MICROBIAL SPOILAGE OF FOODS
Dr. H.A Modi is M.Sc., M.Phil. and Ph.D. in Microbiology. Presently, he is a Reader (Microbiology) at Department of Life Sciences, Gujarat University, Ahmedabad. He is visiting Professor at P.G. Departments of North Gujarat University, Saurastra University, Gujarat Vidyapeeth, South Gujarat University and other Universities of Gujarat. He is a recognized Ph.D. guiding teacher in Microbiology at Gujarat University and North Gujarat University, supervising Ph.D. students in the fields of Enzyme biotechnology, Bioconversion technology, Mushroom technology, Biocontrol and Food microbiology. He has presented several scientific presentations at National as well as International conferences, seminars and symposia. His research papers have been published in journals of national and international repute. He is a member of many prestigious academic and professional organizations. He has published many books. The important ones are Fermentation Technology in 2 vols., Introductory Food Microbiology, Dairy Microbiology, Food Microorganisms, Food-borne Illnesses and Elementary Microbiology in 2 vols. Dr. Modi has widely travelled in countries like Brazil, Germany, England etc. for academic purposes under Group Study Exchange Programmes. He has successfully carried out research projects funded by UGc, DBT, GUJCOST etc.
MICROBIAL SPOILAGE OF FOODS
Dr. H.A. Modi Microbiology Laboratory Department of Life Sciences University School of Sciences Gujarat University Ahrnedabad-380 009 (Guj.)
Aavishkar Publishers, Distributors Jaipur 302 003 (Raj) India
First Published in 2009 by Prern C. Bakliwal for
Aavishkar Publishers, Distributors 807, Vyas Building, Chaura Rasta Jaipur 302 003 (Raj) India Phone: 0141-2578159 e-mail:
[email protected]
© Dr. H.A. Modi
ISBN 978-81-7910-285-5
All rights reserved. No part of this publication may be reproduced or copied for any purp('se by any means, manual, mechanical or electronic, without prior and written permission of the copyright owner and the Publishers.
Printed at
Sheetal Printers Jaipur 302 003 (Raj) India
PREFACE
The loss of food due to microbial spoilage has economic consequences for the producers, processors and consumers. With the increase in population in the world, loss of food due to microbial (as well as non-microbial) spoilage means less food is available for the hungry mouth. To fight against world hunger, efforts should be directed not only to increase food proouction, but also to minimize spoilage so that enough food is available for consumption. Except for sterile foods, all foods harbor microorganisms. Food spoilage stems from the growth of these microorganisms in food or is due to the action of microbial heat-stable enzymes. New marketing trends, the consumer's desire for foods that are not overly processed and preserved, extended shelf-life, and chances of temperature abuse between production and consumption of foods have greatly increased the chances of food spoilage and, in some instances, with new types of microorganisms. The major concerns are the economic loss and wastage of food. New concepts are being studied to reduce contamination as well as control the growth of spoilage microbes in foods. This book includes thirteen chapters. Introduction of Food Microbiology and its horizons are elaborated in Chapter 1. Basics of human nutrition are briefly summarized in Chapter 2. General principles of microbial spoilage of foods covering types of spoilage, microorganisms involved, favourable factors etc. are discussed in Chapter 3. Microbial spoilage of various food categories like meat and meat products (Chapter 4), poultry and eggs (Chapter 5), fish and other seafoods (Chapter 6), dairy products (Chapter 7), vegetables and fruits (Chapter 8) and cereal products (Chapter 9) are dealt in detail. Special
vi chapters on spoilage of canned foods (Chapter 10) and spoilage of frozen foods (Chapter 11) are included in this book due to popularity of such foods in present time. Indicators of microbial foods spoilage are mentioned in Chapter 12. Microbiological testing of food is very important, so it is elaborately explained in final Chapter 13. A list of selected bibliograpby is also given to assist students in expanding their knowledge on the subject. The materials in each chapter are arranged in logical, systematic and concise sequences. The attempt is done that the book should prove to be an useful source of information for the students of food microbiology, food science and technology, food biotechnology, agriculture, horticulhlre, food and nutrition, environmental studies, hotel and catering management and other food-related courses. This book will also be useful for the people who are working in food-processing industries, catering houses, food and agriculture department of Government. I am grateful to Aavishkar Publishers, Distributors, Jaipur for their concern, efforts and encouragement, especially for their excellent cooperation in the task of preparing and publishing this book.
H.A. Modi
CONTENTS Preface ............................................................................................ v 1. Food Microbiology and its Horizons .................................... 1-8 1.1 Prologue................................... .......................................... 1 1.2 Search of Microorganisms ............................................... 2 1.2.1 Spontaneous Generation Theory ...................... 3 1.2.2 Importance of Microorganisms ......................... 3 1.3 Early Developments in Food Microbiology .................. 4 1.4 Significance of Microorganisms in Foods ..................... 6 1.4.1 Foodbome Diseases ............................................ 6 1.4.2 Food Spoilage ...................................................... 6 1.4.3 Food Bioprocessing ............................................ 6 1.4.4 Food Biopreservation .......................................... 7 1.5 Roles of Food Microbiologists ........................................ 7 2. Basics of Human Nutrition ................................................. 9-42 2.1 Prologue ............................................................................. 9 2.2 Importance of Food .......................................................... 9 2.3 Constituents of Foods .................................................... 10 2.3.1 Carbohydrates ................................................... 10 2.3.1.1 Introduction and Classification ....... 10 2.3.1.2 Functions of Carbohydrates in the Diet ................................................ 13
viii Sources of Carbohydrate in the Diet Dietary Guidelines ............................. 2.3.2 ................................................................. Introduction and Chemical Structure .............................................. 2.3.2.2 Properties of Lipids ........................... 2.3.2.3 Functions of Lipids in the Diet ........ 2.3.2.4 Sources of Lipids in the Diet ............ 2.3.2.5 Dietary Guidelines ............................. 2.3.3 Proteins .............................................................. 2.3.3.1 Introduction and Chemical Structure ............... .......................... ..... 2.3.3.2 Functions of Proteins in the Body ... 2.3.3.3 Sources of Protein in the Diet ........... 2.3.3.4 Dietary Guidelines ............................. 2.3.4 Vitamins ............................................................. 2.3.4.1 Introduction and Types ..................... 2.3.4.2 Functions of Vitamins ....................... 2.3.5 Minerals ............................................................. 2.3.5.1 Introduction and Importance ........... 2.3.6 Water .................................................................. 2.3.6.1 Water Balance ..................................... Fate of Major Nutrients in the Body ............................ 2.4.1 Carbohydrates ................................................... 2.4.2 Fats ...................................................................... 2.4.3 Proteins .............................................................. Food and Energy ............................................................ 2.5.1 Energy Value of Nutrients ............................... 2.5.2 Energy Value of Foods ..................................... 2.5.3 Use of Energy by the Body .............................. Dietary Allowances for Indians (ICMR Recommendations) ............................................ Balanced Diets ................................................................ Classes of Natural Food Stuffs and their Nutritional Significance ................................................ 2.3.1.3 2.3.1.4 Lipids 2.3.2.1
2.4
2.5
2.6 2.7 2.8
14 14 14 14 16 17 18 18 18 18 19 19 20 20 20 21 23 23 26 26 27 27 27 27 28 28 28 28 30 32 37
ix
2.8.1 Cereals ................................................................ 37 2.8.2 Pulses ................................................................. 38 2.8.3 Nuts and Oilseeds ............................................ 38 2.8.4 Green Leafy Vegetables .................................... 39 2.8.5 Root Vegetables ................................................. 39 2.8.6 Other Vegetables ............................................... 40 2.8.7 ,Fruits ................................................................... 40 2.8.8 Milk and Milk Products ................................... 40 2.8.9 Sugar and Jaggery ............................................. 41 2.8.10 Fats and Oils ..................................................... 41 2.8.11 Flesh Foods ........................................................ 41 2.8.12 Eggs .................................................................... 41 2.8.13 Condiments and Spices ................................... 42 3. An Introduction to Microbial Spoilage of Foods ........... 43-66 3.1 Prologue ........................................................................... 43 3.2 Major Reasons for Food Spoilage ................................ 43 3.3 Food-spoilage Types ...................................................... 44 3.3.1 Mouldiness and 'whiskers' ............................. 44 3.3.2 Rots ..................................................................... 44 3.3.3 Sliminess ............................................................ 44 3.3.4 Colour Change .................................................. 44 3.3.5 Ropiness ............................................................. 45 3.3.6 Fermentative Spoilage ...................................... 45 3.3.7 Putrefaction ........................................................ 45 3.3.8 Aerobic Hydrolysis ..........: ................................ 46 3.4 The Organisms Involved in Food-spoilage ................. 46 3.4.1 Microbial Load .................................................. 46 3.4.2 Inter-relationships Between Organisms ......... 48 3.4.3 Moulds in Spoilage .......................................... 49 3.4.4 Yeasts in Spoilage ............................................. 51 3.4.5 Bacteria in Spoilage .......................................... 51 3.5 Food Qualities Responsible for Spoilage .................... 54 3.5.1 Water Content ................................................... 55 3.5.1.1 Dry Foods ............................................ 57 3.5.1.2 Deep-frozen Foods ............................. 59
x
3.5.2
3.5.3 3.5.4 3.5.5 3.5.6
3.5.1.3 Salted Foods ........................................ 3.5.1.4 Sweet Foods ........................................ pH ....................................................................... 3.5.2.1 Neutral Foods ..................................... 3.5.2.2 Acid Foods .......................................... 3.5.2.3 Canned Foods ..................................... Gaseous Conditions ......................................... Texture ................................................................ Nutrients ............................................................ Temperature .......................................................
59 60 61. 61 62 63 64 65 65 65
4. Spoilage of Meat and Meat Products .............................. 67-83 4.1 Prologue ........................................................................... 67 4.2 Spoilage of Fresh Meats ................................................ 67 4.2.1 Contamination of Tissues by Microorganisms ................................................ 67 4.2.2 Control of Microbial Growth ........................... 68 4.2.2.1 Initial Contamination ........................ 68 4.2.2.2 Glycogen Reserve ............................... 68 4.2.2.3 Oxidation-Reduction Potential ......... 69 4.2.2.4 The Rate of Cooling ........................... 69 4.2.3 Effect of Storage Temperature .......................... 69 4.2.3.1 Spoilage under Warm Conditions ... 69 4.2.3.2 Spoilage under Cool Conditions ...... 71 4.2.3.3 Spoilage under Refrigeration Conditions ........................................... 71 4.2.4 Chemical Changes Produced by Bacteria in Chilled Meats ................................ 73 4.3 Spoilage of Cured Meats ............................................... 74 4.3.1 Curing Agents ................................................... 74 4.3.2 The Curing Process .......................................... 74 4.3.3 The Microbiology and Spoilage of Bacon and Ham ................................................ 75 4.3.3.1 Unsmoked Bacon ............................... 75 4.3.3.2 Smoked Bacon .................................... 76 4.3.3.3 Ham ..................................................... 77
Xl
4.4
Spoilage of Vacuum-packed Meats .............................. 4.4.1 Types of Packaging Materials ......................... 4.4.2 Influence of Packaging Materials on the Microbiological Flora ........................... 4.4.3 Spoilage of Packed Fresh Meats ..................... 4.4.4 Spoilage of Vacuum-packed Bacon ................
77 77 78 79 81
5. Spoilage of Poultry and Eggs ............................................ 84-89 5.1 Prologue ........................................................................... 84 5.2 Effects of Poultry Processing on the Microbiological Flora ..................................................... 84 5.3 Spoilage of Chickens held at Chill Temperatures ..... 86 5.4 The Chicken's Egg and its Spoilage ............................ 87 5.5 Egg Products ................................................................... 88 6. Spoilage of Fish and other Seafoods ............................... 90-97 6.1 Prologue ........................................................................... 90 6.2 Bacteriology of the Newly Caught Fish ...................... 90 6.3 The Effect of Initial Processing and Storage in ice on Board Ship ...................................................... 91 6.4 The effect of Handling Ashore ..................................... 92 6.5 Chemical Changes induced by Bacteria in Fish ........ 93 6.6 Salted Fish ....................................................................... 94 6.7 Smoked Fish .................................................................... 95 6.8 Shell Fish ......................................................................... 95 6.8.1 Crustaceans ....................................................... 95 6.8.2 Molluscs ............................................................. 96 7. Spoilage of Dairy Products .............................................. 98-105 7.1 Prologue ........................................................................... 98 7.2 Microbiology of Raw Milk ............................................ 98 7.3 Pasteurization ................................................................. 99 7.4 UHT Milk ...................................................................... 100 7.5 Butter .............................................................................. 101 7.6 Cheese ............................................................................ 102 7.7 Yoghurt .......................................................................... 103
xii 8. Spoilage of Vegetables, Fruits and their Products .... 106-113 8.1 Prologue ......................................................................... 106 8.2 Vegetables ...................................................................... 106 8.3 Fruits .............................................................................. 107 8.4 Soft Drinks, Fruit Juices and Preserves, and Vegetable Juices .................................................... 107 8.5 Sauerkraut ..................................................................... 108 8.6 Wine ............................................................................... 109 8.7 Control of Microbial Spoilage ..................................... 109 9. Spoilage of Cereal Products .......................................... 114-118 9.1 Prologue ......................................................................... 114 9.2 Cereal Grains ................................................................ 114 9.3 Bread and Cakes .......................................................... 115 9.4 Refrigerated Dough ...................................................... 115 9.5 Pastas ............................................................................. 115 9.6 Liquid Sweeteners and Confectioneries .................... 116 9.7 Beer ................................................................................. 116 10. Spoilage of Canned Foods ............................................. 119-126 10.1 Prologue ......................................................................... 119 10.2 Leaker Spoilage ............................................................ 120 10.3 Spoilage due to Inadequate Heat Treatment ............ 123 10.3.1 Spoilage of Low Acid Foods ................. ~ ....... 124 10.3.2 Spoilage of High Acid Foods ........................ 125 11. Spoilage of Frozen Foods ............................................... 127-130 11.1 Prologue ......................................................................... 127 11.2 Factors affecting Viability of Microorganisms during Freezing ............................................................ 127 11.3 Effect of Cold Storage ................................................... 129 11.4 Freezing Injury to Cells ............................................... 129 11.5 Thawed Foods and their Spoilage ............................. 129 12. Indicators of Microbial Food Spoilage ........................ 131-137 12.1 Prologue ......................................................................... 131 12.2 Microbiological Criteria ............................................... 133
xiii
12.3 Chemical Criteria ......................................................... 12.4 Assay of Heat-stable Enzymes ................................... 12.4.1 Heat-stable Proteinases in Milk .................... 12.4.2 Heat-stable Lipases in Milk ..........................
135 136 136 136
13. Microbiological Testing of Food ................................... 138-161 13.1 Prologue ......................................................................... 138 13.2 Sampling ....................................................................... 139 13.2.1 Sampling Rate ................................................. 139 13.2.2 The Representative Sample ........................... 139 13.2.3 Sampling Techniques ..................................... 140 13.2.4 Treatment of Sample ....................................... 141 13.3 Microbiological Test Procedures in Common Usage 142 13.3.1 Total Viable Count .......................................... 142 13.3.1.1 The 'Pour Plate' Method ................. 143 13.3.1.2 The 'Spread Plate' Method ............. 143 13.3.1.3 The 'Drop Plate' Method ................. 144 13.3.1.4 The 'Agar Droplet' Method ............ 144 13.3.1.5 The 'Spiral Plate' Method ............... 144 13.3.2 Counting using Electrical Impedance Measurements ............................. 144 13.3.3 Counting by Measurement of Adenosine Triphosphate (ATP) .................... 145 13.3.4 Counting using the Direct Epifluorescent Filter Technique .............................................. 145 13.3.5 Direct Microscopic Count .............................. 146 13.3.6 Indicator Organisms ....................................... 146 13.3.6.1 Coliforms ........................................... 146 13.3.6.2 Enterococci ........................................ 147 13.3.6.3 Enterobacteriaceae ............................ 148 13.3.7 Food Poisoning Organisms ........................... 149 13.3.7.1 Salmonellas ....................................... 150 13.3.7.2 Clostridium perfringens and C. botulinum ............................... 151 13.3.7.3 Staphylococcus aureus ........................ 152 13.3.7.4 Bacillus cereus .................................... 153
xiv
13.3.7.5 Vibrio parahaemolyticus ..................... 153 13.3.8 Food Spoilage Organisms .............................. 154 13.3.8.1 Pseudomonas ....................................... 154 13.3.8.2 Micrococci .......................................... 155 13.3.8.3 Lactobacilli and Leuconostocs ....... 155 13.3.8.4 Streptococci ........................................ 156 13.3.8.5 Spore Formers ................................... 156 13.3.8.6 Yeasts and Moulds .......................... 157 13.3.9 Canned Foods ................................................. 157 13.3.9.1 Dextrose Tryptone Agar .................. 158 13.3.9.2 Reinforced Clostridial Medium ...... 158 13.3.9.3 Tomato Juice Agar ............................ 158 13.3.9.4 Malt Extract Agar ............................. 158 13.3.10 Frozen and Dehydrated Foods ..................... 158 13.3.11 Miscellaneous Tests ........................................ 159 13.3.11.1 Methylene Blue Reduction Test [MBRT] ...................................... 159 13.3.11.2 The Limulus Lysate Test ............... 159 13.3.11.3 Microcalorimetry ............................. 159 13.3.12 Compilation of Specifications ....................... 160 Selected Bibliography ...................................................... 162-178
FOOD MICROBIOLOGY AND ITS HORIZONS
1.1 PROLOGUE The choice of foods available today is extremely wide compared to a few years ago. An increasing amount of food is eaten outside the home. The public has a right to expect food to be of good quality. They do not expect it to make them ill, e.g. by food poisoning. In recent years there has also been more interest in the effect that food has on our health; however, food must still look and taste good. A good place to start is the question, "What is food?" Simply, food is any substance of plant or animal origin that when eaten and absorbed by the body produces energy, promotes the growth and repair of body tissues, or regulates the body processes. The components of food that perform these effects are called nutrients. Nutrition is the study of food (nutrients) and the effect it has on the body. It includes the factors that affect food intake. But food does not just contain nutrients. It contains water which is also essential to life, and various substances that give food colour and flavour. There are other substances that might be put into food such as additives or may just get into food accidentally such as pesticide residues. Many foods also contain a whole range of micro-organisms. The presence of these organisms may be beneficial or they may cause food spoilage or food poisoning.
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MICROBIAL SPOILAGE OF FOODS
Micro-organisms can be deliberately added to food during production, e.g. bread, wine and cheese, or the micro-organisms already present can spoil the food. As spoilage is ultimately inevitable, a whole range of preservation techniques have been developed to try to increase the shelf life (or keeping quality) of our food. Some micro-organisms contaminate our food and cause illness rather than spoilage. Every effort must be made to keep our food free from these hannful micro-organisms. To fully understand the mechanisms by which the processes of spoilage and contamination can be controlled, one needs to understand the basics of microbiology in general and food microbiology in particular.
1.2 SEARCH OF MICROORGANISMS The discovery of microorganisms ran parallel with the invention and improvement of the microscope. Around 1658, Athanasius Kircher reported that, using a microscope, he had seen minute living worms in putrid meat and milk. The magnification power of his microscope was so low that he could not have seen bacteria. In 1664, Robert Hooke described the structure of molds. However, probably the first person to see different types of microorganisms, especially bacteria, was the Dutch businessman turned naturalist Anton van Leeuwenhoek, using a microscope that probably had not above 300x magnification power. He observed bacteria in saliva, rainwater, vinegar, and other materials, sketched the three morphological groups (spheroids or cocci, cylindrical rods or bacilli, and spiral or spirilla), and also described some to be motile. He called them animalcules and in 1675 reported his observations to the newly formed leading scientific organization, The Royal Society of London, where his observations were read with fascination. As fairly good microscopes were not easily available at the time, during the course of the next 100 years, other interested individuals and scientists only confirmed Leeuwenhoek's observations. In the 19th century, as a result of the Industrial Revolution, improved microscopes became more easily available, and that stimulated many inquisitive minds to see and describe creatures they discovered under a microscope. By 1838, Ehrenberg (who introduced the term bacteria) had proposed at least 16 species in four genera and by 1875 Cohn had developed the preliminary classification system of bacteria. Cohn also was the first to discover that some bacteria produced spores. Although, like bacteria, the existence of submicroscopic viruses was recognized in the mid-19th century, they were observed only after the invention of the electron microscope in the 1940s.
