in the destruction of vitamins A, D, E, K, and C, but also in the destruction of essential fatty acids and the development of a pungent and offensive off-flavour. In extreme cases, toxic by-products have resulted from oxidative reactions. The most efficient method of preventing oxidative degradation is the use of antioxidative agents. The antioxidants may be classed as natural and synthetic. Naturally occurring antioxidants exhibit relatively weak antioxidant properties. As a consequence,

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synthetic antioxidants have been developed for use in foods. In order for these substances to be allowed in foods, they must have a low toxicity; should be effective in low concentrations in a wide variety of fats; should contribute no objectionable flavour, order, or colour to the product; and should have approval by the Food and Drug Administration. 7.4.1 L-ASCORBIC ACID (VITAMIN C) L-Ascorbic acid, or vitamin C, is widely present in plants. The structures of ascorbic acid and dehydroascorbic acid are shown in Figure 7.5. Vitamin C is not only an important nutrient but is also used as an antioxidant in various foods. However, it is not soluble in fat and is unstable under basic conitions. Vitamin C reduces cadmium toxicity and excess doses prolong the retention time of an organic mercury compound in a biological system. Overdoses of vitamin C (106 g) induce perspiration, nervous tension, and lowered pulse rate. WHO recommends that daily intake be less than 0.15 mg/kg. Toxicity due to ascorbic acid has not been reported. Although repeated intravenous injections of 80 mg dehydroascorbic acid was reported to be diabetogenic in rats, oral consumption of 1.5 g/day of ascorbic acid for six weeks had no effect on glucose tolerance or glycosuria in 12 normal adult males and produced no change in blood glucose concentrations in 80 diabetics after five days. The same report noted that a 100-mg intravenous dose of dehydroascorbic acid given daily for prolonged periods produced no signs of diabetes. Ascorbic acid is readily oxidized to dehydroascorbic acid, which is reduced by glutathione in blood.

Fig. 7.5 Structures of ascorbic acid and dehydroascorbic acid.

7.4.2 DL- -TOCOPHEROL (VITAMIN E) -Tocopherol is known as vitamin E and exists in many kind of plants, especially in lettuce and alfalfa. Its colour changes from yellow to dark brown when exposed to sunlight. The structure of -tocopherol is shown in Figure 7.6.

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Fig. 7.6 Structure of a-tocopherol (vitamin E)

Natural vegetable oils are not readily oxidized due to the presence of tocopherol. During refining processes, however, tocopherol may be removed from oils; consequently, refined vegetable oils can become unstable towards oxidation. In one experiment, vitamin E appeared to be relatively innocuous, having been given to patients for months both orally and parenterally at a dosage level of 300 mg/day without any observed ill effects. However, in another experiment, 6 out of 13 patients given similar doses complained of headache, nausea, fatigue, dizziness, and blurred vision. Although the chronic toxicity of vitamin E has not been thoroughly studied, WHO recommends 2 mg/kg/day as the maximum daily dose. 7.4.3 PROPYL GALLATE Propyl gallate (n-propyl-3, 4, 5-trihydroxybenzoate, Figure 7.7) is used in vegetable oils and butter. When 1.2 or 2.3% propyl gallate was added to feed for rats, loss of weight was observed. This may be due to the rats’ reluctance to eat food that was contaminated with the bitter taste of propyl gallate. When it was given for 10 to 16 months at the 2 to 3% level, 40% of the rats died within the first month and the remainder showed severe growth inhibition. Autopsies of rats indicated kidney damage resulting from the ingestion of propyl gallate. However, no other animal studies show serious problems and further studies indicated that propyl gallate does not cause serious chronic toxicities

Fig. 7.7 Structure of n-propyl-3,4,5-trihydroxybenzoate (propyl gallate).

7.4.4 BUTYLATED HYDROXYANISOL AND BUTYLATED HYDROXYTOLUENE Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are the most commonly used antioxidants and present constant intractable problems to the food industry. The structures of BHA and BHT are shown in Figure 7.8. BHA produces mild diarrhea in dogs when it is fed continuously for four weeks at the level of 1.4 to 4.7 g/ kg. It also causes chronic allergic reactions, malformations, and damage to the metabolic

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system. When BHT was fed to rats at levels of 0.2, 0.5, and 0.8% mixed with feed for 24 months, no pathological changes were observed. The same results were obtained when the dose was increased to 1% of the feed.

Fig. 7.8 Structure of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA).

Antioxidants—including vitamin C, vitamin E, BHA, and BHT—have some anticarcinogenic activities. If BHT is mixed with a known carcinogen such as N-2fluorenyl acetamide (FAA) or azoxymethane (AOM) in the feed, the rate of tumor induction in rats is diminished. The mechanisms of anti-oxidants in chemical carcinogenesis are not well understood yet, but the relationship between chemical carcinogens and BHT has been intensively investigated.

7.5 SWEETENERS

Naturally occurring sweeteners such as honey and sucrose were known to the ancient Romans. However, sweeteners obtained from natural sources have been limited. To supplement the demand, sweetening agents such as saccharin have been synthesized since the late nineteenth century. Recently, these non-nutritive sweeteners have begun to receive much attention as ingredients in low-calorie soft drinks. The synthetic or non-carbohydrate sweeteners provide sweetened foods for diabetics who must limit sugar intake, for those who wish to limit carbohydrate calorie intake, and for those who desire to reduce food-induced dental caries. 7.5.1 SACCHARIN AND SODIUM SACCHARIN Saccharin, which is 300 to 500 times sweeter than sucrose, is one of the most commonly used artificial sweeteners. The name saccharin is a commercial name of the Fahlberg and List Company. The sodium salt is the form actually used in the formulation of foods and beverages (Figure 7.9). Its acute toxicity is shown in Table 7.5.