FOOD MIRCROBIOLOGY AND ITS HORIZONS
3
1.2.1 Spontaneous Generation Theory Following Leeuwenhoek's discovery, althOl.:gh there were no bursts of activity, some scientific minds did have the curiosity to determine where the animalcules, found to be present in many different objects, were coming from. Society had just emerged from the Renaissance period and science, known as experimental philosophy, was in its infancy. The theory of spontaneous generation, i.e., the generation of some form of life from nonliving objects, had many strong followers among the educated and elite class. Since the time of the Greeks, the emergence of maggots from dead bodies and spoiled flesh was thought to be due to spontaneous generation. But around 1665, Redi disproved that theory by showing that the maggots in spoiled meat and fish could only appear if flies were allowed to contaminate them. The advocates of the spontaneous generation theory argued that the animalcules could not regenerate by themselves (biogenesis), but that they were present in different things only through abiogenesis (spontaneous generation). In 1749, Needham showed that boiled meat and meat broth, following storage in covered flasks, showed the presence of animalcules within a short time. This was used to prove the appearance of these animalcules by spontaneous generation. Spallanzani (1765) showed that boiling meat infusion in broth in a flask and sealing the flask immediately prevented the appearance of these microscopic organisms and thus disproved Needham's theory. This was the time when Antoine-Laurent Lavoisier and his coworkers showed the need of oxygen for life. The believers of abiogenesis rejected Spallanzani's observation, suggesting that there was not enough vital force (oxygen) present in the sealed flask for animalcules to appear through spontaneous generation. Later, Schulze (1830; by passing air through acid), Theodor Schwann (1838; by passing air through red hot tubes), and Schroeder (1854; by passing air through cotton) showed that bacteria failed to appear in boiled meat infusion even in the presence of air. Finally, in 1864, Louis Pasteur demonstrated that, in boiled infusion, bacteria could grow only if the infusions were contaminated with bacteria carried by dust particles in air. His careful and controlled studies proved that bacteria were able to reproduce (biogenesis) and life could not originate by spontaneous generation. John Tyndall, in 1870, showed that in a dust-free box, boiled infusion could be stored in dust-free air without microbial growth. 1.2.2 Importance of Microorganisms The involvement of invisible organisms in many diseases in humans was suspected as early as the 13th century by Roger Bacon. In
4
MICROBIAL SPOILAGE OF FOODS
the 16th century, Francostro of Verona suggested that many human diseases were transmitted by small creahlres from person to person. This was also indicated by Kircher in 1658. In 1762, von Plenciz of Vienna suggested that different invisible organisms were responsible for different diseases. Schawnn (1837) and Hermann Helmholtz (1843) pointed out that putrefaction and fermentation were connected with the presence of the organisms derived from the air. Finally, Pasteur, in 1875, showed that wine fermentation from grapes and souring of wine were caused by microorganisms. He also proved that spoilage of meat and milk was associated with the growth of microorganisms. Later, he showed the association of microorganisms with several diseases in humans, cattle, and sheep, and later developed vaccines against several human and animal diseases, including the rabies virus. Robert Koch, in Germany (in the 1880s and 1890s), isolated bacteria in pure cultures responsible for anthrax, cholera, and tuberculosis. He also developed the famous Koch's postulates to associate a specific bacterium as a causative agent for a specific disease. Along with his associates, he also developed techniques of agar plating methods to isolate bacteria in pure cultures, the petri dish (by Petri in his laboratory), and staining methods for better microscopic observation of bacteria. With time, the importance of microorganisms in human and animal diseases, soil fertility, plant diseases, fermentation, food spoilage and foodborne diseases, and other areas was recognized, and microbiology was developed as a separate discipline. Later, it was divided into several disciplines, such as medical microbiology, soil microbiology, plant pathology, and food microbiology.
1.3 EARLY DEVELOPMENTS IN FOOD MICROBIOLOGY (PRIOR TO 1900 A.D.) It is not known exactly when our ancestors recognized the importance of the invisible creatures, now designated as microorganisms, in food. But it had to be around 8000 B.C. in the Near East after they developed agriculture and animal husbandry. They produced more foods than they could consume within the short growing season, and a portion of the produce was lost due to spoilage. They solved the problems and secured uniform food supplies throughout the year by developing different preservation techniques. Between 8000 and 2000 B.C., they used drying, cooking, smoking, salting, low temperature, baking, modified atmosphere, fermentation, spices, and honey to extend the storage life of different types of raw and processed foods. Although we are not sure if they had perceptions
FOOD MlRCROBIOLOGY AND ITS HORIZONS
5
about the cause of foodbome diseases, they definitely associated food spoilage with some invisible factors and developed successful preventative measures. From the time of the Greeks until the discovery of biogenesis, spoilage of foods, especially of meat and fish, was thought to be due to spontaneous generation, such as the development of maggots. When the presence of different types of bacteria in many foods was diScovered, their appearance through spontaneous generation was explained to be the cause of food spoilage. Schawnn (1837) and Helmholtz (1843) associated the presence of microorganisms (bacteria) in food with both putrefactive and fermentative changes of foods. However, they did not believe in spontaneous generation, but they could not explain how microorganisms could bring about those changes. Finally, Pasteur resolved the mystery by explaining that contamination of foods with microorganisms from the environment and their subsequent metabolic activities and growth were the causes of fermentation of grapes, souring of milk, and putrefaction of meat. Diseases caused by the consumption of certain foods (foodbome disease) was recognized at least during the Middle Ages. Ergot poisoning in Europe was related to the consumption of grains (infested with molds) in the 12th century. In 1857, consumption of raw milk was suspected to be the cause of typhoid fever. In 1870, Selmi related certain food poisoning with ptomaine (histamine). Gaertner was the first to isolate Salmonella from a meat implicated in a foodbome disease in 1888. Denys, in 1894, was able to estClblish Staphylococcus aureus with food poisoning and, in 1896, Ermengem isolated Clostridium botulinum from food. The association of many other pathogenic bacteria and viruses to foodbome diseases was established after 1900 A.D. Pasteur, in the 1860s, recognized the role of yeasts in alcohol fermentation. He also showed that souring of wine was due to growth of acetic acid-producing bacteria (Acetobacter acet!), and developed the pasteurization process (heating at 145°F for 30 min.) to selectively eliminate these undesirable bacteria from wine. Like fermentation, cheese ripening was suggested by Martin in 1867 to be of microbial origin. John Lister, in 1873, was able to isolate milk-souring bacteria (LactOCOCCIlS lactis) by the serial dilution (dilution to extinction) procedure. Cienkowski, in 1878, isolated the bacteria (Leuconostoc mesenteroides) associated with slime formation in sugar. In 1895, microbial enumeration of milk was developed by Von Geuns. After 1900 A. D., the involvement of different microorganisms in food spoilage and food fermentation was demonstrated.
6
MICROBIAL SPOILAGE OF FOODS
1.4 SIGNIFICANCE OF MICROORGANISMS IN FOODS Since 1900 AD., our understanding of the importance of microorganisms in food has increased greatly. Their role in food can be either desirable (food bioprocessing) or undesirable (foodbome diseases and food spoilage), which is briefly discussed here.
1.4.1 Foodbome Diseases Many pathogenic microorganisms (bacteria, molds, and viruses) can contaminate food during various stages of their handling between production and consumption. Consumption of these foods can cause foodbome diseases. Foodbome diseases not only can be fatal, but they can cause large economic losses. Foods of animal origin are associated more with foodbome diseases than foods of plant origin. Mass production of foods, introduction of new technologies in the processing and storage of foods, changes in food consumption patterns, and the increase in imports of food from other countries have increased the chances of large outbreaks as well as the introduction of new pathogens. Effective intervention technologies are being developed to ensure the safety of consumers against foodbome diseases. New methods are also being developed to effectively and rapidly identify the pathogens in contaminated foods.
1.4.2 Food Spoilage Except for sterile foods, all foods harbor microorganisms. Food spoilage stems from the growth of these microorganisms in food or is due to the action of microbial heat-stable enzymes. New marketing trends, the consumers' desire for foods that are not overly processed and preserved, extended shelf life, and chances of temperature abuse between production and consumption of foods have greatly increased the chances of food spoilage and, in some instances, with new types of microorganisms. The major concerns are the economic loss and wastage of food. New concepts are being studied to reduce contamination as well as control the growth of spoilage microbes in foods.
1.4.3 Food Bioprocessing Many food-grade microorganisms are ~d to produce different types of fermented foods using raw materials from animal and plant sources. Consumption of these foods has increased greatly over the last 20 to 30 years and is expected to increase still more in the future. There have been great changes in the production and availability of these microorganisms (starter cultures) to meet the large demand. Also, novel
FOOD MIRCROBIOLOGY AND ITS HORIZONS
7
and better strains are being developed using genetic engineering techniques. Microbial enzymes are also being used to produce food and food additives. Genetic recombination techniques are being used to obtain better enzymes and from diverse sources. Many types of additives from microbial sources are being developed and used in food. 1.4.4 Food Biopreservation Antimicrobial metabolites of desirable microorganisms are being used in foods in place of nonfood preservatives to control pathogenic and spoilage microorganisms in food. Economic production of these antimicrobial compounds and their effectiveness in food systems have generated wide interest.
1.5 ROLES OF FOOD MICROBIOLOGISTS From the above discussion, it becomes apparent what, as a discipline, food microbiology has to offer. Prior to the 1970s, food microbiology was regarded as an applied science mainly involved in the microbiological quality control of food. Since then, the technology used in food production, processing, distribution and retailing, and food consumption patterns have changed dramatically. These changes have introduced new problems that no longer can be solved by just using applied knowledge. Thus, modem-day food microbiology needs to include a great deal of basic science to effectively solve the microbiological problems in food. The discipline not only includes the microbiological aspects of food spoilage and foodbome diseases and their effective control and bioprocessing of foods, it also includes basic information of microbial physiology, metabolism, and genetics. This information is helping to develop methods for rapid and effective detection of spoilage and pathogenic bacteria, to develop desirable microbial strains by recombinant DNA technology, to produce fermented foods of better quality, to develop thermostable enzymes in enzyme processing of food and food additives, to develop methods to remove bacteria from food and equipment surfaces, and to combine several control methods for effective control of spoilage and pathogenic microorganisms in food. An individual who has completed courses in food microbiology (both lecture and laboratory) should gain knowledge in the following areas: 1. Determination of microbiological quality of foods and food ingredients using appropriate techinques.
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MICROBIAL SPOILAGE OF FOODS
2.
Determination of microbial type(s) involved in spoilage, health hazards, and the identification of the sources 3. Design corrective procedures to control the spoilage and pathogenic microorganisms in food 4. Identify how new technologies adapted in food processing can have specific microbiological problem and design methods to overcome the problem 5. Design effective sanitation procedures to control spoilage and pathogen problems in food processing facilities 6. Effective use of desirable microorganisms to produce fermented foods 7. Design methods to produce better starter cuitures for use in fermented foods and probiotics 8. Food regulations (state, federaC international) To be effective, in addition to the knowledge gained, one has to be able to communicate with different groups of people about the subject (food microbiology and its relation to food science).
DDD
BASICS OF HUMAN NUTRITION
2.1 PROLOGUE This chapter describes all the nutrients that we must obtain from food, the types of foods that we get them from and the effect of cooking or processing on nutrients. Although water is not a nutrient, it is essential for life and so water is also included in this section. How our body obtains the nutrients from the food we eat is essential for an understanding of nutrition. The last section introduces you to the concept of food energy or Calories as it is more widely known. Dietary allowances for Indians (ICMR recommendations) as well as balanced diets are also discussed. At the end, various types of natural foodstuffs available in India and their nutrient contents are summarized.
2.2 IMPORTANCE OF FOOD By way of introduction, hunger is one of our strongest drives and we do not need a bell to remind us to eat. We have a physiological signal. Our blood usually contains about one-tenth per cent sugar and draws on the body to maintain this sugar level. A large portion of the sugar obtained from a meal is stored in the liver and slowly released on demand. When the blood sugar falls, we begin to feel sluggish, and the muscular contractions of an empty stomach tells us we must eat. We can eat sugar to boost our blood sugar level temporarily but a stomach must be at least partially filled and coated, even thinly, with fat to overcome hunger.
10
MICROBIAL SPOILAGE OF FOODS
In addition to the physiological aspects, there is a mental response to hunger which stimulates the flow of saliva. Our interest in food is basic to our existence. Food is an absolute need, our energy source. Food should contain the building blocks we need to grow and to repair tissues. Food should contain the nutrients we need to regulate our body systems. Our life process can be seen as a constant use of energy and a constant exchange of materials. While occasionally our own tissues can yield some nutrients to meet a temporary shortage, we cannot do so long. We need food, and there must be a balance between the food we eat and our nutrient needs if we are to achieve a feeling of well being. 2.3 CONSTITUENTS OF FOODS Most people now know that plants and animals as a group contain sugars, proteins, fats, vitamins, minerals and water. Eaten as food, starch and sugars are either burned, stored as fat, or stored as glycogen (animal starch). Protein is broken down into amino acids which are used to build and repair body tissues, burned for energy, changed into sugar, or contribute to the production of fat. Fat is broken down in digestion, passed into the body, recombined into fat, changed into important body chemicals and burned for energy or stored as fat in tissues. All these substances (sugar, protein, and fat) may be used from a variety of food sources as fuel by our bodies. 2.3.1 Carbohydrates
2.3.1.1 Introduction and Classification Carbohydrates are an important group of nutrients in the diet; their main function is providing energy. Thei.r structures are all based on a common lmit called a saccharide unit. This unit is nearly always glucose, and the grouping or classification of carbohydrates depends primarily on the number of saccharide units each carbohydrate contains. This can vary from one to many thousands. Sugars MONOSACCHARIDES one unit (mono meaning one) DISACCHARIDES two lmits (di meaning two) POLYSACCHARIDES NOll-sugars many units (poly meaning many) (A) Monosaccharides (simple sugars) Three types of sugar are important:
BASICS OF HUMAN NUTRITION
11
Glucose (sometimes called dextrose) is t~e most important monosaccharide. It occurs naturally in fruit and plant juices, and in the blood of living animals. Most carbohydrates in food are ultimately converted to glucose during digestion. 2. Fructose occurs naturally in some fruit and vegetables and especially in honey. It is the sweetest sugar known and is often called fruit sugar. 3. Galactose does not exist as such in foods but is produced when lactose (a disaccharide) is broken down during digestion (see below). (B) Disaccharides Disaccharides consist of two monosaccharides linked together: 1. Sucrose occurs naturally in sugar cane and sugar beet and in some roots (e.g. carrots) and fruits. Table sugar, as sucrose is often called, is a chemical combination of glucose and fructose. 2. Maltose is formed during the breakdown of starch by digestion and during the germination or sprouting of barley (important in beer production). It is a combination of two glucose units. 3. Lactose occurs only in milk, including human milk. It is less sweet than sucrose or glucose and is a combination of glucose and galactose. We are being encouraged to eat more fruit and vegetables, hence our intake of intrinsic sugars should rise. However, our intake of the extrinsic sugar sucrose is already extremely high and so most authorities recommend a reduction in this type of sugar. Properties of sugars (Monosaccharides & Disaccharides) 1. All sugars are white crystalline compounds which are soluble in water (i.e. they dissolve in water). 2. All sugars a!e sweet but they do not have the same degree of sweetness. 3. When sugars are heated they caramelise. 4. Sugars can act as preservatives if large amounts are present in a food, e.g. jam. Sugars are also grouped on the basis of where they occur in foods (i.e. inside or outside the cell walls of foods). (Table 2.1) 1.
12
MICROBIAL SPOILAGE OF FOODS
Table 2.1 : Classification of Sugars
Intrinsic (sugars contained inside the cell walls of foods)
1
Sugars Extrinsic (sugars not contained within the cell walls of foods) /Ex as in external "non-milk: milk and milk products
1
.
lactose mostly sucrose (table sugar and walls of fruits and baked foods, also vegetables honey) (C) Polysaccharides These are formed from a varying number of monosaccharide units. They are usually insoluble in cold water (i.e. do not dissolve in cold waterfand are tasteless. Polysaccharides in food fall into three groups: 1. Starch is the most important polysaccharide. It is the major food reserve of plants and is a mixture of two polysaccharides called amylose and amylopectin. Starch is a white powder and does not have a sweet taste. If you heat a mixture of starch in water it eventually thickens; this is the reason why sauces thicken when you heat them. On heating, the starch granules swell and eventually gelatinise. 2. Glycogen is a carbohydrate found only in animals, where small amounts are stored in the liver and muscles, and act as an energy reserve. Glycogen is composed of branched chains of glucose units, but unlike amylopectin it is soluble in water. We do not eat very much glycogen because it breaks down again to glucose after the animal is slaughtered. 3. Non-Starch Polysaccharides (NSP) (or dietary fibre) provide the rigid and fibrous structure of vegetables, fruits and cereal grains. They form the main part of food that is not digested. The term' dietary fibre' was widely used to describe this part of food. However, the amount of fibre
fructose, glucose and sucrose within cell
BASICS OF HUMAN NUI'RITION
13
found in foods appeared to vary depending on which method of chemical analysis had been used. Therefore, to obtain some standardisation, a specific method of chemical analysis has been agreed upon. This measures the amount of non-starch polysaccharide (NSP) in a food (Le. the amount of polysaccharide other than starch). The term NSP may replace dietary fibre in future. NSP is made up of the following: a. Cellulose consists of many thousands of glucose units. It cannot be digested by man because we do not have the necessary enzymes to break it down. Cellulose is important for providing roughage or bulk in the diet and therefore assisting in the passage of digestible materials and waste products through the intestines. b Pectin and other similar polysaccharides are found in many fruits and some root vegetables, e.g. turnips. Apples and the peel of citrus fruits are particularly rich in pectin. Its main importance is as a gelling agent, e.g. in jam making. c. Hemicelluloses and other polysaccharides are found in small amounts. The old term 'dietary fibre' included all the above plus other non-digestible plant material such as the woody material lignin.
2.3.1.2 Functions of carbohydrates in the diet After eating foods containing polysaccharides and disaccharides, they are hydrolysed (broken down) by digestive enzymes. All carbohydrates are absorbed as monosaccharides. As part of the digestive process the monosaccharides fructose and galactose are converted into glucose. Thus almost all digested carbohydrates are eventually converted to glucose. Glucose has two main functions in the body: 1. Energy: Glucose is oxidised in the cells with the release of energy. This energy can be used for physical activity but more usually it is needed by body cells for normal functioning: 19 of carbohydrate provides 3.75 kcal (16 kJ). 2. Converted into body fat: Any carbohydrate you eat that you do not immediately need for energy may be converted into body fat. This conversion takes place in the liver but the fat is stored all over the body, mainly in the adipose tissue under the skin.
14
MICROBIAL SPOILAGE OF FOODS
2.3.1.3 Sources of carbohydrate in the diet 1.
Cereals and Cereal Foods : All cereals contain a high percentage of starch. The main cereals consumed in the world are wheat, rice, maize (corn), oats, rye and barley. Cereal foods account for 40% of the total carbohydra te content of the average British diet. 2. Refined Sugar (sucrose) : Sugar is eaten in large quantities, as table sugar and in manufachlred goods such as biscuits, sweets., ice-cream, jams and soft drinks. Sugar and preserves account for 11 % of the total carbohydrate content of the average British diet. 3. Vegetables: Vegetables contain starch and sugars in varying amounts. Potatoes are the richest source of carbohydrate although pulse vegetables (e.g. beans) also contain significant amounts. They account for 14% of the total carbohydrate content of the average British diet. 4. Fruits: As fruit ripens starch is turned into sugar. Most fruits contain between 5% and 10% sugar. Bananas are the only fruit which contain starch as well as sugar when ripe. Fruits contribute on average 7% of the total carbohydrate content. 5. Milk: Milk and milk products such as yoghurt contain the sugar lactose. They contribute 8% of the total carbohydrate content of the average British diet. Foods such as cheese and butter made from milk do not contain lactose because the whey part of milk which contains lactose is discarded during cheese and butter production.
2.3.1.4 Dietary guidelines We are recommended to eat more starchy foods like cereals and also fruit and vegetables. Starches should provide 39% of our daily energy (Calorie) intake and sugars (mainly sucrose) 11 %. In addition we should aim to eat 18g/day of NSP with a range of 12-24g/ day. Because of their smaller body weight children should eat less.
2.3.2 Lipids [Fats and Oils]
2.3.2.1 Introduction and chemical structure Fats and oils (or lipids) include not only 'visible fats' such as butter and margarine, cooking fats and oils and the fat on meat, but also the 'invisible fats' which occur in milk, nuts, lean meat and other
15
BASICS OF HUMAN NUTRITION
foods. They are a more concentrated source of energy than carbohydrates; much of the energy reserve of animals and some seeds is stored in this form. Although 'lipids' is the correct term, the term 'fats' is often used to mean both fats and oils. Fats and oils found in food consist mainly of mixtures of triglycerides. Each triglyceride is a combination of three fatty acids with a unit of glycerol (glycerine). The differences between one fat or oil and another are the result of different proportions of the various fatty acids in each.
Glycerol 'backbone'
-f
Fatty acid Fatty acid Fatty acid
Fig. 2.1 : Triglyceride structure.
(a) Fatty acids There are many different fatty acids found in nature. They consist of chains of carbon atoms with hydrogen atoms attached (this structure is called a hydrocarbon chain). They differ in the number of carbon atoms and double bonds which they contain. Each carbon atom can make links with a maximum of four other atoms. (b) Saturated fatty acids These have no double bonds and therefore are more stable. This structure is 'saturated' with hydrogen; no more hydrogen atoms can be fitted in. H H H H H H H I
I
I
I
I
I
I
I
I
I
I
I
I
I
- C - C - C - C - C - C - C - COOH (Acid Group) H H H H H H H Fig. 2.2 : Part of the hydrocarbon chain of a saturated fatty acid. (c) Unsaturated fatty acids
Here the carbon chain is not saturated with hydrogen and therefore has one or more double bonds. These react gradually with air making the fat rancid. HHHHHHH I
I
I
I
I
I
I
-C-C=C-C-C=C-C-COOH I
H
I
H
I
H
Fig. 2.3 : Part of the structure of an unsaturated fatty acid.
16
MICROBIAL SPOILAGE OF FOODS
Because each carbon atom can make links with four other atoms, it is possible to fit more hydrogen atoms into this structure by breaking the double bond; hence this structure is 'unsaturated'. Unsaturated fatty acids may be either: Monounsaturated, containing one double bond Polyunsaturated, containing more than one double bond. (d) Cis and trans fatty acids The arrangement of atoms at the double bond may vary and both mono and polyunsaturated fatty acids can be either: 1. CIS fatty acids with the two hydrogen atoms on the same side of the double bond. H
H
I
I
-C=CNaturally occurring unsaturated fatty acids are usually in the CIS configuration. OR
2.
TRANS, fatty acids with the hydrogen atoms on geometrically opposite sides of the double bond. H I
-C=CI
H
Small amounts are found naturally in some foods but larger amounts can occur as a result of certain types of lipid processing. There is some concern about high intakes of TRANS fatty acids and hence current recommendations are that we do not increase our intake of this type of lipid.
2.3.2.2 Properties of lipids 1.
Fats are lipids which are solid at low temperatures and become liquid when they are heated. Oils are lipids which are liquid at room temperature, usually as a result of their higher content of unsaturated fatty acids, and will solidify on refrigeration, e.g. olive oil. 2. Oils and fats do not dissolve in water but may be emulsified with water by vigorous mixing as when butter and margarine are made.
BASICS OF HUMAN NUTRITION
17
3.
Lipids make an important contribution to the texture and palatability of foods. 4. Furthermore, because they are digested comparatively slowly, foods rich in lipids have a high satiety value. If you eat a fatty meal you won't feel hungry as quickly as if you had eaten a high carbohydrate meal. 5. Food lipids usually contain small amounts of other fat soluble substances, e.g. flavour compounds and the fat soluble vitamins.
2.3.2.3 Functions of lipids in the diet 1. Energy: Fat is broken down in the body by oxidation and energy is released. 19 of fat providEs 9kcal (37k1). Fat has more than twice the calorific value of carbohydrates and is therefore a more concentrated source of energy. 2. Formation of adipose tissue : Excess fat, which is not immediately required for energy, is stored in the adipose tissue under the skin where it has three functions. a. an energy reserve; b. it forms an insulating layer and helps to prevent excessive heat loss from the body. It therefore assists in the maintenance of a constant body temperature; and c. when stored around delicate organs such as the kidneys, it protects these organs from physical damage. 3 Essential fatty acids: Some fatty acids are essential in small amounts for the functioning of the body. Linoleic acid and one form of linolenic acid (alpha - linolenic acid) are probably the only truly essential fatty acids. Linoleic acid is needed for the formation of cell membranes. Derivatives of the essential fatty acids are used to form prostaglandins, a group of hormone-like substances which help to regulate many body functions. If the diet provides 1-2% of its energy content as essential fatty acids then deficiency is unlikely. Most diets contain more than 2%. 4. Fat soluble vitamins: The inclusion of certain fats included in the diet help to ensure an adequate intake of the fat soluble vitamins A, 0, E and K.
18
MICROBIAL SPOILAGE OF FOODS
2.3.2.4 Sources of lipids in the diet Fats and oils are obtained from both animals and plants. 1. Meat and fish: All meat contains fat, though the percentage of fat varies from animal to animal, and from one part of an animal to another. Meat provides 25% of the total fat content of the average British diet. Oily or fatty fish such as herring and mackerel contain up to 20% oil, but they contribute little fat to the British diet as they are eaten infrequently. 2. Butter and margarine: Butter contributes 6% and margarine a further 6% of the total fat content of the average diet. 3. Milk, cream and cheese: Full cream milk contains between 3% and 4% fat; some products made from milk, such as cream and cheese contain much larger amounts. Milk and milk products account for 11% of the total fat content of the average British diet, and cheese contributes a further 6%. 4. Other foods : Other important sources of fat are baked goods (cakes, pastries, biscuits) 8%, and vegetable oils, 8%. Many other foods contain a considerable amount of fat. These include ice-cream, chocolates, some sweets, nuts and salad dressings. Most vegetables and fruits do not contain significant amounts of fat except the soya bean (24% fats) and the avocado pear (8% fat). 2.3.2.5 Dietary guidelines It is recommended that, on average, 35% of our energy (Calorie) intake comes from fats and oils with only 11% from saturated fatty acids. At present we eat considerably more than this and hence we are advised to eat less fat, particularly saturated fat.
2.3.3 Proteins
2.3.3.1 Introduction and chemical structure Proteins are found in all living cells of animals and plants. Protein must be provided in the diet for the growth and repair of the body, but any excess is used to provide energy. Proteins consist of chains of hundreds or even thousands of amino acid units. Only about
19
BASICS OF HUMAN NUTRITION
20 different amino acids are involved, but the number of ways in which they can be arranged is almost infinite. It is the unique sequence of these units which gives each protein its characteristic properties. Of the 20 amino acids commonly found in proteins, 8 are essential in the diet (9 for children) and must be supplied by the foods we eat since they cannot be made in the body. The non-essential amino acids can be synthesised in the body by converting one amino a~id into another within the body's cells. (Table 2.2) Table 2.2: Essential and Non-essential Amino Acids Essential Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Histidine (essential for infants)
Non-essential Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Proline Serine Tyrosine
2.3.3.2 Functions of proteins in the body I. Growth and Maintenance : Proteins are the main constituents of the cells of the body. The number of cells in the body increases during periods of growth, therefore during childhood and adolescence, protein requirements are high. In addition, protein in the tissues is constantly being broken down and must be replaced from the amino acids supplied in the diet. Protein is also necessary for the formation of enzymes, antibodies and some hormones. II. Energy: The diet may supply more protein than is required for growth and maintenance. Any excess protein may be used for energy. 19 of protein provides 4kcal (17kJ). 2.3.3.3 Sources of protein in the diet Protein can be obtained from both animal and plant sources.
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MICROBIAL SPOILAGE OF FOODS
1.
2.
3. 4. S.
Meat and fish: Meat makes an important contribution to the protein content of the average British diet. Fish is eaten less frequently and hence makes a smaller contribution to protein intake. Bread and cereals : Bread contains a significant amount of protein and is one of the most important and cheapest sources in the British diet. Other cereal foods such as rice, pasta, breakfast cereals, cakes and biscuits are also significant sources of protein. Milk and cheese : Milk and cheese are valuable sources of good quality protein in the British diet. Eggs: These are an excellent source of high quality protein although their contribution to the diet is small. Nuts: Nuts are a major source of protein in the diets of many vegans and vegetarians.
2.3.3.4 Dietary guidelines In the world most people eat much more protein than they need. It is recommended that we obtain 15% of our energy from protein which, for an adult, is between 45 and SSg protein per day. 2.3.4 Vitamins
2.3.4.1 Introduction and types Vitamins are substances which the body requires in small amounts, yet cannot make for itself, at least in sufficient quantities. They must, therefore, be eaten as part of the diet. As the vitamins were discovered, each was first labelled with a letter, but once a vitamin has been isolated and its structure identified, it was given a specific name. For example, the chemical found in milk which promoted growth was given the name vitamin A. As the vitamins were identified it became possible to divide them into two groups: 1. Fat soluble vitamins Vitamin A - retinol Vitamin D - cholecalciferol Vitamin E - tocopherol Vitamin K - phylloquinone 2. Water soluble vitamins Vitamin B1 - thiamin Vitamin B2 - riboflavin
21
BASICS OF HUMAN NUTRITION
Nicotinic acid Vitamin B6 - pyridoxine Vitamin B12 - cyanocobalamin Folic acid Pantothenic acid Biotin Vitamin C - ascorbic acid Fat soluble vitamins can be stored in the human body, water soluble vitamins cannot. This means that an adequate daily intake of water soluble vitamins such as vitamin C is particularly important. One drawback to the body's ability to store fat soluble vitamins is that toxic levels can accumulate in the body, although this condition is rare. In general, any excess of the water soluble vitamins is immediately excreted in the urine.
2.3.4.2 Functions of vitamins • promote health and help prevent disease • regulate the building and repair of body cells • help regulate the chemical reactions which release energy in body cells. A well-balanced diet will contain all the vitamins in the recommended quantities but as some foods are very poor sources of certain vitamins it is essential to choose appropriate foods. It is rare for people in the developed countries to suffer a severe deficiency of vitamins. Some people may suffer minor deficiencies with symptoms such as tiredness, broken nails, poor condition of skin, h~r and teeth. These symptoms may be due to a shortage of one or of several vitamins. The amount of each vitamin needed to promote health is more than the amount needed to prevent disease. Active form, biochemical functions and deficiency of various fat-soluble and water-soluble vitamins are summarized in Table 2.3. Table 2.3 : Biochemical Functions of Vitamins Vitamin Fat-soluble: Vitamin A [Retinol]
Active Form
Function
ll-cis-retinal retinoic acid
vision, growth factor, xerophthalmia, maintenance of kera tomalacia or blindness epithelial integrity contd ....
Deficiency
22
MICROBIAL SPOILAGE OF FOODS
...contd. / Vitamin Vitamin D [Calciferol] Vitamin E [Tocopherol]
Active Form I, 25-dihydroxycholecalciferol
Function calcium and phosphate metabolism a-tocopherol biological antioxidant, intra-cellular respiration, cell and vascular integrity, central nervous system and muscle integrity 2 methyl-3-phytyl- formation of certain I, 4-naphthoblood clotting factors quinone
Vitamin K [Phylloquinone] Water-soluble: Tniamin (B-1) thiamin pyrophosphate Riboflavin (B-2)
flavin mononucleotide, flavin adenine dinucleotide
Niacin
nicotinamide adenine dinucleotide; nicotinamide adenine dinucleotide phosphate tetrahydrofolic acid and enzymes carboxybiotin and biotin enzymes
Folacin Biotin
Pantothenic acid
co-enzyme A
DeficicncI rickets, osteomalacia microcytic anemia and edema in prematures; creatinuria, red cell hemolysis, ceroid pigment hemorrhage, decreased clotting
aldehyde transfer, metabolism of carbohydrates hydrogen and electron transfer, role in metabolism of macronutrients
beriberi
hydrogen transfer, degradation and synthesis of fatty acids, carbohydrates and amino acids formyl transfer, C 1 metabolism CO transfer, 2 carboxyl group transfer, fatty acid biosynthesis acyl transfer, role in macro-nutrient metabolism
pellagra
glossitis, dermatitis cheilosis, ocular symptoms
macrocytic anemia seborrheic dermatitis
gastrointestinal and nervous disorders contct ....
23
BASICS OF HUMAN NUTRITION
... contd. Vitamin
Active Fonn
Function
Deficien~
Pyridoxine
pyridoxal phosphate, and decarboxy lases, deaminases, transaminases B-12 co-enzymes and enzymes ascorbic acid, dehydroascorbic acid
NH transfer, other
convulsions, dermatitis
(8-6)
Cobalamin (B-12) Vitamin C
Choline
choline, acetylcholine
2
functions in amino acid metabolism
dehydrogena tion, methylation integrity of intercellular substances, oxidation-reduction systems fat metabolism, compound of phospholipids, methyl donor for transmethylation
pernicious anemia scurvy
fatty liver
2.3.5 Minerals
2.3.5.1 Introduction and Importance Apart from hydrogen, carbon and oxygen (the main elements of which protein, fat and carbohydrate are composed) the body also requires around 20 other elements for a variety of reasons. Fifteen of them are known to be essential and a further three or more are necessary for normal life in other animal species and may prove to be necessary for man: no dietary deficiency has yet been shown. Minerals have four main functions: • body building e.g. constituents of bones and teeth • control of body processes, e.g. transmission of nerve impulses • essential part of body fluids and cells • form part of many enzymes and other proteins which are necessary for the release and utilisation of energy. Some mineral elements are required in relatively large amounts and are known as major minerals. These include:
24
MICROBIAL SPOILAGE OF FOODS
1. Major Minerals:
Calcium Sodium Phosphorus Chloride Magnesium Potassium Zinc Iron Sulphur Others are required in minute amounts and are known as trace elements. 2. Trace Elements: Iodine Fluoride Molybdenum Copper Cobalt Sulphur Chromium Selenium Manganese Information about the various major minerals are summarized in Table 2.4. Similarly, main functions and rich sources of trace elements are mentioned in Table 2.5. Table 2.4: Major Minerals Calcium
Iron
Sodium
Phosphorus
Main Functions Growth and development of bones and teeth; blood clotting and hormone secretion Red blood cell formation; oxygen transport and transfer. Deficiency can lead to anaemia Maintenance of constant body water content, muscle and nerve activity. High intakes have been related to high blood pressure Component of all cells; combines with other
Rich Sources Milk, cheese, yoghurt, flou~bread,green
vegetables, canned fish Red meat (particularly offal), bread, flour and cereal products, green leaf vegetables Table and cooking salt, bread, cereal products, meat products
Milk, milk products, bread, cereal products, contd ....
25
BASICS OF HUMAN NUTRITION ... contd.
Magnesium
Chloride
Potassium Zinc
Sulphur
Main Functions minerals (eg. calcium) to give strength to bones and teeth Muscle tone; activation of enzymes-especially in protein synthesis Assists sodium and potassium
Rich Sources meat, meat products
Milk, bread, cereal products, potatoes, other vegetables Table and cooking salt, bread, cereal products, meat products, milk Vegetables, meat, milk, Maintenance of constant fruit and fruit juices body water content Meat and meat Bone metabolism; activation of enzymes; release of products, eggs, fish Vitamin A; growth; immune system; taste; insulin release Component of certain Protein containing foods essential amino acids; metabolism of drugs; bone metabolism Table 2.5 : Trace Elements
Iodine
Copper
Main Functions Constituent of thyroid hormones, which regulate many body processes Growth; component of many enzymes - including those needed for formation of blood and bone - and in the body's defence system; neurotransrnittor function; cell respiration
Rich Sources Milk and milk products, meat, eggs, fish Wholegrain cereals, meat, vegetables
contd ....
26
MICROBIAL SPOILAGE OF FOODS
... contd. Cobalt Chromium
Manganese SeleniuI"
Molybdenum
Fluoride
Main Functions Component of Vitamin BI2 Enhances the action of insulin, which controls the utilisation of glucose Helps to maintain structure of cells; enzymes Part of an enzyme involved in protection of membranes and lipids against oxidative damage Component of several enzymes-including one concerned in the formation of uric acid; possibly in the utilisation of iron Increases the resistance of teeth to decay
Rich Sources Animal products Widely distributed, particularly wholegrain cereals and vegetables Wholegrain cereals, nuts and tea Cereals, fish, offal, meat, cheese, eggs, milk
Widely distributed, particularly in vegetables and pulses
Tea, fish, water
2.3.6 Water By the end of this section you should be able to understand why although water is not a nutrient, it is essential to life.
2.3.6.1 Water balance All living organisms contain water; the human body consists of about 65% water. It is the medium in which nutrients, enzymes and other chemical substances can be dispersed and in which the chemical reactions necessary for maintaining life take place. It is also necessary as a means of transport within the body. Nutrients are carried to cells and waste products are transported from the cells by blood plasma which is 90% water. It is possible to exist for several weeks without food, but the body can only survive a few days without water. Water comes from 'solid' foods as well as from drinks and is lost by evaporation in the breath and sweat as well as in the urine. The balance of water retained in the body is normally very carefully regulated by the kidneys. Excessive losses can occur usually as a result of vomiting or diarrhoea, in illness or from heavy sweating
BASICS OF HUMAN NUTRITION
27
due to strenuous activity or a hot climate. If water intake is not increased, dehydration may result. The amount of water taken into the body is determined mainly by habit and social custom. It is also regulated by thirst which arises as a result of the concentration of sodium in the blood. The body cannot store water and any excess passes into the urine.
2.4 FATE OF MAJOR NUTRIENTS IN THE BODY? 2.4.1 Carbohydrates The simple sugars entering the intestinal wall are carried by the blood stream directly to the liver. They may then be: • passed as glucose to all the cells of the body to be used directly for energy • converted into glycogen and stored in the liver and skeletal muscles as a readily available source of energy • converted into fatty acids and stored in the body fat (adipose tissue) as a source of energy.
2.4.2 Fats Almost all the fatty acids which enter the intestinal wall are immediately rebuilt into triglycerides which are carried to the blood stream by lymph. Fat may be further transformed by the liver and some of it is finally deposited in the adipose tissue. The reservoir of fat is constantly available as a source of energy.
2.4.3 Proteins When the pep tides enter the intestinal wall, they are split into amino acids which are carried in the blood directly to the liver. Then: • they may be passed into the general circulation where they enter the body's 'pool' of essential and non-essential amino acids. These are then built into the structural proteins and specific enzymes which each cell needs • the excess of some amino acids may be converted into those that are lacking • any excess of amino acids will be used as a source of energy or converted to body fat.
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MICROBIAL SPOILAGE OF FOODS
2.5 FOOD AND ENERGY 2.5.1 Energy value of nutrients All living organisms require a source of energy. The chemical energy in food is released in the cells of animals by oxidation. Some of the energy is used to maintain metabolic processes in the cells, some is converted into heat to maintain body temperature and some is converted into mechanical energy which is used for physical activity. The unit of energy is the joule. Since the joule is too small for practical nutrition the kilojoule (kJ) is used. However, traditionally the Calorie or more correctly, the kilocalorie (kcal) unit was used and so normally energy values are given in both types of unit. 1 kilocalorie (lkcal) = 4.18kJ An even larger unit, the megajoule (MJ) is also used: 1MJ = 1,000,000 joules = 1000kJ The three groups of nutrients which provide the body with energy are carbohydrates, fats and proteins. • 19 of carbohydrate provides 3.75kcal (16kJ) • 19 of fat provides 9kcal (37kJ) • 19 of protein provides 4.0kcal (17kJ) 1£ alcohol is consumed this also contributes to the body's energy intake. • 19 of alcohol provides 7kcal (29kJ) 2.5.2 Energy value of foods The energy value of food, often called the Calorie content, depends on the quantities of carbohydrate, fat and protein (and sometimes alcohol) in the food. The total energy value of a food in Calories will be the total of the amount of Carbohydrate (g) X 3.75, Protein (g) X 4, Fat (g) X 9 plus Alcohol(g) X 7.1£ the, energy value is required in kJ thel'. the kJ conversion factors given above will need to be used. 2.5.3 Use of energy by the body 1.
Basal Metabolism: This is the term used to describe the basic metabolic processes which keep the body alive. Energy is needed to keep the heart beating and the lungs
29
BASICS OF HUMAN NUTRITION
functioning, to maintain body temperature and muscle tone and for the numerous chemical reactions taking place in body cells. The rate at which energy is used up in maintaining basal metabolism is called basal metabolic rate (BMR). 2. Physical Activity: In addition to basal metabolism, energy is used by the body for muscular activity. The energy requirements for-various activities have been determined by measuring oxygen uptake during different activities. (Table 2.6). Table 2.6 : Energy consumption for various activities Activity Sitting Writing Standing Washing-up Domestic work Walking Running Tennis Football Swimming
kcallmin 1.4 1.7 1.7 2.4 2.9 3.3-5.0 6.0-15.6 4.S-S.4 4.S-S.4 4.S-12
Although these figures are not accurate for any individual, they provide useful comparisons. 3. Growth, Pregnancy and Lactation: Additional energy is needed during growth to provide for the extra body tissue. During pregnancy and lactation all the infant's needs for energy must be supplied by the mother. Up to SO,OOO kcal of extra food energy may be needed during pregnancy mostly during the final months. 4. Heat: Some of the energy in food will be used as a source of heat, some of which will be used to keep the body warm. The dietary energy required by an individual who is neither gaining nor losing weight exactly equals the energy expended on maintenance and physical activity. In practice this balance is achieved over periods of a few days with remarkable accuracy.
30
MICROBIAL SPOILAGE OF FOODS
2.6 DIETARY ALLOWANCES FOR INDIANS The preceding pages have provided an account of the importance of the different nutrients present in food. In order to prevent the ill-effects due to deficiency of particular nutrients and to sustain a vigorous and healthy life, it is necessary to know in quantitative terms the amounts of the different nutrients needed. Obviously, this need will vary with factors such as age, sex and type of work. A schedule of dietary allowances will have to meet at least the minimum nutritional needs of the majority of persons for whom it is applied and at the same time provide reasonable margin to allow for physiological nonavailability of some nutrients from particular foods. Such a schedule will help a group of persons to select the proper foods to make up a diet that will provide the nutrients in the amounts indicated. Also, on a national level such allowances will be useful in enabling governments to plan their food production policies, to judge the adequacy or otherwise of the national supply of foods, and to point out the areas in which improvements are called for. The recommendations of ICMR (Indian Council of Medical Research) for dietary allowances for Indians are mentioned in Table 2.7. Explanatory Notes on Table 2.7 (1) The dietary allowances suggested for adults are for a reference man weighing 55 kg. and for a reference woman weighing 45 kg. The allowances for calories and proteins and for B-complex vitamins should be increased or decreased depending on the body weight. (2) The allowance for protein recommended by Nutrition Expert Group of Indian Council of Medical Research for adult is about 1 gm. per Kg. body weight per day and it is assumed that the dietary protein is derived from a mixture of vegetable foods. Proteins of animal origin are superior in biological value as compared to vegetable proteins. However, it is possible to improve the biological value of vegetable proteins through a proper admixture of foodstuffs, and it is for this reason that it is not insisted that a certain proportion of the total protein should be derived from animal foods. For infants and children and for pregnant and nursing women, however, it is desirable to supply some part (about 25%) of the total protein from animal foods such as milk, egg, flesh foods, etc. (3) The requirements for fats have not been indicated in the Table and the subject has been discussed in the text. It would appear to be unnecessary to have a fat intake which supplies more than 15% of the calories in the diet. About 15 gms. of vegetable oils, however, should be present in the diet to meet the essential fatty acid requirement.
Table 2.7 : Daily Allowances of Nutrients for Indians
Group
Man
Woman
Infants ChiIdrt'n
Adolescents
Particulars
Sedentary work Moderate work Heavy work Sdedentary work Mod~rate work Heavy work Pragnancy (.ccond half of pregnancy) Lactation (upto 1 year) 0-6 months 7-12 months
2400 2800
0.4 to 0.5
] 55
30
750
40
750
1.2 3000 1.4 2.0 1.0 3000 1.1 1.5 3000 +0.2
30
1150
4600
20
750
3900
1900 2200 3000 +300
J45
0.4 to 05
"0]
i700
+20
120/kg. 100/kg.