Fig. 7.9 Structures of saccharin and sodium saccharin.

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Food Additives Table 7.5 Acute Toxicity of Sodium Saccharin. Animal

Method

LD50 (g/kg)

Mouse Mouse Rat Rat Rat Rabbit

Oral Intraperitoneal Oral Intraperitoneal Oral Oral

17.5 6.3 17.0 7.1 14.2 1.3 5–8 (LD)

It has been questioned whether or not saccharin is a health hazard. In 1972, it was found that 7.5% of saccharin in feed produced bladder cancer in the second generation of rats. Some reports, however, showed contra-dictory results. Consequently, WHO has recommended that daily intake of saccharin be limited to 0 to 0.5 mg/kg. The carcinogenicity of saccharin is still under investigation. When pellets of saccharin and cholesterol (1:4) were placed in the bladders of mice, tumors developed after 40 to 52 weeks. When 2.6 g/kg of a mixture of sodium cyclamate and saccharin (10:1) was given to rats for 80 days, eight rats developed bladder tumors after 105 weeks. When only sodium cyclamate was fed to rats for two years, blad-der cancer also appeared. The main attention, therefore, has been given to the carcinogenicity of sodium cyclamate. 7.5.2 SODIUM CYCLAMATE Sodium cyclamate is an odorless powder. It is about 30 times as sweet as sucrose in dilute solution. The structure of sodium cyclamate is shown in Figure 7.10 and its acute toxicity is shown in Table 7.6. Capillary transitional cell tumors were found in the urinary bladders of 8 out of 80 rats that received 2600 mg/kg body weight per day of a mixture of sodium cyclamate and sodium saccharin (10:1) for up to 105 weeks. When the test mixture was fed at dietary levels designed to furnish 500, 1120, and 2500 mg/ kg body weight to groups of 35 and 45 female rats, the only significant finding was the occurrence of papillar carcinomas in the bladders of 12 of 70 rats fed the maximum dietary level of the mixture (equivalent of about 25 g/kg body weight) for periods ranging from 78 to 105 weeks (except for one earlier death).

Fig. 7.10 Structures of sodium

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Food Hygiene and Toxicology Table 7.6 Acute Toxicity of Sodium Cyclamate. Animal

Method

LD50 (g/kg)

Mouse Mouse Mouse Rat Rat Rat

Oral Intraperitoneal Intramuscular Oral Intraperitoneal Intramuscular

10–15 7 4–5 2–17 6 3–4

In vivo conversion from sodium cyclamate to cyclohexylamine was observed particularly in the higher dosage group. Cyclohexylamine is very toxic (LD50 rat oral ¼ 157 mg/dg) compared to sodium cyclamate (LD50 oral ¼ 12 g/kg). In 1968, the FDA discovered teratogenicity of sodium cyclamate in rats and prohibited its use in food.

7.6 COLOURING AGENTS

Colouring agents have been used to make food more attractive since ancient times. The perception and acceptability of food, in regards to taste and flavour, is strongly influenced by its colour. Nutritionists have long known that without expected colour cues, even experts have difficulty in identifying tastes. Certain varieties of commercial oranges have colouring applied to their peels because the natural appearance—green and blotchy—is rejected by consumers as unripe or defective. Consumers reject orange juice unless it is strongly coloured, even if it is identical in taste and nutritional value. Congress has twice overridden the FDA (in 1956 and 1959) when it proposed a ban of the colouring agents used. The natural pigments of many foods are unstable in heat or oxidation. Thus, storage or processing can lead to variations in colour even when the nutritional value remains unchanged. Changes in the appearance of a product over time may cause consumers to fear that a “bad” or an adulterated product has been purchased, particularly in light of highly publicized incidents of product tampering. The use of food colouring can resolve this problem for retailers and manufacturers. Ripe olives, sweet potatoes, some sauces and syrups, as well as other foods, are coloured mainly to ensure uniformity and consumer acceptability. Candies, pastries, and other products such as pharmaceutical preparations are often brightly coloured. Pet foods are coloured for the benefit of human owners, not their colour-blind pets. Such applications are criticized by some as unnecessary, or even frivolous, even when natural food dyes are involved. Red colour can be produced naturally from a dried sugar beet root (beet-red) or from insects such as cochineal (Central America) and lac dye (South-east Asia). Natural dyes, however, are usually not clear, and their variety is limited. Synthetic colouring agents began to replace them in the late nineteenth century. Twenty-one synthetic chemicals were recognized for use in 1909 at the Second International Red Cross

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Conference. About 80 synthetic dyes were being used in the United States for colouring foods in 1900. At that time, there were no regulations regarding their safety. Many of the same dyes were used to colour cloth and only the acute toxicity of these colouring agents was tested before they were used in foods. The chronology of the addition of new colours is shown in Table 7.7. The number of colouring agents currently permitted as food additives is much fewer than previously and still is being reduced. Table 7.7 Chronology of Newly Developed. Synthetic Colouring Agents Year Agent 1916 1918 1922 1927 1929 1929 1929 1950 1966 1971