2.3-1.8/kg 1.8-1.5/kg
1.3 1.5 2.2 1.0 1.2 1.7 +0.2
+0.4
16] 19
50
100
13] 15 20
50
100
+2
50
150-300]
+5
80
150
26
1.0
1.5
0.5-0.6
1 mg/kg 400 kg. 300
1200
15-20] 250
lOOO
+0.4
25
0.2
1 year]
2 years 3 years 4-6 years 7-9 years 10-12 years
1200 1500 1800 2100
13-15 yrs. Boys Girls 16-18 yrs. Boys Girls
2500 2200 3000 2200
18
17] 20
~
0.4 to 05
0.6
0.7
8
600
1200 0.1l 1600 0.9 2400 1.0
0.8 1.0 1.2
10 12 14
750
3000
1.4 1.2 17 1.2
17 14 21 14
300 400
41 0.6-0.7
0.5-0.6
~J ~J
1.3 1.1
750
3000 1.5 1.1
30-50 50-100
0.5-1.0
32
MICROBIAL SPOILAGE OF FOODS
(4) Figures for carbohydrates are also not given in the Table; but about 70% of the calories in a diet can be from carbohydrates. (5) Most of the ingredients of a diet are rich in phosphorus, and it is for this reason the allowances for this element are not listed. (6) Minerals such as magnesium, copper, iodine, etc., are also essential in nutrition, but they are needed only in small amounts. Normally, if a diet is well-balanced and is adequate with reference to other nutrients, the requirements for these trace elements can be assumed to have been met. (7) Dietary allowances for vitamin A are given both in terms of retinol (preformed vitamin A) and /3-carotene, and the required amounts of vitamin A can be obtained from either or both. Although by definition 1 mg. of /3-carotene is equivalent to more than O. 5 mg. (1666 LU.) of retinol, some studies indicate that for all practical purposes it may be taken as equivalent to 0.25 mg. of retinol because of the inefficiency of utilization of carotene as a source of vitamin A. The total vitamin A value of a diet in terms of retinol can be calculated as follows:Total vitamin A value as Retinol (Ilg) = Retinol (Ilg) + /3 carot:ne(llg) (8) A part of the vitamin D requirement is undoubtedly met by the action of sunlight on the skin. However, it may not be advisable to rely entirely on sunshine for obtaining the vitamin D requirements, especially in the case of children. (9) The requirements for thiamine, riboflavin and nicotinic acid are related to calorie intake and the recommended allowances per 1,000 calories are: thiamine, 0.5 mg., riboflavin, O. 55 mg. and nicotinic acid, 6.6 mg. Nicotinic acid allowances include contribution from dietary tryptophan, 60 mg. of tryptophan being equivalent to 1 mg. of nicotinic acid.
2.7 BALANCED DIETS In the preceding pages, the importance of the various nutrients
in human nutrition has been considered. We shall now consider the planning of diets which would provide these essential nutrients in the needed amounts and proportions. A 'balanced diet' is one which contains different types of foods in such quantities and proportions that the need for calories, minerals, vitamins and other nutrients is adequately met and a small provision is made for extra nutrients to withstand short durations of leanness. Taking into account the foods which commonly form part of the Indian diets, balanced diets have been suggested for various groups of population, and the composition of such diets is given in tables 2.8, 2.9, 2.10 and 2.11.
0:1
Table 2.8 : Balanced Diets for Adult Man Sedentary Work Vegetarian Nonvegetarian
Moderate Work Vegetarian Nonvegetarian
> 5!l n til
Heavy Work 0.., Vegetarian Non::r:: vegetarian ~ > z (gm.) (gm.) Z 7 6 650 650 0 z 80 65 125 125 100 100
(gm.)
(gm.)
(gm.)
(gm.)
1 Cereals Pulses
2 400 70
Green leafy vegetables Other vegetables
100 75
3 400 55 100 75
4 475 80 125 75
5 475 65 125
Roots and tubers Fruits Milk
75 30 200
100
100
100
100
30 200
30 100
30 200
30 100
Fats and oils Meat and fish
35
75 30 100 40
40
40
50
50
Eggs Sugar and jaggery Groundnuts
30 30 30
30
75
~
30 30 40
40
* An additional 30 gm. of fats and oils can be included in the diet in place of groundnuts.
30 30 55 50
55 50* w w
YJ
Table 2.9 : Balanced Diets for Adult Woman Sedentary Work Vegetarian
Cereals Pulses Green leafy vegetables Other vegetables Roots and tubers Fruits Milk Fats and oils Sugar and jaggery Meat and fish Eggs Groundnuts
Moderate Work
~
Heavy Work
Vegetarian NonVegetarian NonNon:· vegetarian vegetarian vegetarian
Additional Allowances During Pregnancy Lactation
(~m.)
(~m.)
(~m.)
(~m.)
(~m.)
(~m.)
(~.)
(~.)
300 60 125
300 45 125
350 70 125
350 55 125
475 70 125
475 55 125
50
100 10 25
75 50 30 200 30 30
75 50 30 100 35 30 30 30
75 75 30 200 35 30
75 75 30 100 40 30 30 30
100 100 30 200 40 40
100 100 30 100 45 40 30 30 40""
40""
25
125 10
125 15 20
~
Q ~
;;
I""
Vl
~>
C) tT1
0
'T1
""An additional 25 gm of fats and oils can be included in place of groundnuts.
8 0
<J'l
c;j
Table 2.10: Balanced Diets for Children Pre-School Children 1-3 Years 4-6 Years Vegetarian
:> :!3
School Children 7-9 Years 10-12 Years
NonNonVegetarian NonNonVegetarian Vegetarian vegetarian vegetarian vegetarian vegetarian
(gm.)
(gm.)
(gm.)
(gm.)
(~m.)
(gm.)
(sm.)
(sm.)
Cereals
150
150
200
200
250
250
320
320
Pulses
50
40
60
50
70
60
70
60
Green leafy
50
50
75
75
75
75
100
100
Other vegetables
30
30
50
50
50
50
75
75
Roots and tubers
30
30
50
50
50
50
75
75
Fruits
50
50
50
50
50
50
50
50
Milk
300
200
250
200
250
200
250
200
Fats and oils
20
20
25
25
30
30
35
35
n <Jl
...,0
::r: ~:> Z
Z
~
::l
0
z
vegetables
Meat and fish
30
30
30
30
Eggs
30
30
30
30
Sugar and jaggery
30
30
40
40
50
50
50
50 c..J V1
w
Table 2.11 : Balanced Diets for Adolescent Boys and Girls Boys 13-15 years NonVegetarian vegetarian Cereals Pulses Green leafy vegetables Other vegetables Roots and tubers Fruits Milk Fats and oils Meat and fish Eggs Sugar and jaggery Groundnuts
0'1
Girls 16-18 years NonVegetarian vegetarian
13-18 years Vegetarian Nonvegetarian
(gm.)
(~.)
(~.)
(~.)
(gm.)
(gm.)
430 70 100 75 75 30 250 35
430 50 100 75 75 30 150 40 30 30 30
450 70 100 75 100 30 250 45
450 50 100 75 100 30 150 50 30 30 40 50*
350 70 150 75 75 30 250 35
350 50 150 75 75 30 150 40 30 30 30
30
40 50*
30
*An additional 30 gm. of fats and oils can be included in the diet in place of groundnuts.
~
Q ~
;>
I"" (J)
g I""
>
C') rT1
~ "Tl
§ til
BASICS OF HUMAN NUTRITION
37
2.8 CLASSES OF NATURAL FOODSTUFFS AND THEIR NUTRITIONAL SIGNIFICANCE Diets with composition shown in the tables supply all the essential nutrients in adequate amounts and keep the majority of individuals consuming them in a state of good health. It may be pertinent then at this stage to consider how each class of foodstuffs suggested in the above diets supplies our daily requirement of the various nutrients. 2.8.1 Cereals Rice, wheat and millets Gowar, bajra, ragi etc.} are the main cereal grains consumed in India. They are the cheapest sources of calories and they contribute as much as 70 to 80% of the calories in the diets of a majority of population in our country. In view of the large amounts in which cereals are included in the diet, they form important sources of nutrients in an average Indian diet. Most cereal grains contain 0 to 12 per cent protein, and in general cereal proteins are somewhat deficient in the essential amino acid lysine which limits the protein quality. Rice protein, however~ is richer in lysine compared to the other cereal proteins and for this reason rice protein is of better quality. Most cereal grains are poor in mineral content and rice is an especially poor source of two important minerals, calcium and iron. However, ragi is very rich in these minerals, especially calcium, and inclusion of this millet in adequate amounts in the diet will go a long way in making up the deficiencies of some of the minerals in the diet. Bajra is also a good source of iron. Whole cereal grains are important sources of B-vitamins, especially thiamine and nicotinic acid. Since these vitamins are present in the cereal grain in the outer bran layers, the vitamin content of the finished product depends on the degree of removal of the outer layers. Particularly in the case of raw rice, the vitamin content decreases with the increase in the degree of milling and polishing given to the grain. Highly poliShed raw rice, therefore, has a very poor content of vitamins. Parboiled rice, on the other hand, contains significant amounts of thiamine because during the course of parboiling in which paddy is subjected to steaming or boiling in water, the vitamin seeps into the inner portions of the grain so that even if the grain is milled and
38
MICROBIAL SPOILAGE OF FOODS
polished, significant amounts of the vitamin are still retained in the grain. Except yellow maize, which contains some amounts of carotene, cereal grains in general do not contain much vitamin A activity and vitamin C.
2.8.2 Pulses Pulses (or legumes as they are also called) are rich in proteins. In diets in which flesh foods are present only in small amounts, pulses are therefore important as a source of protein. Pulse proteins, however, are of relatively low biological value because of the deficiency of the essential amino-acid methionine. Red gram is deficient in tryptophan also. However, pulse proteins are rich in lysine and they are therefore, of good supplementary value to cereal diets. The lysine deficit in cereals is made good by the lysine present in pulses and thus the overall biological value of cereal-pulse diets is better. In the amounts consumed, pulses cannot be considered rich sources of minerals, but they are rich in B-vitamins, especially thiamine and folic acid. Dried pulses do not contain vitamin C in any significant amounts, but when they are germinated, significant amounts of vitamin C are elaborated so that sprouted pulses, especially sprouted green gram and Bengal gram become rich source of this vitamin.
2.8.3 Nuts and Oilseeds Like pulses, nuts and oilseeds are also rich in proteins and in addition they contain fat so that they are rich in calories also. Most of the oilseeds produced in the country are used for extraction of edible oils and the cake left behind is even richer in protein than the original seed. Oilseed cakes were not being used as human food to any significant extent till recently because the methods used so far for extraction of oil were not good enough to produce a wholesome cake. Also, with country 'ghanis' the removal of oil is not complete, and the oil that is retained in the cake turns rancid in course of time and gives rise to off-flavours in addition to posing storage problems. However, improved extraction procedures followed in large mills in recent years have enabled the production of clean products practically free fJ::om offflavours. The meal can be used as such in various ways as food for humans, and procedures are-Cilso available for production of 'protein isolates' from the oil meals.
BASICS OF HUMAN NUTRITION
39
In common with other proteins of plant origin, oilseed proteins
are also low in biological value because of a relative deficiency of the amino-acid methionine, and groundnut protein is particularly poor in methionine. Gingelly (sesame) protein, however, is relatively richer in this amino-acid, as also is sunflower seed protein. Besides protein, oilseeds are rich sources of B-complex vitamins also. Groundnut especially is very rich in thiamine and in nicotinic acid. Some work carried out in recent years in various parts of the world showed that many foodstuffs can become contaminated with fungi (moulds) if they are stored under humid and unhygienic conditions. Some of these fungi produce toxins which are pOSitively deleterious to health. Groundnut is shown to be particularly prone to infestation with a fungus known as Aspergillus flavus which produces aflatoxin, and this toxin has been shown to cause damage to the liver in many experimental animals including monkeys. Only clean and wholesome groundnuts should, therefore, be used as food, and when dealing with the deoiled cake it should be seen to it that it does not contain aflatoxin in amounts above the accepted safe and permissible limits. 2.8.4 Green Leafy Vegetables
Many types of green leaves such as palak, amaranth, fenugreek leaves, drumstick leaves, mint etc., are consumed all over the country as vegetables, and most of them are rich sources of calcium, iron, carotene, vitamin C, riboflavin and folic acid. These vegetables are, therefore, inexpensive sources of many nutrients which are essential for growth and maintenance of normal health. Deficiency of these nutrients is commonly seen in our country and steps should, therefore, be taken to encourage cultivation of green leafy vegetables in kitchen gardens and school gardens. Consumption of such vegetables in adequate amounts especially by pregnant and nursing women and by children should also be encouraged. 2.8.5 Root Vegetables
Some of the important foodstuffs belonging to the group of root vegetables are tapioca, potato, sweet potato, carrots, yam and colocasia. They are all rich in carbohydrates and hence they yield mainly energy. Foods like carrots and yellow varieties of yam are also rich in carotene,
40
MICROBIAL SPOILAGE OF FOODS
and foods like potato contain significant amounts of vitamin C. Some root vegetables like tapioca, which is consumed commonly in Kerala, are such high yielders per acre of land that they have served as emergency or famine foods in times of cereal shortage. 2.8.6 Other Vegetables Other vegetables are those which do not fall under the category of leafy and root vegetables. Many such vegetables like brinjals (eggplant), ladies fingers (okra), French beans, various gourds, etc., are consumed mainly to add variety to the diet. Some of them are also fair sources of vitamins and minerals. 2.8.7 Fruits Fruits are in general good sources of vitamin C, and amla is an especially rich source of this vitamin. Yellow fruits like mango and papaya contain carotene in addition and dried fruits like dates and raisins are sources of iron. The commonly used banana is a fruit rich in carbohydrate, and it therefore yields energy also. If green leafy vegetables are included in the diet in adequate amounts, the need for fruit as an essential item in the diet is much reduced. 2.8.8 Milk and Milk Products Milk is an ideal food for infants and children, and it is a good supplementary food for adults. It contains proteins of good quality and also other nutrients in proper proportion and it is thus a complete food. It is, however, deficient in vitamin C and in iron. With only minor exceptions, the overall nutritive value of milk of different species can be said to be similar. Human milk contains more lactose (milk sugar) and buffalo milk contains more fat as compared to cow's milk. Cow's milk contains more protein than does human milk. Unless the whey is thrown away, the products derived from milk retain most of the nutrients contained originally in milk. For example, curd, which is the form in which milk is consumed to a significant extent in India, is for all practical purposes the same in nutritive value as milk is. The nutrient composition of dried milk (milk powder) is more or less the same, as that of milk on a moisture-free basis. The requirement for milk by persons of different age groups are given in the suggested balanced diets. It may be noted that these
BASICS OF HUMAN N1ITRITION
41
amounts are low, but it should be pointed out that these low figures are suggested as practical levels in the context of the prevailing low per capita availability of milk in the country. It should be our aim in food planning to achieve a much higher figure than this. In the more advanced countries and also in some regions in our country, the daily intake of milk is nearly 600 ml. per person. Renewed and vigorous efforts should be made to increase the average level of milk consumption, and in the meantime the available milk should be channelized to meet the priority needs of infants, growing children and pregnant and nursing women. 2.8.9 Sugar and Jaggery Sugar and jaggery are used as sweetening agents in beverages and other foods to increase their palatability. They are mainly sources of energy although jaggery contains in addition, iron. 2.8.10 Fats and Oils The visible fats that enter the diet are fats such as butter and ghee and the various vegetable oils and sometimes also vanaspati made by hydrogenation of oils. Irrespective of the type, all fats and oils yield the same amount a energy. However, vegetable oils have necessarily to be included in the die to the extent of 15 gms. per day to obtain the necessary amounts of essential fatty acids required by the body. Vegetable oils, especially safflower oil, are rich in polyunsaturated rated fatty acids.
2.8.11 Flesh Foods Flesh foods such as fish and meat are rich in proteins of high biological value and in B-vitamins. Especially vitamin B12 is contained only in foods of animal origin and not in plant foods. Flesh foods are generally not good sources of vitamin A, but liver, which is very rich in vitamin A, is an exception. Fish is a good source of calcium, especially the small varieties which are consumed whole.
2.8.12 Eggs Egg is a rich source of all nutrients except vitamin C. The protein contained in egg is considered to be a perfect protein, and because of its high biological value and digestibility, egg protein is used in nutrition work as a reference protein for comparison with other proteins. Egg of different species of birds can be said to be similar in
42
MICROBIAL SPOILAGE OF FOODS
nutritive value. Raw egg-white, however, contains a protein known as avidin which renders the vitamin biotin unavailable to the body. Duck egg-white contains in addition a substance known as trypsin-inhibitor which inhibits the action of trypsin on protein. Heating egg, as, for instance, in the preparation of boiled egg destroys both avidin and the trypsin-inhibitor. 2.8.13 Condiments and Spices These are accessory foodstuffs mainly used for flavouring food preparations. Some of the condiments like chillies and coriander are good sources of carotene. Green chillies supply vitamin C, and turmeric and tamarind are fair sources of iron. However, because of the small amounts in which many of the condiments and spices are used, they do not add substantially to the l\utritive value of the diet. Some spices like garlic and asafoetida are believed to contain active principles which inhibit the growth of putrefactive bacteria in the intestinal tract.
DOD
AN INTRODUCTION TO MICROBIAL SPOILAGE OF FOODS
3.1 PROLOGUE Spoilage of food involves any change which renders food unacceptable for human consumption and may result from a variety of causes. It is often difficult to decide when a food is actually spoiled since views differ on what is and is not acceptable and fit or unfit to eat. These differences of opinion are particularly evident when viewed on a worldwide basis as can be illustrated by the following well-known example. The British prefer game meat to be 'hung' for several days to allow organoleptic changes to take place which encourage the development of a 'strong' flavour. Whilst the British consider such flavoured meat to be a delicacy other nationalities, including Americans, regard it as spoiled and unacceptable.
3.2 MAJOR REASONS FOR FOOD SPOILAGE The main causes of food spoilage are: 1. Physical damage in transporting, storage, etc., resulting in changes in texture such as bruising. 2. Insect, rodent or other animal activity. 3. Chemical breakdown or chemical contamination resulting in deterioration in quality. 4. Autolytic enzymes which catalyze reactions within the food resulting in texture breakdown and the food becoming
44
MICROBIAL SPOILAGE OF Fboos
soft and pulpy. It may also be more vulnerable to attack by insects, rodents and microorganisms. 5. Micro-organisms, whose entry into the food is aided by I, 2, 3 and 4 above, which grow and change the texture, colour, taste, smell and quality of the food. Spoilage caused by micro-organisms is recognized by changes in foods which are often given common names - such as 'slime', 'rots' and 'putrefaction'. The main features of microbial spoilage are that the texture of the food degenerates and it gradually becomes soft and sticky and eventually fluid. These changes are often accompanied by odours which become more marked as time passes-some odours are very distinct, of which the 'sulphur stinker' spoilage of canned foods is an extreme example. Spoiled food can also change in colour, although this is dependant on the type of organism present. The characteristics of these microorganisms which cause them to spoil foods are those which also make them beneficial in the normal decay of organic material in the soil and in water. They break down the organic components of foods for their own use and in so doing convert them to simpler compounds. 3.3 FOOD-SPOILAGE TYPES The main types of spoilage are: 3.3.1 Mouldiness and 'whiskers' Moulds, being aerobic, grow mainly on the outside surfaces of the affected foods, initially as small separate colonies-'spots' which may later merge. Foods become sticky, 'whiskery' and locally coloured. 3.3.2 Rots A general word used to refer to spoilage of fruit, vegetables, eggs and other foods, for example, black rot of eggs, watery soft rots of fruit and vegetables. 3.3.3 Sliminess Growth of bacteria on moist surfaces of vegetables, meat, fish, etc., may cause taints and odours and can result in such deterioration of the food that it degenerates into slime. Pigmentation may occur at the same time. 3.3.4 Colour Change Many microbes produce brightly coloured colonies or pigments which give colour to the spoiling food, for example Serratia marcescens-
AN INTRODUCTION TO MICROBIAL SPOILAGE OF FOODS
45
red, Sarcina lutea-yellow, Pseudomonas fluorescens-green with fluorescence, Aspergillus niger-black, Penicillium species-green. 3.3.5 Ropiness Rope is the formation of a viscous sticky material closely allied to slime and caused by a wide variety of organisms such as LeUconostoc
mesenteroides, Leuconostoc dextranicum, Bacillus subtilis, Lactobacillus plantarum and others. In some foods, especially high sugar foods, the rope organisms produce copious capsules, and as the number of cells increases, the rope appears. Rope is also caused by microbial hydrolysis of starch and protein to produce glutinous non-capsular materials. Rope can affect soft drinks, wine, pickling brine, vinegar, milk and bread. 3.3.6 Fermentative Spoilage Many types of organisms, especially yeasts, aerobic and anaerobic sporing bacteria, and lactobacilli are able to ferment carbohydrates. Yeasts usually convert sugars into alcohols and carbon dioxide, 'homofermentative' lactic acid bacteria convert sugars into lactic acid, while the heterofermentative bacteria produce several acids, such as butyric and propionic acid, in addition to lactic acid, and the gases carbon dioxide and hydrogen. Bone taint refers to fermentative spoilage which arises close to the bone in meat. Flat sours occur in canned foods in non-gas producing fermentative spoilage. Blown cans occur as a result of gas producing fermentation in which such copious quantities of gas are evolved that the pressure within the can distorts the sides and ends of the can and it may eventually blow. Fermentative spoilage may occur in foods which are produced by fermentation'wild' organisms flourishing to the detriment of the product. This can be a problem in beer manufacture, for example. 3.3.7 Putrefaction The anaerobic decomposition of proteins into pep tides or amino acids causes the production of foul odours in the food due to hydrogen sulphide, ammonia, methyl and ethyl sulphides, amines and other strong smelling products. Foods which are likely to deteriorate in this way are those which have been poorly processed and packed to provide anaerobic conditions-for example improperly processed canned meat and vegetables.