Tartrazine Yellow AB & OB Guinea Green Fast Green Ponceau SX Sunset Yellow Brilliant Blue Violet No. 1 Orange B FD&C Red No. 40

A large class of synthetic organic dyes is the azo group. Many commercial dyes belonged to this class, particularly due to their vivid colours, especially reds, oranges, and yellows. Azo compounds contained the functional group (R—N = N—R’), in which R and R’ can be either aryl or alkyl. There are two kinds of azo dyes, those that are water-soluble and those that are not. In general, the water-soluble azo dyes are less toxic because they are more readily excreted from the body. Either type, however, can be reduced to form the toxic amino group, —NH2, in the body in conjunction with the action of microorganisms such as Streptococcus, Bacillus pyocyaneus, and Proteus sp. One particular bioassay report on azo compounds showed that only 12 out of 102 azo compounds did not reduce into amine. In 1937, dimethyl amino azobenzene or butter yellow was found to induce malignant tumors in rats. The carcinogenicity of butter yellow was later confirmed. 7.6.1 AMARANTH (FD&C RED NO. 2) Amaranth is 1-(4-sulfo-1-paphthylazo)-9-naphthol-3,6-disulfonic acid, trisodium salt (Figure 7.11) and is an azo dye. It is a reddish-brown powder with a water solubility of 12 g/100 ml at 30C. Before it was prohibited by the FDA in 1976 following indications that it induced malignant tumors in rats, amaranth had been used in almost every processed food with a reddish or brownish colour, including soft drinks, ice creams, salad dressings, cereals, cake mixes, wines, jams, chewing gums, chocolate, and coffee

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as well as a variety of drugs and cosmetics at the level of 0.01 to 0.0005%. In 1973, an estimated $2.9 million worth of amaranth was added to more than $10 billion worth of products.

Fig. 7.11 Structure of amaranth.

In toxicity studies, amaranth (0.5 ml of a 0.1% solution) was injected under the skin of rats twice a week for 365 days, in which no tumor growth was observed. When 0.2% of amaranth in feed (average 0.1 g/kg/day) was fed to rats for 417 days, no induction of tumors was observed. However, one case of intestinal cancer was observed when feeding was continued for an additional 830 days. The FAO/WHO special committee determined its ADI as 0 to 1.5 mg/kg. It was determined that amaranth is metabolized into amine derivatives in vivo. Amaranth is reduced by aqueous D-fructose and D-glucose at elevated temperatures to form a mixture of hydrazo and amine species, which may have toxicological significance. The interactions between additives such as amaranth and other food components should be considered from the viewpoint of toxicology. 7.6.2 TARTRAZINE (FD&C YELLOW NO. 4) This colouring agent is 5-hydroxy-1-p-sulfophenyl-4-(p-sulfophenylazo)-pyrazol-3carboxylic acid, trisodium salt (Figure 7.12). It is a yellow powder and has been used as food colouring additive since 1916. Tartrazine is known as the least toxic colouring agent among synthetic colouring chemicals. The median acute oral lethal dose of tartrazine in mice is 12.17 g/kg. Beagle dogs received tartrazine as 2% of the diet for two years without adverse effects, with the possible exception of pyloric gastritis in one dog. Tumor incidence was unchanged relative to controls, in rats receiving tartrazine at 1.5% of the diet for 64 weeks, and in rats administered this dye at 5.0% of the diet for two years. Human sensitivity to tartrazine has been reported with some frequency and has been estimated to occur in 1/10,000 persons. Anaphylactic shock, potentially life-threatening, has been reported but symptoms more commonly cited are urticaria (hives), asthma, and purpura (blue or purple spots on the skin or mucous membrane). Of 97 persons with allergic symptoms in one trial, 32 had adverse reactions to challenge with 50 mg tartrazine. Physicians use 0.1 to 10 mg tartrazine to test for its sensitivity.

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Fig. 7.12 Structure of tartrazine

In the United States, tartrazine can be used in foods only as a colouring agent (FDA Regulations 8.275, 8.501). It is also permitted in Great Brit- ain for use as a foodcolouring agent. An ADI was set in 1966 at 0 to 7.5 mg/kg by the FDA. Although many synthetic colouring agents are toxic if used in large enough amounts, and many are suspected carcinogens as well, natural colouring agents are not always safe. Caramel, which gives a light brown colour, contains carcinogenic benzo[a]pyrene in small amounts; curcumin, which gives the yellow colour to curry, is 15 times more toxic than tartrazine.

7.7 FLAVOURING AGENTS

For many years, natural or synthetic flavour and fragrance chemicals have been used to enhance the palatability, and thus, increase the acceptability of foods. They also are used to produce imitation flavours for various food products including ice cream, jam, soft drinks, and cookies. Since the mid-nineteenth century, numerous flavour chemicals have been synthesized. Coumarin was synthesized in 1968; vanilla flavour, vanillin, was synthesized in 1874; cinnamon flavour was made in 1884 (cinnamic aldehyde). By the twentieth century, nearly 1000 flavouring chemicals had been developed. At the present time, over 3000 synthetic chemicals are used as flavour ingredients. As was the case with colouring agents, the toxicity of flavouring agents began to receive attention in the 1960s. Most natural flavours used in the United States are generally recognized as safe (GRAS) on the basis of their long time occurrence in foods and wide consumption with no apparent ill effects. These chemicals have been used in large quantities in most food products with little regard for safety. Food industries even attempt to produce flavour ingredient chemicals that are found naturally in plants (so-called natural identical substances). Since general attention to food safety has focused on incidental additives, such as pesticides, it has been assumed that the natural flavouring chemicals are not health hazards. However, there are many toxic chemicals in natural products and their chronic toxicity should be carefully reviewed.