46
MICROBIAL SPOILAGE OF FOODS
- 3.3.8 Aerobic Hydrolysis Aerobic hydrolysis of proteins leads to the development of bitter flavours in foods-which are not necessarily unpleasant and sometimes enhance the flavour. 3.4 THE ORGANISMS INVOLVED IN FOOD-SPOILAGE The way that spoilage develops in a food. depends on the types of organisms present and whether the food, under its existing conditions of storage, can support the growth of any or all of them. 3.4.1 Microbial load The microbial load-the f,umbers and species of organisms which a food carries is initially determined by the food type and its origins. Later it will be altered by the handling and processing to which the food is subjected. Raw foods carry their own characteristic flora. Soil crops carry on their surfaces organisms which are saprophytic or parasitic, as well as soil organisms; meat carries organisms derived from the animal bowel, skin and fur; fish, organisms derived from the fish skin, intestine and from the water in which it lived; milk, organisms from the udder; etc. Subsequent treatment of the food will either reduce or increase the total load. Procedures such as the removal of soil from root vegetables, peeling fruit or vegetables, washing foods and heat treatments, such as pasteurization, cooking or canning, tend to reduce the microbial load. Storage of food under warm conditions-such as grain in inadequately aerated silos, or fish or other food in a warm shop window-tends to increase the microbial load. Table 3.1 lists some treatments of food and their probable effect on the microbial load of the food. Table 3.1 : Some Treatments of Foods and their Probable Effect on the Microbial Load Food type
Treatment
Grain
Harvesting Milling-removes outer layers
Probable effect on microbial load Adds organisms in dust to the natural flora Reduces flora contct ....
AN INTRODUCfION TO MICROBIAL SPOILAGE OF FOODS
47
... contd. Food type Flour
Milk
Bulk egg
Vegetables
Fruit
Probable effect on microbial load Storage under dry conditions Dormant organisms gradually reduce in numbers Storage under damp conditions Moulds and yeasts very likely to proliferate Numbers of bacteria Kept warm increase Chilled immediately after Numbers of bacteria increase slowly. Psychrotrophic milking organisms increase Pasteurized Numbers of organisms reduced-heat sensitive organisms killed Sterilized Cells destroyed-no growth Pasteurized, chilled Very slow growth of remaining organisms Pasteurized, frozen No growth while frozen Harvesting Adds organisms from dust and soil Washing Removes some surface organisms Washing, followed by storage Organisms multiply leading to rapid wet spoilage or damp Bruising Aids penetration of organisms-leads to rapid spoilage Treatment
contd. ...
48
MICROBIAL SPOILAGE OF FOODS
... contd. Food type
Treatment Storage wet
Meat
Warm storage Prolonged chilling
Frozen storage
Fish
Warm storage Wet cool storage
Probable effect on microbial load Rapid increase in numbers of organisms Rapid increase in numbers of organisms Increase in numbers of organisms especially of psychrotrophs ~o increase in numbers of organisms while frozen Very rapid increase in numbers of organisms Increase in numbers of organisms
3.4.2 Inter-relationships Between Organisms If a food contains a single species of organism, or only very closely related species, then their growth will not be in competition with any others and the growth rate will be determined by the environmental conditions. If spoilage occurs it will probably be very characteristic, such as the flat souring of canned foods by Bacillus species. In a mixed population of organisms the different species affect each other's growth in several ways. The rate at which an organism is able to multiply in a food determines whether it will achieve dominance, the fastest growing organisms having the greatest opportunity. When bacteria, yeasts and moulds are present in a food which is capable of supporting the growth of all three it is most likely that the bacteria will become dominant first. Mould or yeast spoilage may occur at a later stage if the conditions in the food at that time permit. The waste products that the dominant organisms produce may either stimulate or inhibit the growth of other organisms present. For example, some moulds of the Penicillium species may produce antibiotics in their growth which are inhibitory to other organisms; some bacteria may produce acids which favour the growth of acidophiles.
AN INTRODUCfION TO MICROBIAL SPOILAGE OF FOODS
49
Sequential spoilage occurs when the initial wave of growth due to one or several species of organism dies down due to factors such as overcrowding, depletion of food supply and build up of waste products to toxic levels. The conditions now existing may favour the rapid growth of a second group of organisms whose growth up to this point has been repressed. In a similar way to the first wave, the second wave may later die down to be replaced by a third and also perhaps a fourth wave of growth. The spoilage of the food which results from these population changes may be distinctive-as when mould follows bacterial growth. The conditions of storage and the treatment of a food affect which categories of organisms can become dominant, for example: 1. Pasteurization destroys heat sensitive bacteria, yeasts and moulds, and leaves heat resistant spoilage organisms. 2. Storaee of food under chilled conditions may discourage mesophiles but allow psychrophiles to grow unchecked. 3. Vacuum-packed food can spoil anaerobically whereas packed aerobically it would spoil in a different way. 3.4.3 Moulds in Spoilage Mould growth is initiated when a ripe spore is able to germinate and start mycelium growth. The affected food becomes coloured, musty, softer and sticky or slimy. Because moulds are aerobic, spoilage generally begins at the surface, although the mycelium later penetrates deep into the food. As well as spoiling the more perishable foods, moulds are often associated with the spoilage of 'dry' foods especially those stored under damp conditions and those foods containing high concentrations of sugar or salt. Moulds important in food spoilage
1.
Non-septate moulds-reproduce by asexual and sexual
spores. (a)
(b)
(c)
Genus Rhizopus: Widespread. Fluffy, luxuriant
mycelium. 'Pin head' sporangia which become dark as they ripen. Spoilage: 'bread mould', soft rots in fruit and vegetables, spoil chilled meat. Genus Mucor: Widespread. Approximately 150 species. Hyphae pale; sporangia become greyish as they ripen. Spoilage: wide range of foods affected. Genus Thaminidium: Not common. Greyish brown sporangia. Psychrophilic. Spoilage: chilled meat.
50
MICROBIAL SPOILAGE OF FOODS
2. Septate moulds-usually reproduce by asexual spores only. (a) Genus Aspergillus: Widespread. Compact colonies - white, buff, green, black. Bear conidia in 'globose' heads. Two important groups: Aspergillus glaucus group grey, green. Grow well in a low ~. Spoilage: dried foods and those preserved in sugar and in salt. Optimal temperature range for growth 1520°C. Aspergillus niger group Black conidia. Spoilage: bread, black rots of fruit and vegetables. Optimal temperature for growth 30°C. (b) Genus Penicillium: Widespread. Compact velvety grey-green, or white colonies. Bear conidia in 'brushes'. Spoilage: soft rots in citrus fruits, 'blue rot'; greenish patches on stored meat, yellow or green spots in eggs, greenish spoilage of cheddar and other cheese, bread, and so on. Optimal temperature for growth 20-25°C. (c) Genus Trichothecum. Trichothecum roseum causes spoilage of stored moist fruits. (d) Genus Geotrichum: Commonly found in dairy produce; compact felt-like colonies-white, yellow, red or orange. Hyphae break to form arthrospores. Spoilage: dairy produce-yoghurt, cheese, bread, stored citrus fruits and chilled meat. (e) Genus Monilia: Associated with the spoilage of bread-pink loose textured growth requiring moist conditions. (f) Genus Sporotrichum: Compact white colonies; requires high aw • Spoilage: stored chilled meats. (g) Genus Cladosporium: Common. Dark colonies. Spoilage: green rot of fruit, vegetables; black spot of meat, eggs, cheese. Wide temperature range for growth, favouring low temperatures. (h) Genus Alternaria: Dirty green mycelium, brown multicellular conidia. Spoilage: fruit and vegetables. (i) Genus Fusarium: Produce sickle-shaped multicellular conidia. Spoilage: rot fruit and vegetables; cause discolouration in butter.
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51
3.4.4 Yeasts in Spoilage Yeasts tend to grow in acid conditions and where the sugar concentration is high. They grow both in aerobic and anaerobic conditions. Fermentative yeasts break down sugars to produce carbon dioxide, alcohols and acids. Oxidative or film yeasts oxidize sugars, organic acids and alcohol, and in their growth raise the pH; they tend to grow on the surface of liquors forming a skin or film. Osmophilic yeasts tolerate conditions of low <\v and are associated with the spoilage of dried fruits, honey, concentrated fruit juices and so on. Salt tolerant yeasts may contribute to the spoilage of brines and salted foods.
Yeasts important in spoilage 1
Saccharomycetales-true yeasts. (a) Genus Saccharomyces. In addition to their many industrial uses some strains which are fermentative and osmophilic are spoilage organisms; for example
Saccharomyces rouxii, Saccharomyces mellis-spoilage
2.
of jams, syrups, pickles, brines, and alcoholic beverages. (b) Byssochlamys fulva: This is a 'mould' which also produces ascospores and is classified with the true yeasts. The ascospores are heat resistant causing spoilage of canned fruits. Cryptococcales-false yeasts. (a) Genus Candida. Some are 'film' yeasts, some of which are acid tolerant, and some osmophilic. Spoilage: high acid foods and brines; some fats such as butter and margarine are attacked by lipolytic strains. (b) Genus Rhodotorula Spoilage: 'spotting' of meats. (c) Genus Torulopsis. Some of these are fermentative and some are salt tolerant. They may cause trouble in brewing.
3.4.5 Bacteria in Spoilage Bacterial spoilage starts when vegetative cells are able to grow because the food provides a suitable nutrient environment and the physical qualities of the medium permit growth. Bacteria cause spoilage under many conditions, the main limiting factors to bacterial spoilage being the availability of water and the pH. They require a high <\v and cannot therefore contribute to the spoilage of dry foods while they remain dry.
52
MICROBIAL SPOILAGE OF FOODS
Bacteria important in food spoilage Gram positive aerobic or facultatively anaerobic cocci: In regular or irregular groups. Some form non-water soluble pigments. (a) Genus Micrococcus: Widespread, often isolated from dust and water; some are salt tolerant, some thermoduric, and some psychrophilic. Spoilage: salted foods,. pasteurized milk, chilled foods. Optimal growth temperature of 25-30°C for most species. (b) Genus Staphylococcus Isolated from the skin. Staphylococcus aureus causes a wide range of infections and intoxications including food poisoning. Staphylococcus epidermidis is also associated with skin infections. Both groups are salt tolerant-optimal growth temperature 37"C but grow well at lower temperatures and can be associated with food spoilage. (c) Genus Streptococcus: Widespread. Salt tolerant up to 6.5 per cent w Iv solution. Facultatively anaerobic. Need complex vitamin rich foods for growth. Ferment sugars to produce lactic acid. Streptococcus pyogenes group-pathogens. Streptococcus lactis group-used in the manufacture of dairy produce. Streptococcus faecalis group-derived from animal intestine (for example Streptococcus faecalis, Streptococcus faecium, Streptococcus durans) and associated with the spoilage of raw meat, fresh and pasteurized dairy produce. Wide temperature range for growth of 1O-45°C. (d) Genus Leuconostoc: Fermentative-produce copious capsules and slimes in favourable environments. Found in slimy sugars, fermenting vegetables, milk and dairy products. Some are salt tolerant, some produce distinct flavours in foods due to diacetyl. 2. Gram-positive cocci - anaerobic (e) Genus Sarcina: Large cocci occurring in packets. Isolated from soil and found on grairis. 3. Gram-positive rods - non-spore forming (f) Genus Lactobacillus: Varying from long and slender straight rods to coccobacilli. Anaerobic or facultative.
1
AN INTRODUCfION TO MICROBIAL SPOILAGE OF FOODS
4.
5.
53
Ferment glucose to form lactic acid. Lactose not fermented. Aciduric-optimum pH close to 5. Some thermoduric strains with growth optima above 40"C; many mesophilic strains and some psychrophilic strains. Found in dairy products and effluents, grains and meat products, water, sewage, beer, wine, fruits, fruit juices and pickled vegetables. Gram-positive rods - spore forming (g) Genus Bacillus: Aerobic, occur in dust and soil. Some are mesophilic, such as Bacillus subtilis; some are thermophilic for example Bacillus stearothennophilus, Bacillus coagulans with growth optima 37-55°C. All are active biochemically: saccharolytic, proteolytic and lipolytic strains. Spoilage: aerobic and microaerophilic-some strains cause flat sours in canned foods; some saccharolytic strains cause rope, for example Bacillus subtilis in bread. (h) Genus Clostridium: Anaerobic. Habitat-soil, organic material, animal bowel and excreta. Saccharolytic and putrefactive. The thermophilic species are of importance in spoilage of foods stored at high temperatures. The mesophilic species are important in canning for example Clostridium botulinum. Some are proteolytic and putrefactive-for example Clostridium histolyticum, Clostridium sporogenes; some are saccharolytic-for example Clostridium butyricum, Clostridium perfringens. Gram-negative aerobic rods: non-sporing. Produce water and non-water soluble pigments. (a) Genus Pseudomonas: Widely distributed, biochemically active, aerobic. Source-soil, fresh and sea water, decomposing organic material. Tend not to use carbohydrates but grow well in proteinaceous foods with the production of slime, pigments and odours. They prefer a high a w ' Many are psychrotrophic although the optimum temperature for growth ranges between 15 and 40°C. Spoilage: meat, fish, poultry, eggs. (b) Genus Halobacterium: Obligate halophiles spoiling foods high in salt. (c) Genus Acetobacter: Oxidize ethyl alcohol to acetic acid. Spoilage: alcoholic beverages.
54
MICROBIAL SPOILAGE OF FOODS
(d)
6.
Genus Alcaligenes: From manure, soil, water, dust. Produce alkaline reaction in some foods. Slimes. (e) Genus Flavobacterium: Pigmented colonies yellow to orange shades, some psychrophilic. Spoilage: discolouration of shellfish, butter, eggs, milk. Gram-negative facultative anaerobic rods (f) Genus Escherichia: Derived from soil or from the intestine. Their presence in food can indicate faecal pollution. Some species spoil food, fermenting the carbohydrate to acid and gas, and also causing 'off' odours. (g) Genus Envinia: Plant pathogens-causing rots. (h) Genus Serratia: Cause red colourations in foods. (i) Genus Shigella, genus Salmonella, genus Proteus: Pathogenic organisms which may be carried by foods.
3.5 FOOD QUALITIES RESPONSIBLE FOR SPOILAGE Of all the organisms present in a food only certain species will be able to grow and spoil it. The physical qualities of the food, listed below, determine which those organisms are. 1. Water content 2. pH 3. Temperature 4. Gaseous conditions 5. Texture 6. Nutrients In practice this means that foods can be classified according to their tendency to spoil. If the physical characteristics of the food change, or are changed, it is probable that the potential of the food to spoil will alter (see Table 3.2). Table 3.2 : Food Spoilage Potential
1.
Food category Non-perishable
Examples Foods in stable preservation-deepfrozen, canned foods (excluding pasteurized ones), sugars, jams, syrups, dry foods. contd ....
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AN INTRODUCTION TO MICROBIAL SPOILAGE OF FOODS
... contd. Examples Root vegetables, brined and salted foods, 2. semi-moist foods. 3. Perishable foods Fresh fruit, vegetables, meat, fish, dairy produce-all foods with high moisture content, or held at high relative humidity. In order to understand the principles of spoilage, each of the physical qualities of the food must be considered in tum. Food category Semi-perishable
3.5.1 Water Content The more water that a food contains, the more likely it is to spoil and conversely, the less water that a food contains the less likely it is to spoil. Factors which alter the water status of a food alter its spoilage status too. For example-a moist food is liable to be spoiled by a wide range of organisms. If that food is dried, salted, deep-frozen or preserved in sugar, its water status alters and it becomes less liable to certain categories of spoilage and more liable to others. Foods which have a high water content are very perishable and likely to spoil rapidly because micro-organisms grow best when water is plentiful. The conditions of storage together with autolytic changes may increase the levels of available water, the quantity and location of which will determine which organisms are able to grow and cause spoilage. Table 3.3 gives examples of foods with high water contents. Table 3.3 : Examples of Foods with High Water Content Food Milk, fresh skimmed Milk, fresh whole Eggs, fresh, whole Fruit, grapefruit oranges apricots, raw peaches, raw apples, raw (English eating) pears, raw
gm water/tOO gIn food 90.9 87.6 74.8 90.7 86.1 86.6 86.2 84.3 83.2 contd ....
56
MICROBIAL SPOILAGE OF FOODS
... contd. Food gm waterl100 gm food Vegetables - tomatoes, raw 93.4 90.3 cabbage, raw 87.1 beetroot, raw 78.5 peas, fresh, raw 75.8 potatoes, old, raw 74.4 Meat - chicken breast, raw 66.7 beef steak, raw chicken, boiled 63.4 beef, corned, canned 58.5 57.1 beef steak, stewed Bacon, dressed carcase, raw 48.8 63.9 Fish - herring~ raw 64.9 salmon, smoked 68.0 salmon, fresh 82.1 cod, fresh fillets 81.2 lemon sole Shellfish - crab (boiled) 72.5 72.4 lobster (boiled) shrimps (boiled) 62.5 oysters (boiled) 85.7 84.1 mussels (boiled) Source: A. A. Paul and D. A. T. Southgate, McCance and Widdowson's 'The composition offoods', 4th edition of Medical Research Council Special Report, no. 297 (HMSO 1978). Note: The analyses refer to the analYSis of the edible portion of the food, i.e. excluding parts such as the skin of fruit, bones in fish and meat.
If the moisture content of the atmosphere surrounding a food is in equilibrium with the moisture in the food neither will lose water to the other - a point known as the ERH (equilibrium relative humidity). If they are in a state of imbalance the water content of the food will alter. In a dry atmosphere a moist food will tend to dry out - for example the drying of bread and cakes - whereas in a moist atmosphere a food may absorb water with the result that its water content increases. Local moisture levels can be altered - for example a sealed polythene packing will not allow water vapour to escape from the atmosphere surrounding the food, with the result that the food surfaces can become very moist and liable to spoilage. The practice of putting
57
AN INTRODUCfION TO MICROBIAL SPOILAGE OF FOODS
hot food into a refrigerator causes condensation which will affect the surfaces of cool foods resulting in risk of surface spoilage of those chilled foods by moulds, yeasts or bacteria. These vulnerable foods with high water contents can be protected from spoilage in several ways. The reduction of their water content has the effect of decreasing the number of types of organism which can grow in or on the food at its new, lower a w ' The limiting a w for growth of some groups of spoilage organisms, is shown below: 0.91 Normal bacteria Normal yeasts 0.88 0.80 Normal moulds Halophilic bacteria 0.75 0.62 Osmophilic moulds 0.60 Osmophilic yeasts No organisms proliferate 0.50 3.5.1.1 Dry foods
Dry foods are classed as 'non-perishable' because their water content is too low to allow much microbial growth. Table 3.4 lists a range of dry foods. Table 3.4 Examples of Foods with Low Water Content
Food Cereal foods biscuits, cream crackers biscuits, sweet mixed oatmeal, raw spaghetti, macaroni flour, English (100% whole wheat) Milk, dried whole Eggs, dried Fruit and vegetables dried vegetables dehydrated fruits and vegetables dried fruits
gm waterllOO gm food 3.5-5.0 3.5-5.0 8.9 12.4 15.0
1.3 5.0-7.0
14.0-20.0 5.0 18.0-25.0
contd ....
58
MlrnOBIAL SPOILAGE OF FOODS
... contd. Food gm waterl100 gm food butter beans, raw 11.6 beans, haricot 11.3 Nuts almonds 4.7 barcelona 5.7 brazil nuts 8.5 4.5 peanuts Meat dehydrated meat 7.5 Dry foods are not usually sterile but their microbial flora is unlikely to be able to grow while the dry conditions persist (see Table 3.5)
Table 3.5 : Flora of Some Dried Foods Food Grain
Dried fruit and vegetables Dried egg
Dried milk
Flora Natural flora plus organisms from soil and other sources - wide range of bacteria, yeasts, moulds and their spores. Natural flora plus contaminants from processing and retailing. Many spores. Organisms from faecal material on shells; organisms from handling - hundreds to millions per gram. Salmonella can be present. Organisms from udder and from handling; heat resistant strains survive pasteurization - for example species of Streptococcus.
There are some organisms known as xerophytes which are able to grow at very low moisture contents - these are usually moulds. Prolonged storage under dry conditions usually results in a decline in the number of viable cells and spores present. On the reconstitution of the dried food viable cells are able to multiply and spores are able to germinate. Spray-drying, drum-drying and freeze-drying are methods of preserving foods which tend to allow the deposition of a protective
AN INTRODUCfION TO MICROBIAL SPOILAGE OF FOODS
59
layer of food material around the organisms present enabling them to survive better than unprotected cells. If a dried food is held under humid conditions it is able to absorb water at its surfaces and will be able to support the growth of moulds; if the water absorption continues to rise yeasts and bacteria will be able to grow. For example when flour becomes damp it is first liable to mould spoilage, the flour amylases convert the starch to sugars and as the a w rises acid formers such as members of the genus Acetobacter, coliforms, lactic acid bacteria and aerobic spore-bearing bacteria, or alcohol formers such as yeasts grow. The flour develops distinctive odours and suffers changes in appearance and quality.
3.5.1.2 Deep-frozen foods Deep-frozen foods do not support the growth of microorganisms because the water which they contain is not available while in the crystalline state. The process of freezing tends to reduce the number of viable cells but leaves a significant number which will become reactive when the food is thawed out. There are many foods which are protected to a certain extent from spoilage by the addition of either sugar or salt in high concentrations. In this way the aw of the food is lowered. In high concentrations these solutes exert a high osmotic pressure which has an adverse effect on most organisms and destroys them by the withdrawal of water from their cells. A few strains of organism can withstand the osmotic pressures or even prefer these conditions and contribute to the spoilage of foods otherwise protected.
3.5.1.3 Salted foods The addition of salt in high concentration to foods protects them from spoilage by a wide range of organisms but halophilic organisms with a preference for such conditions- can cause trouble. Table 3.6 indicates the salt preference of organisms. Table 3.6 : Salt Preferences of Organisms Organisms Range of tolerance Non-halophilic organisms up to 2% Slightly halophilic 2-5% Moderately halophilic 5-20% Extremely halophilic over 20% Note: At lOoC saturated salt solution (NaCl) contains 26.3 gm/IOO gm.