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Fig. 7.13 Structure of methyl anthranilate.

7.7.1 METHYL ANTHRANILATE Methyl anthranilate (Figure 7.13) is a colourless liquid that has a sweet, fruity, grapelike flavour. It is found in the essential oils of orange, lemon, and jasmine and has been widely used to create imitation Concord grape flavour. Table 7.8 shows the acute toxicity of methyl anthranilate. Methyl anthranilate promotes some allergic reactions on human skin, which has led to it being prohibited for use in cosmetic products. Table 7.8 Acute Toxicity of Methyl Anthranilate. Animal

LD50 (oral, mg/kg)

Mouse Rat Guinea pig

3,900 2,910 2,780

7.7.2 SAFROLE (1-ALLYL-3,4- METHYLENEDIOXYBENZENE) Safrole is a colourless oily liquid possessing a sweet, warm-spicy flavour. It has been used as a flavouring agent for more than 60 years. Oil of sassafras, which contains 80% safrole, also has been used as a spice.

Fig. 7.14 Structure of safrole and its metabolite, 1’-hydroxysafrole

In the United States, the FDA banned the use of safrole in 1958 and many other countries followed this lead and also banned the use of safrole in flavours. Safrole, either naturally occurring in sassafras oil or the synthetic chemical, has been shown to induce liver tumors in rats. The continuous administration of safrole at 5,000 ppm in

Toxicants Formed during Food Processing

195

the total diet of rats caused liver tumors. Studies in dogs showed extensive liver damage at 80 and 40 mg/kg, lesser damage at lower levels, but no tumors. In vivo, safrole metabolites into 1’-hydroxysafrole. The structures of safrole and 1’-hydroxysafrole are shown in Figure 7.14. 7.7.3 DIACETYL (2, 3-BUTANE DIONE) Diacetyl is an intensely yellowish or greenish-yellow mobile liquid. It has a very powerful and diffusive, pungent, buttery odour and typically used in flavour compositions, including butter, milk, cream, and cheese. Diacetyl was found to be mutagenic in Ames test conducted under various different conditions with Salmonella typhimurium strains. For example, diacetyl was mutagenic by TA100 in the absence of S9 metabolic activation at doses up to 40 mM/plate. It was mutagenic in a modified Ames assay in Salmonella typhimurium strains TA100 with and without S9 activation. The acute oral LD50 of diacetyl in guinea pigs was calculated to be 990 mg/kg. The acute oral LD50 of diacetyl in male rats was calculated to be 3400 mg/kg, and in female rats, the LD50 was calculated to be 3000 mg/ kg. When male and female rats were administered via gavage a daily dose of 1, 30, 90, or 540 mg/kg/day of diacetyl in water for 90 days, the high-dose produced anemia, decreased weight gain, increased water consumption, increased leukocyte count, and an increase in the relative weights of liver, kidneys, and adrenal and pituitary glands. The data for teratogenicity and carcinogenicity are not available. Although the FDA has affirmed diacetyl GRAS as a flavouring agent, low molecular weight carbonyls, such as formaldehyde, acetaldehyde, and glyoxal have been reported to possess a certain chronic toxicity. Therefore, diacetyl should be reviewed carefully for its toxicity.

7.8 FLAVOUR ENHANCERS

A small number of food additives are used to modify the taste of natural and synthetic flavours even though they do not directly contribute to flavour; such substances are known as flavour enhancers. Most people are familiar with the use of table salt to enhance the flavour of a wide variety of foods. Salt can be an effective enhancer even at levels far below the threshold for salty taste and is widely used in processed foods such as canned vegetables and soups. Another well-known enhancer is monosodium glutamate (MSG). Its use as an enhancer has periodically aroused concern about the potential toxicity of a quantity of free glutamate ingested at once. MSG became controversial because of its association with the so-called Chinese restaurant syndrome. Symptoms of the syndrome, which is usually self-diagnosed, include headache and drowsiness. Because of these concerns, both the acute and chronic toxicity of MSG have been widely studied. After a review of the available data, the FDA affirmed the GRAS status of MSG in the United States. Although some individuals are susceptible to transient discomfort following ingestion of MSG, it does not pose any risk of lasting injury.

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8 Toxicants Formed during Food Processing

8.1 INTRODUCTION

Food processing practices have been used since the Ancient Era. Roasting and drying were applied to eggs and honey in the Old Stone Age (15,000 BCE). Smoking techniques were used to preserve milk and wine in the New Stone Age (9,000 BCE). Fermentation of rice and onions was recorded in the Bronze Age (35,000 BCE). Flavouring food started in the Iron Age (1,500 BCE) and adulteration methods are observed in the Roman era ( 600 BCE). And, in the Modern Era, the development of food processing technology—which includes evaporation, smoking, sterilization, pasteurization, irradiation, pickling, freezing, and canning—greatly expanded the longevity of food storage. For example, smoke treatment made a year-round supply of fish possible; and canned foods could be sent anywhere in the world. Another important method of food processing is home cooking. Cooking techniques such as frying, toasting, roasting, baking, broiling, steaming, and boiling increases the palatability—flavour, appearance, and texture. Cooking also improves the stability as well as digestibility of foods. Moreover, it kills toxic microorganisms and deactivates such toxic substances as enzyme inhibitors. Since antiquity, people appreciated home-cooked food. In the United States, commercial food processing is subject to regulation by the FDA and must meet specified standards of cleanliness and safety. Some particular methods of food processing are considered under the category of food additives, since they may intentionally alter the form or nature of food. Chemical changes in food components, including amino acids, proteins, sugars, carbohydrates, vitamins, and lipids,