60
MICROBIAL SPOILAGE OF FOODS
If too little salt has been added to a 'salted' food such as bacon or salt fish, spoilage can result from the growth of both salt sensitive and slightly halophilic organisms. Conversely, in foods where moderate quantities of salt are required in the manufacture, the addition of excess salt can suppress the growth of the desired moderate halophiles and allow the growth of spoilage organisms such as halophilic slime producing yeasts. The salt itself can introduce halophilic spoilage organismsthe pink slime which is accompanied by unpleasant odours in wet salted fish is due to the growth of pigment producing strains of Serratia, Sarcina, and Micrococcus. Wet fish in the holds of trawlers at sea can be spoiled by halophiles derived from the sea water; and brining solutions for smoked fish can become colonized by halophiles which have an adverse effect on the keeping quality of the smoked product. Examples of salted foods are: 1. Vegetable preservation for pickle production 8-11 per cent w Iv NaC!. This inhibits spoilage organisms but allows the growth of the desired fermentative lactobacilli and Aerobacter. 2 Ham and bacon-sides are pumped with pickle - 25 per cent w Iv salt which contains a proportion of sodium and potassium nitrate, and sodium and potassium nitrite. It is then dry salted (sprinkled with salt), or tank cured during which time the meat takes up the salt. The final product must not contain more than 500 ppm sodium or potassium nitrate, or 200 ppm sodium or potassium nitrite. 3 Fish is either dry salted or brined during which it takes up salt. The final salt concentration in the fish may reach 8 per cent.
3.5.1.4 Sweet foods In a similar way to salt, sugar is used to protect foods from spoilage. To reduce the a w to a protective level, very high sugar concentrations are required - nearly 70 per cent, as in jams, preserves, syrups and dried fruits. However osmoplillic organisms may be able to grow and cause spoilage. Moulds such as strains of Aspergillus and Penicillium tend to grow on the surface of the affected foods. Yeasts such as strains of Candida, Saccharomyces and Rhodotorula ferment the food with the production of alcohol and CO2 and bacteria can produce ropiness (for example Leuconostoc species in syrups), cloudiness (strains of Bacillus) or colouration (Pseudomonas species) in the affected foods.
61
AN INTRODUCTION TO MICROBIAL SPOILAGE OF FOODS
Examples of foods containing high levels of sugar are: preserves with nearly 68% w Iv sugar (jam, curds, mincemeat, fruit fillings), syrups, honey.
3.5.2 pH The pH value of a food limits the range of organisms which it can support (see Table 3.7 for the pH values of some foods). Foods may be spoiled by the growth of a wide range of organisms whose effect is to alter the flavour, texture and appearance of the affected item, and at the same time pave the way for the growth of other species. Foods manufactured by fermentative processes may be spoiled by contaminating, acid producing organisms during the growth of which unwanted acids and other substances with distinctive flavours are evolved. A food which has a neutral pH can be protected from some spoilage by neutrophiles by decreasing its pH to such an extent that only a limited range of acidophiles could grow if present. It has long been the practice to preserve perishable foods by the addition of acetic acid (vinegar) - for example, onions, gherkins and pickles. Canned foods can be subject to microbial spoilage - the type of spoilage is dependant, after initial heat processing, on the pH of the contained food.
3.5.2.1 Neutral foods Examples of the spoilage of neutral foods in which acid is produced and the pH falls are given below. (See Table 3.7)
Table 3.7: pH Values of Some Foods Food lemons vinegar gooseberries prunes, apples, grapefruit rhubarb apricots strawberries peaches raspberries oranges cherries
pH value
2.2 2.9 3.0 3.1
3.2 3.3 3.4 3.5 3.6 3.7 3.8 contd ....
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MI(]{OBIAL SPOILAGE OF FOODS
... contd. Food
pears tomatoes bananas egg albumin carrots cucumbers cabbage bread meat, ripened tuna fish potatoes peas egg yolk milk shrimp meat, unripened egg white
pH value 3.9
4.2 4.6 4.6 5.0 5.1 5.2 5.4 5.8 6.0
6.1 6.2 6.4
6.6 6.9
7.0 7.0-9.0
Note: The minimum process applied to canned foods with pH values above 4.5 must provide an adequate safeguard against the risk of botulism.
Meat (pH 7.0-5.5) may be spoiled by the anaerobic growth of lactobacilli on its surfaces-resulting in the development of surface slime and a drop in surface pH. Under aerobic conditions proteolytic organisms such as pseudomonads may attack the meat protein and release strong smelling compounds associated with spoilage, and as a result the pH of the affected areas may rise. Milk (pH 6.6) contains a range of lactose fermenting organisms which can sour milk rapidly if it is held between 10 and 37°C. Pasteurization destroys the more heat sensitive strains of organisms, leaving the heat resistant lactobacilli and streptococci which are also capable of souring the warm milk. Under chilled conditions pasteurized milk sours slowly because of the reduced ability of these organisms to produce acid at lower temperatures. Later proteolytic organisms may attack the clotted milk proteins and the pH may rise.
3.5.2.2 Acid foods Acid foods can be spoiled by aciduric organisms. An example of this is in the spoilage of wine and beer (wine pH 3.5-4.0) which are
AN INTRODUCTION TO MICROBIAL SPOILAGE OF FOODS
63
manufactured by yeast fermentation processes producing alcohol from carbohydrate. They can be spoiled by the growth of lactobacilli producing lactic acid, members of genus Acetobacter producing acetic acid, and other fermentative aciduric organisms such as wild yeasts, Clostridium butyricum and members of genus Achromobacter, producing a whole range of end products. More commonly acid foods are spoiled by acidophilic organisms. Fruit and vegetables have low pH values-fruits range from pH 2.0 to 4.2 and vegetables, slightly higher - from 4.5 to 7.0. The more acid fruits and vegetables clIe liable to be spoiled by acidophilic organisms - moulds such as species of Penicillium, Rhizopus and Aspergillus which grow on their surfaces. They may be rotted by saprophytic bacteria such as the slime producing members of genus Erwinia, for example, Erwinia carotovora, and members of genus Pseudomonas such as Pseudomonas marginalis. Those acid fruits which contain increasing amounts of sugar as they ripen may be fermented by wild yeasts - damsons may be spoiled in this way.
3.5.2.3 Canned foods Canned foods are divided into two groups based on their acidity. The separation of the two groups is based on the fact that the dangerous food poisoning organism Clostridium botulinum is inhibited below pH 4.5. Thus foods which have a pH below this - that is are more acid - do not need to be as rigorously processed as those with a pH above 4.5 (less acid) in order to be safe from this hazard. Canned foods can be classified into two groups: Group 1 : Medium and low acid foods - pH 4.5 and above. For example, meat, sea-foods, milk, meat and vegetable mixtures. Group 2 : Acid and high acid foods - pH below 4.5. For example, tomatoes, fruit, pickles, citrus fruits, rhubarb. Group 1 Because of the risk of toxin production in the low acid, anaerobic conditions inside the can, medium and low acid foods are processed by heating to above 100°C. Reference indicates the exposure is necessary to destroy the spores of Clostridium botulinum. The type of spoilage to which such foods are subject is due to the survival of spores of the genera Bacillus and Clostridium which have a greater heat resistance than those of Clostridium botulinum. Thermophilic strains of these two genera grow well at 55°C and only with difficulty at 37°C. Their heat resistant spores can germinate in the canned foods and cause spoilage if the can is stored under warm conditions. For this reason they do not often cause spoilage in cold
64
MICROBIAL SPOILAGE OF FOODS
country as it is rare for ambient conditions to favour their growth. In hot countries they frequently cause trouble. This type of spoilage can be avoided by the storage of canned foods under cool conditions. Bacillus stearothermopilus and Bacillus coagulans ferment carbohydrates and cause flat sours; saccharolytic strains of Clostridia not only produce acid but also copious quantities of gas (carbon dioxide and hydrogen) with the result that the can swells and distorts. Those Clostridia which produce hydrogen sulphide may give rise to 'sulphur stinker' spoilage - which is, fortunately, rare. Mesophilic bacilli such as Bacillus 5ubtilis, Bacillus cereus, Bacillus megaterium and Bacillus polymixa may cause spoilage of canned milk, fish or cured meat causing either flat or gassy sours. When large hams are canned they are normally only pasteurized - treatment which is inadequate to destroy the spores of Clostridium botulinum. The curing salts are inhibitory but only if the can is kept under chilled storage will the product be both safe and unspoiled. Its shelf life is, however, short. Other mesophilic Clostridia can spoil canned foods - for example, the putrefactive Clostridium sporogenes. Group 2 These foods do not require the rigorous processing to which the low and medium acid foods are subjected when canned. Because of this it is possible for heat resistant aciduric bacteria, yeasts and tue111ds to survive processing and to grow inside the can. For example, Clostridia and Bacilli can cause gaseous spoilage and flat sours in canned fruits. The non-sporing lactobacilli can ferment the foods and strains of Leuconostoc can cause slime and ropiness. This type of spoilage can be avoided by slightly more rigorous heat treatment of the product. The mould Byssochlamys fulva being heat resistant can cause spoilage of canned soft fruits, causing them to go soft and pulpy. Exposure to 90°C for a few minutes is an adequate heat treatment.
3.5.3 Gaseous Conditions The oxygen tension and the oxidation-reduction (O-R) potential of a food influences the type of organisms which can grow in it. Spoilage by aerobic organisms occurs at the surfaces of foods; most fresh plant and animal foods have a low O-R throughout and they are aerobic at the surface only. Heating destroys this, as oxygen can diffuse into the food more easily and the food is the more readily spoiled. Facultative organisms grow both on the surface of the foods and within them, as is shown when cans of food are spoiled by members of the genus
AN INTRODUCTION TO MICROBIAL SPOILAGE OF FOODS
6S
Bacillus. Anaerobic organisms grow within foods held under anaerobic conditions such as inside cans; similarly vacuum-packs are liable to fermentative spoilage by bacteria and yeasts.
3.5.4 Texture Fluid foods spoil rapidly because the organisms can easily spread throughout the food by means of their own motility or by convection currents. Semi-solid foods such as meat stews, soup and tinned fruits can spoil as rapidly as fluid foods. Solid foods tend to spoil from their outside surfaces inwards, these being the first surfaces to become contaminated. In other words, the time taken for the whole food to become spoiled depends on how easily the organisms can penetrate the food - thus mincing or otherwise dividing foods aids rapid spoilage because a greater number of surfaces are contaminated by the organisms.
3.5.5 Nutrient;; All foods suitable for human consumption are liable to be spoiled by some micro-organisms. The composition of the food limits which spoilage organisms it can support - most foods supporting a variety. Protein foods such as meat, fish, and eggs are liable to be attacked by proteolytic organisms such as members of the genera Pseudomonas, Achromobacter, Flavobacterium; 'carbohydrate' foods such as bread, flour, pasta, syrups and jams are more liable to attack by fermentative organisms; fats are liable to be attacked by lipolytic organisms.
3.5.6 Temperature Any non-sterile food is liable to spoil in time if it is held between the temperatures of -SoC and +70°C - the holding temperature dictating to a certain extent the type of spoilage- although other factors such as the pH and the water content obviously exercise considerable influence. Every type of organism is able to grow within a certain temperature range, maximum growth occurring at and around the optimum temperature. So organisms are divided on the basis of their temperature preferences into rough categories-psychrophiles, mesophiles and thermophiles. Moulds and yeasts tend to grow best at room temperature and below, and therefore assume greater importance in the spoilage of foods held at cool and chilled temperatures. The holding temperature range favours the growth of certain species of the entire flora present-that is those whose optimum for growth is near to the holding temperature. Provided that other environmental factors will allow growth, foods will be subject to spoilage as shown in Table 3.8.
Table 3.8 : The Storage Conditions of Foods which Affect the Type of Microorganisms which can Grow and Cause Spoilage Food Chilled storage Fresh meat, fish, vegetables, milk, etc. (a 1.0-0.95) w Dry foods - noodles, whole egg powder, biscuits, etc. (a below 0.5) w Salted or sugared
Psychrophiles grow
Psychrophilic osmophiles and halophiles may grow
Acid foods Canned foods
Pasteurized canned hams can spoil slowly
Conditions of storage Non-chilled ambient Warm storage vel)' hot weather, heated cabinet, etc. winter, shade Mesophiles grow, thermophiles Mesophiles grow may grow Too dry to spoil
Mesophilic osmophiles As 'non-chilled' but spoilage and halophiles may more rapid grow Acidophilic organisms may grow Mesophilic or thermophilic spoilage is possible if improperly processed.
~
Q~
,. Vl
~ ~
~
Note: The terms psychrophiles, mesophiles and thermophiles relate to bacteria, yeasts and moulds.
DOD
~
SPOILAGE OF MEAT AND MEAT PRODUCTS
4.1 PROLOGUE Originally meat was a term used to describe any solid food, but has now come to be applied solely to animal flesh. As such, it has played a significant role in the human diet since the days of hunting and gathering, and animals (sheep) were first domesticated at the beginning of the Neolithic revolution around 9000 B.c. Though abjured by some on moral or religious grounds, meat eating remains widely popular today. In the main, this is due to its desirable texture and flavour characteristics, although meat protein does also have a high biological value. Various aspects on microbial spoilage of fresh meats, cured meats and vacuum-packed meats are discussed elaborately in this chapter.
4.2 SPOILAGE OF FRESH MEATS 4.2.1 Contamination of Tissues by Microorganisms Large numbers of microorganisms and a great variety of types are found on the exterior surface and in the intestinal tract of cattle, sheep and pigs before death. Counts in excess of 105 per cm2 are common on the hides of cattle and substantially higher recoveries (l08 per cm2 ) have been observed for pigs and from the unwashed wool of sheep. However, the underlying muscle tissue is generally assumed to be sterile except in infected animals.
68
MICROBIAL SPOILAGE OF FOODS
The slaughter of cattle with a captive bolt pistol and the subsequent procedures such as sticking, skinning, evisceration and butchery common to all animals have the effect of contaminating the previously sterile underlying muscle tissues (sheep and pigs conventionally are stunned electrically and this does not involve microbial contamination). Obviously it is the exposed newly cut surface flesh which is going to carry the majority of the contaminating organisms but deep tissue may in due time become contaminated via the visceral blood supply. Total bacterial counts for freshly cut meat surfaces are likely to vary between 103 and 105 organisms per cm2• These organisms are derived mainly from the exterior and the gut of the animal but also from knives, other utensils, butchery tables, etc. so that variations in count often reflect the hygienic conditions in the abattoir.
4.2.2 Control of Microbial Growth Meat is an ideal environment for the growth of microorganisms, particularly bacteria, and rapid growth can be expected unless control is effected. The numbers of microorganisms on meats can be controlled in a variety of ways which are briefly considered below. It should be pointed out that most studies on the spoilage of meats have involved beef but the spoilage characteristics of lamb, pork and other meats are essentially similar.
4.2.2.1 Initial contamination As will be seen later, the onset of off-odours and other spoilage characteristics in meats are associated with a particular level of bacteria. As the rate of bacterial growth on meats at a given temperature follows a prescribed pattern, the lower the initial contamination of the meat the longer it will take for the bacterial flora to achieve spoilafe levels. Thus, with beef stored at 5°C, if the initial count exceeds 10 per cm2 spoilage can be detected within 6 days whereas with a count of 103 per cm2 spoilage would not occur until the 10th or 11th day. Since the hide is probably the source of most microorganisms on dressed carcasses, animals brought to the abattoir should be freed of adhering dirt by washing before slaughter. Further reductions in the microbial load of the freshly killed animal can be achieved by spraying the dressed carcasses with hot water and by implementing a rigorous sanitation programme in the abattoir; this should include thorough cleaning of items such as walls, floors, cutting tables, knives and other utensils, operatives clothes, etc.
4.2.2.2 Glycogen reserve When animals are slaughtered, glycogen stored in the muscles is converted to lactic acid. Under normal conditions this causes a fall in the pH of the muscle from about 7 to 5.6 and this drop is important
SPOILAGE OF MEATIAND MEAT PRODUcrS
69
since it is responsible for a reduction in the growth rate of the bacterial contaminants. However, if the animal is stressed before slaughter (e.g. by excitement, fatigue or starvation) the glycogen reserves become depleted so that a reduced amount of lactic acid is produced and the ultimate pH of the meat is nearer to neutrality; such meat spoils more rapidly and therefore it is imperative that animals are in a sound physiological condition immediately before slaughter.
4.2.2.3 Oxidation-Reduction (OR) potential After slaughter oxygen stored in the muscle is depleted thus causing a fall in OR potential to extremely low levels. The strong reducing capacity of the medium together with the high initial temperature (38°C) create an ideal environment for the growth of anaerobic bacteria. The predominant spoilage bacteria are Clostridium spp. which grow within rather than on the meats breaking down the tissues with the production of offensive decomposition products such as hydrogen sulphide and ammonia. This process is known as putrefaction and must be avoided by cooling the meat rapidly before the OR potential falls sufficiently to allow the growth of these organisms. Furthermore, it is now recognised that the presence of certain putrefactive anaerobes in large numbers can be a cause of food poisoning. One of the organisms predominant in the early stages of putrefadion is C. perfringens, and C. botulinum can be isolated occasionally from putrefying meats. The rapid cooling of meat postslaughter is also advantageous in that it reduces the growth of other food poisoning bacteria such as salmonellas which are also frequent contaminants. 4.2.2.4 The rate of cooling It is obvious from the foregoing that rapid cooling of carcasses is essential to reduce the early bacterial growth and thus increase potential storage times. Subsequent storage temperatures have a profound effect upon the microbial spoilage pattern and this aspect is now discussed in some detail. 4.2.3 Effect of Storage Temperature
4.2.3.1 Spoilage under warm conditions Carcass meat and meat joints held at temperatures of 20°C or over will inevitably undergo putrefactive spoilage as indicated above. However, if the surface area to volume ratio is increased by mincing or slicing the raw meat the OR potential also increases thus creating conditions that are less favourable for the growth of putrefactive anaerobes. Growth at or near the surface of the meat is now most rapid
70
MICROBIAL SPOILAGE OF FOODS
but the raised OR potential enables a mixed flora to develop. The bacterial flora at the time of spoilage still contains clostridia but it is now dominated by mesophilic, facultatively anaerobic, Gram-negative rods. Most of these organisms are enteric in origin and will include genera such as Escherichia, Aeromonas, Proteus and Enterobacter. Other genera also represented should include Staphylococcus and Micrococcus (Gram-positive cocci) and Bacillus (aerobic and facultatively anaerobic spore forming bacteria). Spoilage of sliced or minced fresh meats at 20°C is rapid and maximum numbers are reached within 3 to 4 days (Fig. 4.1). The first signs of spoilage (Le. off-odours) are detected within 2 days and surface slime is evident at 3 days. It is interesting to note that, whatever the storage temperature, off-odours and slime production are alwals first evident when the total counts have reached 107 per cm2 and 10 per g, respectively; in fact, this relationship holds true for meats in general. The higher OR potential of minced or sliced meat favours proteolytic rather than putrefactive spoilage. The off-odours that appear are often referred to as 'sour' and are due to the formation of volatile acids such as formic and acetic acids; the surface slime is caused by heavy bacterial growth and the softening of meat structural proteins. The nature of the biochemical changes occurring at these higher temperatures has been little studied and far more work has been done on these changes at the chill storage temperatures used commercially. 20°C
3
10
102~~~~~__~__~~____~__________~
1 2 3 4 5 7 10 Storage period (days)
14
Fig. 4.1 : Numbers of bacteria on fresh minced beef stored at different temperatures.
21
SPOILAGE OF MEAT AND MEAT PRODUcrS
71
4.2.3.2 Spoilage under cool conditions With storage temperatures below 20°C there is a tendency for the mesophilic bacteria to be overgrown by psychrotrophs although a small proportion of the former may still be capable of growth at 5°C. Sliced and minced meats held at 15 or lOoe develop off-odours after 4 to 5 days' storage and surface slime is evident at about 7 days (Fig. 4.1); the bacterial flora becomes progressively dominated by Pseudomonas spp. which represent over 95 % of the flora at the time of spoilage.
4.2.3.3 Spoilage under refrigeration conditions At temperatures of 5°C and below a definite lag phase is apparent. The length of this phase depends on the storage temperature and extends for 24 h at 5°C and for 2 to 3 days at O°C. In addition, at temperatures close to O°C there is an initial fall in the numbers of viable bacteria which is probably due to the death or injury of many types at these low temperatures. As temperatures approach O°C bacterial growth, once initiated, is much slower and progressively fewer types are capable of growing. Thus the period before the onset of the first signs of spoilage is extended and off-odours and slime production take 8 and 12 days, respectively, to develop at SoC and 16 and 22 days at O°C (Fig. 4.1). Qualitatively, the spoilage flora is again dominated by Pseudomonas spp. in the later stages, due to their ability to grow faster than all other competing species at these low temperatures; the pseudomonads present at this stage are mainly (80 %) of the nonfluorescent types. On the other hand, true mesophiles now only represent a small fraction of the total flora but the fact that the numbers of bacteria appearing on media incubated at 37°C increase during storage indicates that some mesophilic types must be growing on meats held at 5°C (Fig. 4.2). Because of the strongly aerobic character of pseudomonads, growth is limited to the surface and to a depth of 3-4 mm in the underlying tissues. The spoilage pattern is thus largely independent of the size of cut or joint of meat and the spoilage of carcasses is likewise limited to the superficial layers; the growth of clostridia is inhibited at these low temperatures so that anaerobic putrefaction does not occur.
72
MICROBIAL SPOILAGE OF FOODS
(a)
••.•.••...•.....
.. •....'
~ ..# ..
'
//
../
...
#,l
.' .',.'.'
.'"
.'
............•....•.•.••• 7 Storage time (days at 5°C)
14
Storage time~days at 5°C)
14
Fig. 4.2 : Growth of bacteria on fresh meat stored at SOC showing: (a) increasing proportion of pseudomonads (--, total count; - - - -, Pseudomonas), and (b) changes in total psychrotrophs (- - - -) and total mesophiles (--).