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197

caused by high-heat treatment have raised questions about the consequence of reducing nutritive values and even the formation of some toxic chemicals such as poly-cyclic aromatic hydrocarbons (PAHs), amino acid or protein pyrolysates, and N-nitrosamines. Among the many reactions that occur in processed foods, the Maillard Reaction plays the most important role in the formation of various chemicals, including toxic ones. During processing, undesirable foreign materials may accidentally be mixed into foods. Although most modern food factories are engineered to avoid any occurrence of food contamination during processing, low-level contamination is hard to eliminate entirely. Many instances of accidental contamination by toxic materials have been reported. In 1955 in Japan, sodium phosphate, a neutralizing agent, was contaminated with sodium arsenite and added to milk during a drying process. The final commercial dried milk contained 10 to 50 ppm of arsenic. Subsequently, many serious cases of arsenic poisoning were reported. It is a common misunderstanding that gamma irradiation, which is most often used for food irradiation, produces radioactive materials in foods. In fact, although the electromagnetic energy used for irradiation is sufficient to penetrate deep into foods and can kill a wide range of micro-organisms, it is far below the range required to produce radioactivity in the target material. However, there are still uncertainties about the toxicity of chemicals that may be produced during irradiation. The energies used are sufficient to produce free radicals, which may in turn produce toxic chemicals.

8.2 POLYCYCLIC AROMATIC HYDROCARBONS (PAHS)

Polycyclic aromatic hydrocarbons (PAHs) occur widely in the environment and in everyday products such as water, soil, dust, cigarette smoke, rubber tires, gasoline, roasted coffee, baking bread, charred meat, and in many other foods. Typical PAHs are shown in Figure 8.1. For over 200 years, carcinogenic effects have been ascribed to PAHs. In 1775, Percival Pott, an English physician, made the association between the high incidence of scrotal cancer in chimney sweeps and their continual contact with chimney soot. Research on the toxicity of PAHs, however, progressed somewhat slowly. In 1932, benzo[a]pyrene was isolated from coal tar and found to be highly carcinogenic in experimental animals. 8.2.1 OCCURRENCE One of the most abundant food sources of PAHs is vegetable oil. However, it is possible that the high levels of PAHs occurring in vegetable oils are due to endogenous production, with environmental contamination playing only a minor role. Some PAHs in vegetables are apparently due to environmental contamination because PAH levels decrease in vegetables cultivated farther from industrialized centers and freeways. The occurrence of PAHs in margarine and mayonnaise appears to be due to contamination of the oils

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used to make these products. Levels of PAHs in soil can also be quite high, even in areas distant from industrialized centers. Levels of PAHs of 100 to 200 ppm in the soil were found in some locations distant from human populations. It is thought that these levels result primarily as a residue from decaying vegetation. The significance of these relatively high levels of potentially carcinogenic substances in the soil is not fully understood.

Fig. 8.1 Structures of typical PAHs.

Charcoal broiling or smoking of food also causes PAH contamination (Table 8.1). PAHs are formed mainly from carbohydrates cooked at high temperatures in the absence of oxygen. Broiling meat over hot ceramic or charcoal briquettes allows the melted fat to come into contact with a very hot surface. PAHs are produced in the ensuing reactions.

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Toxicants Formed during Food Processing

These products rise with a resulting cooking fumes and are deposited on the meat. Similarly, the presence of PAHs in smoked meats is due to the presence of these substances in smoke. PAH levels in meat that is cooked at a greater distance from the coals are lower than in meat that is cooked close to the coals. Obviously, food processing produces certain levels of PAHs. It is of major importance to be aware of the presence of carcinogenic PAHs in our foods, and the overall public health hazard should be evaluated and controlled. Table 8.1 Polycyclic Aromatic Hydrocarbons Found in Smoked Foods (ppb). Food

Benzo[a]anthracene

Benzo[a]pyrene

Benzo[e]pyrene

Fluoranthene Pyrene

Beef Cheese Herring Dried herring Salmon Sturgeon Frankfurters Ham

0.4

—

—

1.7 0.5 — — 2.8

1.0 — 0.8 — 3.2

1.2 0.4 — — 1.2

0.6 2.8 3.0 1.8 3.2 2.4 6.4 14.0

0.5 2.6 2.2 1.8 2.0 4.4 3.8 11.2

8.2.2 BENZO[A]PYRENE (BP) The most commonly known carcinogenic PAH is benzo[a]pyrene (BP), which is widely distributed in various foods (Table 8.2). BP was reportedly formed at a level of 0.7 and 17 ppb at 370 to 390 and 650 C, respectively, when starch was heated. Amino acids and fatty acids also produced BP upon high-temperature treatment (Table 8.3). Many cooking processes utilize the 370 to 390 C range; for example, the surface temperature of baking bread may approach 400 C and deep fat frying is 400 to 600 C, suggesting that cooking produces some PAHs, including BP. The meat inspection division of the USDA and FDA analyzed 60 assorted food-stuffs and related materials for BP. Samples that contain relatively high levels of BP are shown in Table 8.1. Table 8.2 Benzo[a]pyrene Found in Various Foods. Food

Concentration (ppb)

Fresh vegetables Vegetable oil Coffee Tea Cooked sausage Smoked hot sausage Smoked turkey fat Charcoal-broiled steak Barbecued ribs

2.85–24.5 0.41–1.4 0.31–1.3 3.9 12.5–18.8 0.8 1.2 0.8 10.5

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Food Hygiene and Toxicology Table 8.3 Amounts of PAHs Produced from Carbohydrates, Amino Acids, and Fatty Acids Heated at 500 and 700 C (mg/50 g). Starch

PAH Pyrene Fluoranthene Benzo[a]pyrene

500 41 13 7

700 965 790 179

D-Glucose

L-Leucine

Stearic Acid

500 23 19 6

500 — — —

500 0.7 — —

700 1,680 1,200 345

700 1,200 320 58

700 18,700 6,590 4,440

8.2.2.1 Toxicity BP is a reasonably potent contact carcinogen, and therefore has been sub-jected to extensive carcinogenic testing. Table 8.4 shows the relative carci-nogenicity of BP and other PAHs. A diet containing 25 ppm of BP fed to mice for 140 days produced leukemia and lung adenomas in addition to stomach tumors. Skin tumors developed in over 60% of the rats treated topically with approximately 10 mg of benzo[a]pyrene three times per week. The incidence of skin tumors dropped to about 20% when treatment was about 3 mg 3 per week. Above the 10 mg range, however, the incidence of skin tumors increased dramatically to nearly 100%. BP is also carcinogenic when administered orally. In one experiment, weekly doses of greater than 10 mg administered for 10 weeks induced stomach cancers, although no stomach cancers were produced at the dose of 10 mg or less. At 100 mg doses, nearly 79% of the animals had developed stomach tumors by the completion of the experiment. When 15 ppm of BP in feed was orally administered to mice, production of leukemia, lung adenomas, and stomach tumors were observed after 140 days. Table 8.5 shows the stomach tumor incidence (%) occurred in mice by BP. Table 8.4 Relative Carcinogenicity of Typical Polycyclic Aromatic. PAH

Relative Activity

Benzo[a]pyrene 5-Methylchrysene Dibenzo[a,h]anthracene Dibenzo[a,i]pyrene Benzo[b]fluoranthene Benzo[a]anthracene Benzo[c]phenanthrene Chrysene þþþ, high; þþ, moderate; þ, weak.

þþþ þþþ þþ þþ þþ þ þ þ

Table 8.5 Stomach Tumor Incidence Caused by BP in Mice Dose (oral, ppm) 30 40–45 50–250

Duration (days)

Incidence (%)

110 110 122–197

0 10 70

201

Toxicants Formed during Food Processing 250 250 250 250

1 2–4 5–7 30

0 10 30–40 100

8.2.2.2 Mode of Toxic Action BP is transported across the placenta and produces tumors in the offspring of animals treated during pregnancy. Skin and lung tumors appear to be the primary lesions in the offspring. The biochemical mechanisms by which benzo[a]pyrene initiates cancer have been studied in some detail. Benzo[a]pyrene is not mutagenic and carcinogenic by itself, but first must be converted to active metabolites. The metabolic conversion initially involves a cytochrome P450-mediated oxidation, producing a 7,8-epoxide. The 7,8epoxide, in turn, undergoes an epoxide hydrolase-mediated hydration, producing the 7,8-diol, which, upon further oxidation by cytochrome P450, produces the corresponding diolepoxide. This diolepoxide is highly mutagenic without metabolic activation and is also highly carcinogenic at the site of administration. The benzo[a]pyrene diolepoxide can react with various components in the cells, in which case it is possible that mutation will occur. This is thought to be the primary event in benzo[a]pyrene-induced carcinogenesis. The hypothe-sized chemical mechanisms of BP toxicity are shown in Figure 8.2.

Fig. 8.2 Hypothesized chemical mechanisms of alkylating agent from benzo[a]pyrene.

The criterial factors for PAHs to be carcinogenic are (1) the entire polycyclic aromatic hydrocarbon must be coplanar; (2) a phenanthrene nucleus must be present together with some substituents, preferably at least one additional benzene ring; and (3) the covex edge (C4 – C5) of the phenanthrene moiety must be free of substituents.

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8.3 MAILLARD REACTION PRODUCTS

In 1912, the French chemist L. C. Maillard hypothesized the reaction that accounts for the brown pigments and polymers produced from the reaction of the amino group of an amino acid and the carbonyl group of a sugar. Maillard also proposed that the reaction between amines and carbonyls was implicated in in vivo damage; in fact, the Maillard reaction later was proved to initiate certain types of damage in biological systems. The Maillard reaction is also called non-enzymatic browning reaction. It occurs readily in any matrix containing amines and carbonyls under heat treatment. A summary of the Maillard reaction is shown in Figure 8.3.