Under normal conditions of carcass meat storage the humidity is high and the surface layers remain moist. Over prolonged storage periods or at lower humidity levels drying of the surface layers becomes more pronounced and the consequent drop in a w renders conditions more favourable for ftmgal growth. When ftmgal growth is induced in this way it is largely localized and only involves the most superficial
SPOILAGE OF MEAT AND MEAT PRODUcrS
73
layers; it can be trimmed away without any harm to the rest of the meat. Spoilage associated with the growth of moulds includes: 1. 'Whiskers': members of the genera Mucor, Rhizopus and Thamnidium produce mycelia giving a whiskery, white co grey appearance to the surface of beef carcasses. 2. 'Black spot': Cladosporium herbarum and C. cladosporoides can grow on a variety of meats even at temperatures as low as -soc. It produces black spots of growth due to the darkly pigmented mycelium. 3. Penicillium spp. and Cladosporium spp. produce large numbers of yellow to green spores when growing 00 meats; these cause similarly coloured spoilage patches on the meat. 4. 'White spot': normally caused by the growth of Sporotrichum earn is. 4.2.4 Chemical Changes Produced by Bacteria in Chilled Meats In considering the chemical changes which occur during spoilage at low temperatures it is necessary to differentiate between changes induced by enzymes naturally present in animal tissues and those induced by bacterial enzymes. In fact, such a differentiation is difficult to make and this has meant that until the 1970s the changes induced by bacteria per se were poorly understood. Changes occur in free amino acid levels in stored meats and these are brought about by either or both of these sets of enzymes. Bacteria initially attack glucose, amino acids and other low molecular weight compounds such as nucleotides rather than the primary meat proteins. These changes are accompanied by a marked rise in pH from 5.6 to as high as pH 8.5 due, primarily, to the formation of ammonia by bacterial degradation of amino acids; in consequence pH values have found a use in the assessment of the keeping qualities of meats. Proteolysis, the breakdown of primary meat proteins, only occurs at a relatively late stage of storage and only becomes evident after the onset of other signs of spoilage. This protein breakdown results from t.l-tc activities of bacterial proteases and is first noted near the surface of the meat; in time, however, these enzymes penetrate deeper into the tissues. The pseudomonads are primarily responsible for this proteolysis which occurs when their numbers are already in excess of 109 per cm 2 . Ldrge numbers of volatile compounds are
74
MICROBIAL SPOILAGE OF FOODS
probably produced as a result of bacterial growth; of these, acetone, methyl ethylketone, dimethyl sulphide and dimethyl disulphide probably correlate most closely with the extent of spoilage. Many pseudomonads are also active lipase producers at chill temperatures and they are therefore often implicated in the hydrolysis of fats, a process which results in the production of undesirable flavours consequent upon the formation of fatty acids. Spoilage bacteria may also produce lipoxidases which accelerate the oxidation of unsaturated fatty acids to produce aldehydes and in this way they contribute to the problem known as 'oxidative rancidity'. Oxidative rancidity is caused normally by the slow uptake of oxygen and is not primarily of microbiological origin. However, it is known that the bacterial spoilage of surface fat tissues on fresh meat follows a similar pattern to that of protein breakdown with bacterial attack on lipids again being delayed until an advanced stage of spoilage. 4.3 SPOILAGE OF CURED MEATS 4.3.1 Curing Agents The principal ingredients of any cure are salt, sodium nitrate and sodium nitrite. Salt is present as a preservative agent which acts by reducing the aw of the meat. Pseudomonas spp., important in the spoilage of refrigerated meats, are particularly sensitive to lowered ~ levels and this partly accounts for the relative stability of cured meats. The role of nitrate in spoilage control is not clear although it is useful in the development of the red colour of meats and it is reduced by bacteria to nitrite. The nitrite also helps to maintain the colour of meat but its principal role is to prevent growth of germinating spores. On its own nitrite is not very active but its effectiveness is enhanced by factors such as the salt concentration, pH and storage temperature which are all important in determining the stability of cured meats. 4.3.2 The Curing Process Curing can be performed by one of three basic procedures. In the first, dry curing, the curing agents are rubbed onto the surface of the meat whilst in the second process, pickling, the meats are immersed in a pickle of the curing agents in water. In both these methods the meats are held at 3-4°C until the agents have penetrated into the centre of the meats. The low temperatures involved reduce the chances of putrefactive anaerobes growing but nevertheless spoilage problems can
SPOILAGE OF MEAT AND MEAT PRODUcrS
75
occur due to the slow penetration of the brine. These problems are largely overcome in the third procedure, stitch pumping, introduced in recent years. In this process the pickle is injected into the deeper tissues by means of long needles, with several holes along their length, which are arranged in rows so that hundreds of separate injections are made. A variation of this technique is to pump the pickle into the vascular system which channels it to the various parts of the body. In both these injection procedures the meats are subsequently immersed in a pickling brine. To increase the shelf-life of the bacon a dry salting process has been developed more recently whereby bacon sides are passed through a cloud of dry salt after removal from the curing brine. Bacon, ham, salted beef and salted pork are the principal products of the curing process, and of these only the first will be considered in any detail. 4.3.3 The Microbiology and Spoilage of Bacon and Ham
4.3.3.1 Unsmoked bacon Bacon is conventionally cured as whole sides and when cured in this way the term 'Wiltshire bacon' is used. Brines used in the curing of bacon usually contain 20-27 % salt and this has a profound effect on the types of organisms present. The microbiological flora of a typical Wiltshire curing brine is dominated by micrococci which are able to tolerate the low a w environment. These organisms also become predominant on the sides during the curing process so the normal heterogeneous flora of fresh meat is largely replaced by this group. As well as growing on bacteriological media containing 20% NaCl, these Micrococcus spp. are psychrotrophic growing at 4°C and usually at O°c. Another of their important characteristics is their ability to reduce nitrate to nitrite and they therefore play an important part in the curing process. After curing, which lasts from 4 to 14 days, the sides of bacon are drained and allowed to mature for a further 5-10 days at 4°C; during these processes there is a gradual reduction in the salt concentration of the bacon down to levels of well below 10%. In fact, higher salt concentration bacons may be regarded as bacons with a final salt concentration of > 5 %; at maturation with such bacons bacterial counts range from 104 to 106 per cm2 and whilst the predominance (.>60%) of micrococci is maintained there is an increase in the proportion of Gramnegative bacteria, particularly. Acinetobacter and Vibrio spp., on the sides.
76
MICROBIAL SPOILAGE OF FOODS
This change in £lora does not occur on the rind probably due to its lower ~. During subsequent storage of the bacon there is a gradual increase in bacterial numbers up to a maximum of 108 microorganisms per cm2 after 2-3 weeks at lO°e. Qualitatively, the flora now consists of approxnnately equal proportions of members of the genera Micrococcus, Vibrio and Acinetobacter although if the bacon is held under chill conditions vibrios tend to predominate, particularly on the meat surface. The high count on the surface of a side of bacon is associated with slime formation and is commonly caused by halophilic vibrios but there should be no obvious deterioration in the quality of the bacon as changes within the meat are normally minimal. One such change is that of bone taint which is caused mainly by vibrios and micrococci. It is characterized by an unpleasant smell noticed when de-boning the product; it results from faulty curing or curing meats of too high a pH. When spoilage does eventually occur it is commonly caused by micrococci and vibrios together with a variety of yeasts and moulds, including Torulopsis and Aspergillus spp., respectively. Off-odours and flavours are mainly associated with the fat rather than the lean meat although in the latter micrococci can induce proteolytic changes. Hydrolysis of fats is caused by bacterial and tissue lipases whilst oxidative rancidity causes yellowing of the fat. With lower salt « 5% NaCI) bacons, popular in Europe and in the United States, the microbiological flora is more varied and less salt tolerant forms figure more extensively. With such cures and bacon, bacteria from the genera Lactobacillus, Leuconostoc and Streptococcus (i.e. the 'lactic acid bacteria') are more readily isolated and it is these organisms together with micrococci that are now responsible for the microbiological spoilage. With the so-called lactic acid bacteria dominating the spoilage flora, the characteristic spoilage is now souring resulting from the acid produced by these organisms.
4.3.3.2 Smoked bacon As well as providing a desirable flavour and colour, smoking also contributes to the preservation of bacons. The effect is both bacteriostatic (i.e. stopping bacterial growth) and bactericidal (i.e. killing bacteria) although fungi are also affected to some degree. Smoke acts in two ways: first, by drying the surface layers it further reduces the awand accentuates the effects of salt; second, it impregnates the tissues with chemical preservatives such as formaldehyde an~ phenols which inhibit bacterial growth. In addition, large numbers of bacteria are killed on the bacon during the smoking
SPOILAGE OF MEAT AND MEAT PRODUCTS
77
process, the numbers depending on the time and type of smoking. Micrococci, yeasts and moulds are most frequently isolated on smoked bacon although lactic acid bacteria are more likely to predominate where liquid smoke is used. As these 'lac tics' are responsible for a less offensive souring spoilage than that associated with micrococci, and at a later stage, the shelf-life of the product is extended.
4.3.3.3 Ham The processes involved in the curing of hams are similar to those employed for bacon except that sugar is often added to the cures. This can be attacked by bacteria, particularly lactobacilli, and the fermentations produce souring of various types; however, it has been suggested that lactobacilli may be useful in maintaining the stability of curing brines by preventing excessive pH rise. In general the microorganisms found on hams are similar to those on bacon and the flora consists mainly of micrococci, streptococci and lactobacilli, the proportions depending on the salt concentration and period of storage. Higher salt concentration hams also tend to support the growth of a greater proportion of yeasts and possibly moulds.
4.4 SPOILAGE OF VACUUM-PACKED MEATS 4.4.1 Types of Packaging Materials The packaging materials used by the food industry vary from the highly impermeable, required for vacuum-packaging to the highly permeable and from the opaque to the transparent. Materials may consist either of single components such as polyethylene (polythene) or polyvinyl chloride (PVC), or of multiple components. In the latter case the materials are made up of layers of different components to give more favourable packaging characteristics. Coatings made of nitrocellulose and wax may be applied to one or both sides of a single component film such as cellulose; alternatively multi-layers can be built up using, for example, ethylene vinyl acetate with two layers of PVC (i.e. Cryovac). From a microbiological standpoint the key properties of packaging materials are their permeability to water vapour and to certain gases including oxygen. Permeability to water vapour can vary according to the packaging material and may range from 500 g per m 2 per 24 h for a film thickness of 2.5 x 103 cm to under 1 g. Relative permeabilities to oxygen and water vapour are often similar as in the
78
MICROBIAL SPOILAGE OF FOODS
case of cellulose MSAT (low) and cellulose QSAT (high) but there may be gross differences as with the polythenes (see Table 4.1). Table 4.1 : Permeabilities of Packaging Materials to Water Vapour and Oxygena Material Cellulose MSAT Cellulose QSAT Polyethylene (low density) Polyethylene (high density) Polyvinyl chloride
Permeability to Water vapourb Oxygene 3 1.5 2000 150-350 5 3000-10000 1.5 500-3000 10 200
From Hannan (1962). b Results expressed as g/m2/24 h at 25°C and 75% relative humidity. 2 C Results expressed as mllm /24h at 20°C.
a
4.4.2 Influence of Packaging Materials on the Microbiological Flora Fresh meats are normally packed in oxygen-permeable films in order to conserve the bright red colour of oxygenated myoglobin. Conversely, cured meats are packed in oxygen-impermeable films in order to prevent fading of the colour due to oxidation. In recent years the possibility of distributing fresh beef in the form of refrigerated vacuum-packed prime joints rather than as carcasses has been examined and this method is now becoming more popular due to the improved shelf-life obtained; an additional advantage is that weight losses due to surface drying are restricted. Vacuum-packaging is also becoming more popular in the retail trade in spite of justifiable criticisms of loss of colour. The compensations are that the meat has a considerably extended shelf-life and a rapid regeneration of the normal red colour occurs once the pack is opened or the meat is re-packed in an oxygen-permeable film. The availability of oxygen within the pack has some effect on the microbial flora; meats have a high demand for oxygen as do many microorganisms, so oxygen levels can be readily depleted in the more impermeable packs without a vacuum being applied. At the same time, carbon dioxide (C02) levels tend to increase in such packs at a rate dependent upon the permeability of the film. However, packaging materials are more permeable to CO2 than to oxygen so that a film of
SPOILAGE OF MEAT AND MEAT PRODUCTS
79
low permeability may exclude oxygen but still ~llow <;02 to escape and maintain the vacuum in the pack. There are advantages in increasing the concentration of CO2 within the pack as it is inhibitory to many microorganisms includin5 moulds and pseudomonads, the latter group being the dominant flora on spoiled fresh meats. The lactic acid bacteria and yeasts are much more resistant to higher CO2 levels and so may be expected to figure in the spoilage pattern associated with packaged meats. Another factor affecting the bacterial spoilage pattern is that of a w which is likely to be high within the pack wrapped in an impermeable film. Since there is no water loss from the package, bacterial growth is not reduced by a drop in aw but the effects of aw ' are subordinate to those of carbon dioxide and oxygen within the pack. 4.4.3 Spoilage of Packed Fresh Meats When packaged meat is stored at warm temperatures it undergoes normal putrefactive spoilage. Such meat is therefore always stored at refrigeration temperatures and only such storage will be considered. The growth of microorganisms is retarded on vacuum-packed fresh meats stored at 3-5°C and a lag phase extending for 3 to 5 days is usually observed. Subsequent growth is slow and continues for up to about 10 days by which time the final total count of 107 per cm2 (or per gram in minced meat) will be reached; this is about 1% of that obtained with permeable films (Fig. 4.3). Qualitatively the microbial flora in the impermeable pack becomes dominated by lactic acid bacteria (mainly lactobacilli and leuconostocs) which represent 50-90 % of the totd flora at the end of storage. This reflects their inherent resistance to the accumulating carbon dioxide and their ability to grow in the anaerobic conditions; the pseudomonads are unable to thrive in the presence of carbon dioxide and in the depleted oxygen conditions (Fig 4.4). The lactic acid bacteria attack carbohydrates preferentially but due to the small amount present in meat relatively little acid can be formed and, as a consequence, only a slight fall in pH is evident (Fig. 4.3). This means that even at the maximum cell densities of these organisms relatively little spoilage is apparent; spoilage is associated with sour or cheesy odours due to the formation of fatty acids, these principally being acetic and butyric acids. With increasing permeability of the package film there is a gradual change in the spoilage flora to one that comprises a high proportion of pseudomonads; thus the spoilage changes are more
80
MICROBIAL SPOILAGE OF FOODS
typical of unwrapped fresh meat (Fig. 4.3). The relative proportions of pseudomonads, lactic acid bacteria and less significant groups are dependent principally on the concentration of carbon dioxide within the pack. Included amongst the less significant groups is a bacterium, Brochothrix thermosphacta (formally Microbacterium thermosphactum), which is a Gram-positive, small, non-motile rod. Like the pseudomonads it decreases in numbers in impermeable packs but in permeable packs it often represents 20-30% of the total bacterial count at spoilage. Unlike the pseudomonads, however, B. thermosphacta is unaffected by the presence of carbon dioxide and it often grows to high numbers with lamb and pork in particular when wrapped in intermediate permeability films which accumulate some carbon dioxide but still contain low levels of oxygen.
104
t::'""------Impenneable pack
1d~----------------~----------------~S Storage time relays at SOC)
_ _--+. 14
Fig 4.3 : Comparison of numbers of bacteria and pH in fresh minced meat stored at SoC under penneable and impenneable conditions.
The deliberate addition of carbon dioxide to impermeable meat packs has been used to extend the shelf-life and the problems of colour loss have been minimized by using CO2: 02 mixtures. This principle is utilized in controlled atmospheric packaging (CAP) and there are indications that at the concentrations used (e.g. 40% CO2 : 60% 02) the mixture is somewhat inhibitory to microorganisms although lactic acid bacteria become predominant in the later stages. Other mixtures incorporating carbon dioxide and nitrogen (e.g. 20% CO2: 80% N 2 ) also extend the shelf-life of the pack.
81
SPOILAGE OF MEAT AND MEAT PRODUcrS
10
10
bacteria
7
Storage time (days at SoC) Fig. 4.4 : Growth of bacteria in vacuum-packed fresh meat.
4.4.4 Spoilage of Vacuum-packed Bacon Mature bacon normally carries 104-106 bacteria per cm2 which are subsequently distributed over the cut surfaces at the time of slicing before packaging. Because of this the bacon has a rather limited shelflife but it can be stored at ambient temperatures without loss of colour. As previously mentioned, micrococci are the main types of bacteria developing on the higher salt concentration bacons when stored at ambient temperatures (20°C). When such bacons are stored under vacuum-packaged conditions, micrococci still predominate and rise in numbers to about 107 per gram after about 9 days' storage. Obvious spoilage is apparent at about 2 weeks and is characterized by a rancid odour. With lower salt concentration bacons the flora is initially more mixed but counts again reach a peak at about 9 days and spoilage follows a few days later. At this time, the flora consists of approximately equal proportions of micrococci, streptococci (i.e. enterococci) and other lactic acid bacteria (Le. lactobacilli and leuconostocs); with high storage temperatures (e.g. 25°C). Gram-negative forms (e. g. Vibrio spp. and Proteus spp.) may be responsible for putrefactive spoilage. The advantage of chill storage is that it takes between 3 and 4 weeks for bacterial counts to reach a maximum and hence obvious spoilage may be delayed for up to 5 weeks. Qualitatively the changes in the microbial flora during storage reflect the influence of salt
82
MICROBIAL SPOILAGE OF FOODS
concentration and the flora is essentially similar to that for vacuumpacked bacon stored at higher temperatures. Microbial spoilage of red meats and their products are summarized in Table 4.2. Table 4.2: Microbial Spoilage of Red Meats and their Products Product Fresh, refrigerated (0°-5°C) meat
Fresh (15°-40°C) meat
Vacuum-packaged meat Cured meats Bacon
Vacuum-packaged
Spoilage Off-odour, slime, discolora tion
Lipolysis, pungent odour Moldy Whiskers Black spot White spot Bone taint Gassy Foul odour Acid, sweet, rancid
Orsanism
Pseudomonas, Aeromonas, Alcaligenes, Acinetobacter, Microbacterium, Moraxella, Proteus, Flavobacterium, Alteromonas, Sacchilromyces Pseudomonas, yeasts Penicillium Thamnidium Cladosporium Sporotrichum Clostridium C. perfringens C. bifermentans, C. histolyticum, Lactobacillus, Microbacterium, Enterobacter, Hafnia
Cheesy, sour, rancid Micrococcus Discoloration Molds Lactobacillus, Micrococcus Slight souring
Putrefaction Cabbage odour Tainted
Vibrio, Alcaligenes, Corynebacterium Clostridium sporogenes Proteus inconstans Vibrio contd ....
83
SPOILAGE OF MEAT AND MEAT PRODucrS ... contd. Product Brines Ham
Spoilase Turbid Surface slime Gassy or puffy Green discoloration Bone and meat sours" Surface growth (Dry-cured) Slime on surface Gas production (vacuum-packed) Greenish discoloration Slime II
Sausages
Fermented sausage
SEots
Orsanism
Debaryomyces, Kloeckera Micrococcus, Microbacterium, Yeasts Clostridium lActobacillus, Streptococcus, Leuconostoc Clostridium Molds
Micrococcus, yeasts lActobacillus lActobacillas viridescens, Leuconostoc Yeasts Molds
DOD
SPOILAGE OF POULTRY AND EGGS
5.1 PROLOGUE The tt!rm 'poultry' applies to a range of domestic fowls and whilst the following paragraphs deal exclusively with chickens, the general principles apply to other commonly eaten fowls such as hlrkeys and ducks. In addition, the discussion will be limited to commercially dressed chickens since this is now accepted as the normal method of marketing. Spoilage of eggs and egg products are also discussed in this chapter.
5.2 EFFECTS OF POULTRY PROCESSING ON THE MICROBIOLOGICAL FLORA When live birds are brought into the processing plant they are harbouring large numbers of microorganisms of many different types on their feathers and feet and in their intestines. The various stages in processing are outlined in Fig. 5.1 and only those that have a significant effect on the microbiological condition of the carcass will be discussed. Scalding to loosen the feathers is performed by immersing birds for 30s in a tank of hot water (55°C). There is a reduction in the numbers of organisms on the carcass due to the washing effect and to the destruction of heat-sensitive bacteria including, in particular, psychrotrophic spoilage bacteria. Even where lowt!r scalding temperatures are needed (50°C for birds to be air chilled; see below) psychrotrophic bacteria are still destroyed. The mechanical feather pluckers increase the bacterial load on the skin of birds and also cause cross-contamination and 'aerosol'
85
SPOILAGE OF POULlRY AND EGGS
problems. Evisceration also increases the bacterial load on the skin by spreading faecal types onto the surface; such bacteria can be easily transferred to other carcasses again causing cross-contamination problems. r-------,
Reception of live birds
Removing head and feet
I
Spray washing
H
Chilling
I
Chill storage Cold storage
Fig. 5.1 : Stages in the processing of poultry.
The microbial load reaches a maximum immediately prior to spray washing. This process, which produces an approximately 90% reduction of microorganisms per carcass, is followed by chilling which is performed by one of three methods. In small processing operations chilling is frequently performed in static tanks containing equal quantities of carcasses, ice and water. The carcasses may be held in these tanks for many hours and those towards the bottom can become heavily contaminated by bacteria washed off carcasses situated towards the top; furthermore, growth of psychrotrophic bacteria is encouraged by these conditions. In larger operations mechanised 'spin' chillers are commonly used. The system utilizes one, two or three units in series, each consisting of a large tank through which chlorinated water flows continuously in one direction whilst carcasses flow on a helical screw principle in the opposite direction; additional cooling is obtained by the addition of ice to one or more of the units. In addition to effectively cooling the birds, the chiller system, if properly operated, further reduces the bacterial load on the carcass by some 90%. Efficiency is dependent upon a controlled flow of chlorinated water.