Fig. 8.3 Systematic diagram of the Maillard reaction

Many chemicals formed from this reaction in addition to the brown pigments and polymers. Because of the large variety of constituents, a mixture obtained from a Maillard reaction shows many different chemical and biological properties: brown colour, characteristic roasted or smoky odors, pro- and antioxidants, and mutagens and carcinogens, or perhaps antimutagens and anticarcinogens. It is common practice to use the so-called Mail-lard browning model system consisting of a single sugar and an amino acid to investigate complex, actual food systems. The results of much mutagenicity testing on the products of Maillard browning model systems have been reported. Some Maillard model systems that produced mutagenic materials are shown in Table 8.6. Table 8.6 Mutagenic Materials Produced from the Maillard Model System. Model System

Salmonella typhimurium Strains

D-Glucose/cysteamine

TA 100 without S9 a TA 98 with S9 TA 98 without S9

Cyclotene/NH3

203

Toxicants Formed during Food Processing TA TA TA TA TA TA TA TA TA TA

L-rhamnose/NH3/H2S Maltole/NH3 Starch/glycine Lactose/casein Potato starch/(NH4)3CO3 Diacetyl/NH3 aMetabolic

1538 without S9 98 with S9 98 with S9 100 with S9 98 with S9 98 with S9 98 with S9 100 with S9 98 with S9 100 with S9

activation.

8.4 POLYCYCLIC AROMATIC AMINES (PAA) 8.4.1 OCCURRENCE In the late 1970s, mutagenicity of pyrolysates obtained from various foods was reported. However, PAHs that could not be accounted for were those formed on the charred surface of certain foods such as broiled fish and beef. Table 8.7 shows the mutagenic activity of pyrolysates obtained from some foods. Some classes of cooked protein-rich foods, such as beef, chicken, and dried seafood, tended to be more mutagenic than others and the extent of heating influenced the level of mutagenic activity. The most highly heated samples of milk, cheese, grains, and several varieties of beans, although heavily charred, were only weakly mutagenic or not mutagenic. Hamburger cooked at high temperatures was reported as mutagenic. The mutagenicity, however, was limited to the surface layers where most pyrolysates are found. On the other hand, no mutagenic activity was found in comparable samples of uncooked hamburger meat. The formation of these mutagenic substances seems to depend on temperature. For example, in heated beef stock, temperature dependent mutagen formation has been determined quantitatively. Table 8.7 Mutagenicity of Pyrolysates Obtained from Select Foods Heated at Various Temperatures. Food Beef Chicken Egg, whole Hairtail (or beltfish), raw Eel, raw Squid, dried Skipjack tuna, dried Sea weed, Nori

Revertants of TA98 þ S-9 Mix/0.1 g Sample 250 C 300 C 400 C

269 1,220

178 661 121 849 309 8,000 24,300 260

11,400 15,120 4,750 12,320 6,540 4,490 6,200 3,040

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Fig. 8.4 Structures of typical PAAs.

The mutagenic principles of the tryptophan pyrolysates were later identified as nitrogen-containing heterocyclic compounds. A group of polycyclic aromatic amines is produced primarily during the cooking of protein-rich foods. Their structures are shown in Figure 8.4. PAA are divided into two groups. Heating a mixture of creatine/creatinine, amino acids, and sugars produces the imidazoquinoline (IQ)-type. The IQ-type possesses an imidazole ring (i.e., a ring that is formed from creatine). The IQ-type PAAs are IQ, MeIQ, IQx (structure not shown), MeIQx, 4,8-DiMeIQx (structure not shown), and PhIP. The formation of IQ-type PAA is a result of heat treatment that causes cyclization of creatinine to form the imidazole moiety. The remaining moieties of the structure arise from pyridines and pyrazines formed through the Strecker degradation of amino

Toxicants Formed during Food Processing

205

acids and breakdown products of b-dicarbonyl products formed by the Maillard reaction. The other category of PAAs, the so-called non-IQ type, is composed of pyrolysis products formed from tryptophan: Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2, AaC, and MeAaC. 8.4.2 TOXICITY The acute toxicities of Trp-P-1 and Trp-P-2 are toxic to animals: LD50 (intragastric intubation) of Trp-P-1 are 200 mg/kg in mice, 380 mg/kg in Syrian golden hamsters, and 100 mg/kg in rats. Trp-P-2 is slightly more toxic than Trp-P-1. Animals that received over the LD50 usually died in convulsions within an hour. Trp-P-1 and Trp-P-2 induce a local inflammatory reaction when injected subcutaneously. The early work on the isolation and production of these substances was based on their mutagenicity. They are also minor components of fried beef. Several other mutagens of this class are also present in cooked meat. Beef extracts, which contain IQ and MeIQx, are metabolically converted to active mutagens by liver tissue from several animal species and humans. Although these substances are highly potent mutagens, they are fairly weak carcinogens in rats. Following the mutagenicity studies on these pyrolysates, the carcinogenicity of tryptophan (Trp-P-1 and Trp-P-2) and glutamine (Glu-P-1) was demonstrated using animals such as rats, hamsters, and mice. For example, a high percentage of tumor incidences were observed in mice fed a diet containing Trp-P-1 or Trp-P-2. High incidences of hepatoma were found in mice treated with Trp-P-1, Trp-P2, Glu-P-1, and Glu-P-2. Female mice were more sensitive than males. The various reports indicate that both amino acid and protein pyrolysates may act as carcinogens in the alimentary tracts of experimental animals. Extensive research is presently being conducted to determine whether PAAs produced during the cooking process are hazardous to humans. The identities of the mutagens produced under normal cooking conditions have been established in some cases. For example, the major mutagens in broiled fish are imidazoquinoline (IQ) and methylimidazoqui-noline (MeIQx) (Figure 8.4). Table 8.8 shows the mutagenicity of a typical PAA along with that of well-known carcinogens in S. typhimurium TA98 with S9 microsomal activation. Some PAAs, such as IQ and MeIQ, exhibit strong mutagenic activity. Although TrpP-1, Trp-P-2, and Glu-P-1 are highly mutagenic—being more mutagenic than the wellknown carcinogen aflatoxin B1, they are much less carcinogenic than aflatoxin B1. This may be the result of their high initiating activities but low promoting activities. Carcinogenicity of these potent PAA mutagens is accounted for by hypothesizing that PAA can be metabolically activated by humans both through N-oxidation and Oacetylation, to produce highly reactive metabolites that form DNA adducts. The human enzyme activities for some substrates are comparable to those of the rat, a species that readily develops tumors when fed these PAA as part of the daily diet. Therefore, these PAAs should be regarded as potential human carcinogens.

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Food Hygiene and Toxicology

Table 8.8 Mutagenic Activity of Typical PAA Found in Foods in Salmonella typhimurium Strain TA98 with S9 Microsomal Activation. PAA

Found in

Revertants/mg

MeIQ IQ MeIQx Trp-P-1 Glu-P-1 Trp-P-1 Glu-P-2 Aflatoxin B1 Benzo[a]pyrene

Broiled sardine Fried beef Fried beef Broiled sardine Glutamic acid pyrolysate Broiled sardine, broiled beef Broiled, dried cuttlefish Corn Broiled beef

661,000 433,000 145,000 104,000 49,000 39,000 1,900 6,000 320

8.5 N-NITROSAMINES

Mixtures of inorganic salts, such as sodium chloride and sodium nitrite, have been used to cure meat and certain fish products for centuries. Some countries, not including the United States, also permit the addition of nitrate in the production of some varieties of cheese. The nitrite ion plays at least three important roles in the curing of meat. First, it has an antimicrobial action. In particular, it inhibits the growth of the microorganisms that produce botulism toxin, Clostridium botulinum. It recently has been recognized that the bacterial reduction or curing action results from the nitrite ion. However, the mechanism and cofactors of this antimicrobial action are not clearly understood. Nitrite also imparts an appealing red colour to meats during curing. This arises from nitrosomyoglobin and nitrosylhemoglobin pigments. These pigments are formed when nitrite is reduced to nitric oxide, which then reacts with myoglobin and hemoglobin. If these pigments did not form, cured meats would have an unappetizing grayish colour. Finally, nitrite gives a desirable “cured” flavour to bacon, frankfurters, ham, and other meat products. In general, since cured meats often are stored under anaerobic conditions for extended periods, curing is very important in ensuring the safety of these foods. 8.5.1 PRECURSORS Nitrosation of secondary and tertiary amines produces stable nitrosamines. Unstable nitrosocompounds are produced with primary amines. The reaction rate is pH-dependent and peaks near pH 3.4. The nitrosation of weakly basic amines is more rapid than that of more strongly basic amines. Several anions, such as halogens and thiocyanate, promote the nitrosation process; on the other hand, antioxidants, such as ascorbate and vitamin E, inhibit the reaction of destroying nitrite. Diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) occur in the gastric juice of experimental animals and

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207

humans fed diets containing amines and nitrite. The nitrosation reaction is also known to occur during the high-temperature heating of foods, such as bacon, which contains nitrite and certain amines. Figure 8.5 shows typical structures of N-nitrosamines.

Fig. 8.5 Structure of dimethylnitrosamine

8.5.2 OCCURRENCE IN VARIOUS FOODS Nitrates are found in a wide variety of foods, both cured and uncured. In cured foods, nitrates vary in levels ranging from 10 to 200 ppm, depending on the country. Cured meats all have been shown to contain nitrosamines (Table 8.9), the higher levels appearing in cured meats that have been subjected to relatively high heating. It is important to note that the levels of nitrosamines detected in various foods are quite variable. The reasons for this variability are not clear but seem to be dependent on the type of food and conditions in the laboratory conducting the examination. The levels of volatile nitrosamines in spice premixes, such as those used in sausage preparation, were found to be extraordinarily high. Premixes contained spices with secondary amines and curing mixtures included nitrite. Volatile nitrosamines formed spontaneously in these premixes during long periods of storage. The problem was solved simply by combining the spices and the curing mixture just prior to use. In vegetables, nitrate is encountered often in relatively high levels (1000–3000 ppm). In produce such as cabbage, cauliflower, carrots, celery, and spinach, the nitrate levels are variable and the exact causes are uncertain. The dietary intake of nitrates and nitites for adult Americans has been estimated at 100 mg per day. Vegetables, especially leaf and root vegetables, account for over 85% of the total, and cured meats contribute about 9%. In certain areas, well water contains high levels of nitrate. Although exposure from meat products may have dropped in recent years, the use of nitrate in fertilizers means that vegetables continue to be significant sources of nitrates. However, significant amounts of reduced nitrites are not found in most foods. An additional source of nitrites in humans results from the intestinal tract. Most ingested nitrite comes from saliva, which is estimated to con-tribute 8.6 mg of the total daily intake of 11.2 mg from the diet.

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Food Hygiene and Toxicology Table 8.9 Nitrosamine Content in Typical Cured Meats. Meat

Nitrosamine

Level (ppb)

Smoked sausage

Dimethylnitrosamine Diethylnitrosamine Dimethylnitrosamine Dimethylnitrosamine Dimethylnitrosamine Nitrosoproline