86
MICROBIAL SPOILAGE OF FOODS
The final method of chilling is by air. This process, used where chickens are to be retailed chilled, tends to dry the skin and hence retard the growth of psychrotriophic spoilage bacteria. After the preliminary chilling, microbial counts on chicken skin range from 5 x 103 to 1 x 1()5 per cm2whilst counts in the visceral cavity are usually < 1 x 104 per cm2; the lowest counts will be found in hygienically and efficiently operated processing lines. Qualitatively the microbial flora is extremely mixed at this stage and amongst the more commonly isolated bacterial groups will be micrococci, flavobacteria, various intestinal types, such as Escherichia, Enterobacter and Streptococcus spp., and Acinetobacter spp. 5.3 SPOILAGE OF CHICKENS HELD AT CHILL TEMPERATURES When chickens are stored at chill temperatures most of the microbial growth occurs on the skin and, to a lesser extent, in the lining of the visceral cavity. Over a period of 10 days the number of bacteria increases to a maximum on the skin of between 109 and 1010 per cm2. This increase in numbers is accompanied by off-odours (107 bacteria per cm2) and copious slime production (108 per cm2) and arise in pH to 7.5 and so, as might be expected, there are many similarities between the spoilage of raw chicken and other meats. Furthermore, as with chilled meats, the spoilage flora of refrigerated chickens becomes dominated by Pseudomonas spp. (both fluorescent and non-fluorescent types). In fact at advanced stages of refrigerated spoilage chicken skin often fluoresces when illuminated with ultraviolet light due to the presence of large numbers of fluorescent pseudomonads. At the time of spoilage pseudomonads represent 7080 % of the flora but also present in smaller numbers are Acinetobacter spp. (10% of the flora) and Alteromonas (Pseudomonas) putrefaciens. The latter organism is particularly interesting as its growth is much faster on chicken leg tissue (pH 6.5) than on breast (pH 5.8); thus the predominance of pseudomonads is less pronounced on the former tissue. Using gas chromatography and mass spectrometry, attempts have been made to identify the off-odours produced during the refrigerated spoilage of chickens. Freeman et al. (1976) found that 22 volatiles were produced during spoilage under chill conditions and 15 of these were produced as a result of microbial attack on muscle
87
SPOILAGE OF POULTRY AND EGGS
tissue and were responsible for or associated with the characteristic off-odours of the later stages of spoilage. Amongst these 15 compounds were hydrogen sulphide, methyl mercaptan, dimethyl sulphide, methyl acetate, ethyl acetate, methanol, ethanol and benzaldehyde; the large variety of compounds identified clearly illustrates the complexity of this problem in terms of the role of microorganisms in the spoilage process. 5.4 THE CHICKEN'S EGG AND ITS SPOILAGE
The chicken's egg is generally regarded as being sterile at the time of laying unless it has been infected congenitally, usually by certain salmonellas. Contamination of the egg occurs after laying and access of microorganisms into the egg is most common through cracks in the shell. The shell, covered with a water-repelling cnticie, acts as a mechanical barrier if intact, but an alternative means of entry for microorganisms is through pores which perforate the egg shell (see Fig. 5.2). These pores should be filled with plugs but in the largest pores the plugs may become dislodged. Penetration is aided by moisture setting up capillary effects in the pores. Beneath the shell are two membranes which further retard invasion by bacteria for limited periods but probably offer no barrier to the infiltrating hyphae of moulds. (a)
f-t'----- (b) -+-_ _ _ _ (c)
(e) (f)
Fig. 5.2 : Simplified diagram of a section through an egg shell showing: (a) cuticle, (b) cuticular plug, (c) pore, (d) matrix, (e) outer shell membrane, and (f) inner shell membrane
88
MICROBIAL SPOILAGE OF FOODS
The white (albumen) of eggs contains a battery of antimicrobial agents which restrict or totally inhibit the growth of invading microorganisms provided levels of contamination are low. Lysozyme is particularly effective against Grampositive bacteria causing lysis of cell walls whilst conalbumin, effective against both Grampositive and Gramnegative bacteria, acts as a chelating agent, removing iron which is essential for growth. The yolk, however, is a rich source of nutrients and contains no inhibitory agents; thus rapid growth of invading organisms is possible if the yolk is involved. This can occur when the yolk comes to rest in the uppermost part of the egg about 10 days after laying. If penetration of the egg has occurred in the membrane area impinging on the yolk the defence mechanisms of the egg are shortcircuited and heavy growth of invading bacteria is likely. Spoilage of eggs is caused principally by Gram-negative bacteria which produce characteristic rots.
5.5 EGG PRODUCTS Although the contents of fresh eggs are usually sterile, commercially produced egg products (liquid, frozen and dried) used to be heavily contaminated with bacteria. In particular, evidence accumulated that liquid whole egg was frequently contaminated with salmonellas that were well able to withstand the comparatively low temperatures frequently used in the baking industry. Heat treatment regulations were introduced in the UK in 1963 and since then all liquid whole egg intended for distribution in the chilled state, for freezing or for spray drying, has been pasteurized at 64.4°C for 2Vz min and then cooled immediately. The heat-resistant salmonellas should be destroyed by this treatment and the baking properties of the egg are not impaired. Furthermore, in the chilled liquid state pasteurized whole egg can be safely stored for at least 6 days without significant increases in bacterial counts although post-pasteurization contamination, mainly caused by coliforms, must be avoided. In Britain, liquid egg albumen is now conventionally pasteurized although heat-treatment regulations now pending have yet to be implemented. Untreated chilled albumen is normally spoiled by pseudomonads and related Gram-negative rods whilst faecal streptococci and lactobacilli are predominant in pasteurized albumen. More severe heat treatment is required to destroy salmonellas in liquid egg yolk but again no heating regulations have been introduced. Microbial spoilage of poultry and poultry products like eggs have been summarized in Table. 5.1.
89
SPOILAGE OF POULTRY AND EGGS Table 5.1 : Microbial Spoilage of Poultry and Poultry Products Product Poultry meat
Spoilase Off -odour, slime
Shell eggs
Black rot White rot (colourless) Sour Green white Musty Moldy Red rot Custard rot Yellow and green rot
Liquid whole egg
Fishy
Off -odour, sour
Liquid albumen
Off-odour
Microorganism
Pseudomonas, Acinetobacter, Moraxella, Alicaligenes, Aeromonas, Alteromonas Proteus, Aeromonas Citrobacter, Alcaligenes Pseudomonas P. fluorescens Pseudomonas Many types of molds
Serratia marcescens Citrobacter, Proteus, Enterobacter Alcaligenes, Flavobacterium, Cytophaga Pseudomonas, Flavobacterium, Chromobacterium Proteus, Alcaligenes, Escherichia, Flavobacterium, Pseudomonas, Bacillus Pseudomonas, Acinetobacter, Enterobacter [J[J[J
SPOILAGE OF FISH AND OTHER SEAFOODS
6.1 PROLOGUE The fishes principally include free swimming teleosts and elasmobranchs. The same term can also encompass all seafoods including crustaceans with a chitinous exoskeleton such as lobsters, crabs and shrimp, and molluscs such as mussels, clams and oysters. Historically the extreme perishability of fish has restricted its consumption in a reasonably fresh state to the immediate vicinity of where the catch was landed. This has detracted only slightly from it playing a significant role in human nutrition as, throughout the world, traditional curing techniques based on combinations of salting, drying and smoking were developed which allowed more widespread fish consumption. Various aspects on spoilage of fish and other seafoods are discussed in this chapter. 6.2 BACTERIOLOGY OF THE NEWLY CAUGHT FISH Most of the microbiological studies dealing with fish have concentrated on marine varieties and only these types will be considered. It is generally accepted that the flesh of newly caught healthy fish is sterile but bacteria are found in variable numbers in three sites on the fish: the slime coat, the gills and the intestines. Numbers on skin have been reported to range from 102 to 107 per cm2,
SPOILAGE OF FISH AND
OrnER SEAFOODS
91
on gills from 103 to 106 per g tissue and in the intestines from 103 to 108 per ml contents. However, it has been claimed that numbers of bacteria on fish in unpolluted waters are at the lower end of the ranges quoted and that the higher numbers result from the poor hygienic standards on board ship during initial handling. Variations in marine environments affect the types of bacteria present on the skin and gill sllrfaces of newly caught fish. Thus in the cooler seas of the northern hemisphere the bacterial flora is dominated by psychrotrophic Gram-negative rods. Liston found the flora of North Sea flatfish (skate and lemon ~ole) to comprise Pseudomonas spp. (60%), AcinetobacterlMoraxeIla (14 %) with the majority of the remainder being other types of Gram- negative rods; Georgala found that the bacterial flora of North Sea cod consisted of pseudomopads (44%) and acinetobacters (32 %) together with a variety of miscellaneous types. In the warmer waters off India, the east coast of South Africa, Australia and in the Adriatic the proportion of mesophiles increases and micrococci and coryneforms become more important. Thus Gillespie & Macrae found micrococci (49%) to predominate on newly caught fish from Australian waters; pseudomonads (only 18%), coryneforms (12%) and acinetobacters (only 9 %) were the other main groups isolated.
6.3 THE EFFECT OF INITIAL PROCESSING AND STORAGE IN ICE ON BOARD SHIP Gutting of the fish on board ship tends to spread the intestinal flora over the surface of the fish. The principal organisms found in the intestines are Vibrio spp. although many other genera are represented. Fish are then washed in sea water and either packed in crushed ice or possibly frozen. The conventional method is to pack in ice and the effects of this form of storage will be considered in some detail. The ice in which the fish are to be preserved is itself usually contaminated (103 per ml of ice melt water) and, in addition, the holds of the fishing vessels normally have an indigenous flora composed of Pseudomonas and Acinetobacter spp. Thus when fish, which already contain a fairly high proportion of pseudomonads, are placed in ice they are likely to be further contaminated with these organisms. Partly because of the relatively high proportion of pseudomonads on the skin of fish initially and in the storage environment and partly because of the relatively high pH of many fish species, spoilage in ice is relatively rapid. Even under the best conditions with a temperature of DoC total counts may reach a maximum of > 108 per g after 12 to 14 days' storage
92
MICROBIAL SPOILAGE OF FOODS
(Fig. 6.1). Occasionally in the holds of vessels temperatures may reach 6 or 7°C and under these conditions maximum numbers are reached within 5 or 6 days. Spoilage of fish is thus much faster than spoilage
of raw meat, maximum numbers of bacteria in the latter being reached only after 9 or 10 days' storage at 7°C. The actual quality of fish landed at the ports is dependent upon the time it has been held in ice and the hygienic conditions on board the fishing vessels. Average bacterial counts on fish taken from trawler holds on docking are 1(Ji per cm2 of skin surface and, qualitatively, the proportion of Pseudomonas spp. is higher than in the newly caught fish. During prolonged storage in ice pseudomonads become the dominant group and represent 80-90% of the spoilage flora at peak numbers. Even with fish caught in warmer waters the pseudomonads are the predominant group at the time of spoilage and therefore overgrow the initial mainly Gram-positive flora. b Q)
8
""0
~ El 7
~ !ib6 "....
g5
u
I II
'\:1
.2:l u 4
III ,.0
,33 Time in days Fig. 6.1 : Numbers of bacteria on fresh fish stored at: (a) 25°C, (b) 7°C, (e) QOC, and (d) 4°C.
6.4 THE EFFECT OF HANDLING ASHORE After landing, the fish may be left on the quay for many hours uniced in boxes or kits. Under such conditions the temperature of the
SPOILAGE OF FISH AND OrnER SEAFOODS
93
fish will rise and growth of the psychrotrophic bacteria will become more rapid so that a la-fold increase in numbers in a few hours can be anticipated. The wooden fish boxes still widely used ashore harbour vast numbers of bacteria, counts often being in excess of 106 per cm2 even on 'clean' boxes; however, it is probable that these bacteria have little effect on the spoilage of fish since the time spent by fish in boxes is usually limited to less than 12 h. The fish may then be re-boxed, often in ice, and transported to the processor. Here, depending on the type of fish, they may be filleted or otherwise processed. All these events, including filleting and final transport to the retailer, affect the bacteriological flora which becomes more varied and more mesophilic in character with increasing degrees of handling. 6.5 CHEMICAL CHANGES INDUCED BY BACTERIA IN FISH Like meat, fish may be spoilt by naturally occurring autolytic enzymes or by bacterial activity. Spoilage is principally due to the activities of enzymes produced by Gram-negative rods, particularly pseudomonads. These organisms, invariably become predominant during prolonged storage of fish whether or not stored in ice. The role of pseudomonads in the spoilage of fish has been studied in some detail. Adams et al. characterized potential spoilers by inoculating them as pure cultures into sterile muscle press juice from sole and measuring their ability to produce off-odours, volatile reducing substances and trimethylamine. They found that only about 10 % of the initial flora were spoilers using the above criteria and that the proportion of spoilers was never more than 30 % of the total flora during storage at 5°C. The spoiling bacteria were later identified as being mainly pseudomonads (some of these would now be classed as Alteromonas spp.), acinetobacters or vibrios; it was stressed that even though pseudomonads were the predominant group only a small proportion were active spoilers. Similar results were obtained with cod by Shaw & Shewan who found that the proportion of active spoilers to the total viable population did not change significantly during spoilage and always remained below 25%, again Pseudomonas spp. were the principal group but in this work Acinetobacter/Moraxella spp. and Vibrio spp. were not implicated as spoilers. Spoilage bacteria first utilize low molecular weight compounds such as nucleotides and amino acids present in fish muscle and it is the breakdown of these materials which is reasonable for off-odours and other spoilage effects; thus, as with meats, protein plays a minor role in spoilage. Studies in which pure cultures of spoilage-inducing
94
MICROBIAL SPOILAGE OF FOODS
pseudomonads have been inoculated into blocks of sterile fish muscle indicate that different strains generate totally different odours. Some of the volatiles produced were 'fruity' and probably esters whilst others were 'sulphidy'. More definitive studies using gas-liquid chromatography have identified some of the principal volatiles in spoiling fish as methyl mercaptan, dimethyl sulphide, dimethyl disulphide, hydrogen sulphide, trimethylamine, ethyl acetate and ethanol; it should be noted that many of these volatiles have also been associated with meat spoilage and that they need only be present in very low concentrations to produce obvious off-odours.
6.6 SALTED FISH Two basic techniques may be used in the salting of fish, either 'dry' or 'wet' salting. In the former, used mainly in the barrel salting of herring, salt is spread over the surface of the fish and layers of fish are interspersed with salt layers. In wet salting the fish are placed in a salt solution; this method is often adopted when fish are to be smoked. A combined 'dry' and 'wet' salting method is sometimes used. Irrespective of the method used, the added salt lowers the '\v of the fish and, as has already been discussed with cured meats, this has a profound effect on normal spoilage patterns. The normal predominantly Gram-negative flora of fish is relatively sensitive to high salt concentrations and so bacterial numbers decline. The final flora depends on the strength of the cure. In many instances micrococci become predominant but in the highest salt concentrations a specialized group of bacteria can sometimes cause spoilage problems. These bacteria, represented by the genera Halobacterium and Halococcus are termed 'extreme halophiles' and tolerate salt concentrations in excess of 20% NaCl; indeed, they require 10-15% NaCl in culture media before they will grow. The spoilage produced is a red discolouration on the surface of the fish ('pink' fish) and is caused by the growth of these organisms which are pigmented red. Another type of spoilage known as 'dun' is caused by a halophilic mould, Sporendonema expizoum; it is associated with the formation of peppered spots which are visible on the fleshy side of salted fish, particularly cod, the spots ranging in colour from chocolate to brown and fawn. Lightly salted cod is also susceptible to 'sliming' which is caused by the growth of the indigenous Gram-negative flora (primarily pseudomonads); this condition is characterized by a slimy beige coloured layer on the surface of the fish.
SPOILAGE OF FISH AND OruER SEAFOODS
95
6.7 SMOKED FISH Before smoking, fish are gutted and given a preliminary salting treatment, the NaCl concentration depending on the leve1 desired in the fish. The salting is often comparatively light and the preservative effect is therefore minimal as with, for example, finnan haddock, kippers and smoked cod. With certain fish such as 'red' herring and smoked salmon the salting is more extreme and may playa significant part in preserving the fish. Following brining the fish are either 'cold' or 'hot' smoked. In the commonly used cold smoking process the temperature of the fish should not exceed that at which protein is denatured (30°C). The process, which includes both drying of the fish and impregnation with wood smoke, results in some reduction in the numbers of bacteria caused mainly by phenolic substances present in the smoke; overall, however, the effects of this process on the microbial flora are insubstantial. In hot smoking, used for specialized canned products such as brisling (sprats) and sild (small herring) the temperature of the fish is raised to 70°C for 30 min so only the more heat-resistant bacteria should survive; the microbial flora therefore consists mainly of micrococci and Bacillus spp. The microbiological changes occurring during the storage of smoked fish have not been studied in any detail but the predominant microorganisms at the time of spoilage depend largely on the processing conditions. With lightly brined cold smoked fish pseudomonads may become the major group but a slight increase in salt concentration could result in micrococci predominating. One of the most common causes of spoilage of smoked fish is due to mould contamination; both Penicillium and Aspergillus spp., which grow readily at chill temperatures, are present in the sawdust used for smoke production and may subsequently develop in the stored fish. With the higher salt concentration smoked fish storage life is prolonged for several weeks or months even at high ambient temperatures and little change in the microbial flora is likely to occur.
6.8 SHELL FISH 6.8.1 Crustaceans Freshly caught shrimps are highly perishable due to bacterial and enzymatic activity. Bacterial activity is enhanced by the high content of amino acids but autolytic enzymes (proteases) cause rapid breakdown of protein providing bacteria with an ideal growth substrate.
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MICROBIAL SPOILAGE OF FOODS
Because of their perishable nature, shrimps are preferably either frozen or boiled as soon as possible after catching but storage in ice is also common. The initial flora is similar to that of freshly caught fish but storage in ice results in an increase in the proportion of Acinetobacter/ Moraxella species which account for over 80 % of the flora at the time of spoilage. This group does not appear to be responsible for bacterial spoilage, however. The active spoilers are Alteramonas spp. and pseudomonads playa relatively minor role in the spoilage of this food; spoilage is accompanied by increases in ammonia, trimethylamine, hypoxanthine and acetic acid. Autolytic enzymes are particularly active in lobsters and this makes them another highly perishable food. Use is made of these enzymes in conditioning which involves storage in ice for 2 to 4 days. As with shrimps, further storage results in a pronounced increase in the proportion of acinetobacters and moraxellas which are predominant at the time of spoilage. Whether or not these organisms are responsible for spoilage, characterized by increasing concentrations of ammonia and trimethylamine, has yet to be determined. Crab meat, too, spoils rapidly and crabs are therefore cooked in boiling water immediately after capture. Studies on crabs have concentrated on the bacteriology of the cooked meat: it is likely that the flora at the time of spoilage is again dominated by acinetobacters and related species. 6.8.2 Molluscs Of the bivalve shellfish eaten by humans, oysters, scallops and mussels are probably most frequently consumed. The main microbiological problem associated with these foods is the hazard of food poisoning resulting from the not infrequent pollution of their growth habitat. It is therefore a necessary practice to cleanse these foods in clean chlorinated water. The natural flora of the bivalves may thus change extensively during treatment, and spoilage characteristics may vary depending on the efficiency of the cleansing process. An important feature of bivalves is the significant amount of ca:t:bohydrate (3-6 %) present in their flesh and this can influence the type of spoilage obtained. If fermentative bacteria such as Escherichia coli and other coliforms are not removed during the cleansing process, spoilage is primarily one of souring, acids being formed by the dissimilation of the carbohydrate. With properly cleansed molluscs held at chill temperatures the spoilage is totally different and is associated with increases in volatile bases and hypoxanthine, and the flora is now dominated by Acinetobacter/Moraxella spp.
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SPOILAGE OF FISH AND OrnER SEAFOODS
Microbial spoilage of fish and other seafoods are summarized in Table 6.l. Table 6.1 : Some Microbial Spoilage of Seafoods Product Fish Fresh
Salted
Crayfish Oysters Shrimp Squid
Spoilage
Microorganism
Off-odour
Pseudomonas, Alteromonas, Acinetobacter, Vibrio, Aeromonas, Moraxella, Proteus Pseudomonas Pseudomonas, Alteromonas Pseudomonas, Alteromonas Halobacterium, Halococcus Hemispora stellata, Sporendonema epizoum Red halophilic bacteria Pseudomonas, Lactobacillus, Coryneforms Yeasts (Rhodotorula) Pseudomonas P. putida Serratia marcescens
Fruity Ammonical H 2S odour Pink Dun (red growth) Cheesy, putrefactive Sweet to foul odour Pink Off-odour Yellow discoloration Red discoloration
DOD
SPOILAGE OF DAIRY PRODUCTS
7.1 PROLOGUE Milk is the fluid secreted by mammals for the nourishment of their young. A number of animals are used to produce milk for human consumption, although the cow is by far the most important in commercial terms. Milk, when obtained from the animal, may not be sterile. During and after milking, the milk is subjected to microorganisms from various sources, although contaminated equipments used to handle, transport, store and process the milk seems to be the main source of organisms. With extened storage of milk products at refrigerated temperatures, psychrophilic or psychrotrophic organisms are a cause of spoilage. The organisms associated with spoilage generally do not survive the heat treatment used to pasteurise milk. Hence, spoilage is usually caused by organisms recontaminating the milk after pas turisa tion. The spoilage that can occur in milk due to microbial growth are: (1) off flavours; (2) lipolysis with development of rancidity; (3) gas production; (4) fermentation to lactic acid with souring; (5) coagulation of milk proteins; (6) viscous or ropy texture; and (7) discolouration etc. Various aspects on spoilage of milk and other dairy products are discussed in this chapter.
7.2 MICROBIOLOGY OF RAW MILK Even when drawn under aseptic conditions, milk always contains microorganisms which are derived from the milk ducts in the cow's udder. Numbers vary from quarter to quarter and from cow to
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99
COW but are roughly in the range 102-10 3 organisms per ml. In practice, freshly drawn milk contains 5 x 103 to 5 X 104 organisms per ml, contaminants coming from the outside of the udder, from milking equipment and from human handlers. Many different microorganisms may be present including species of Pseudomonas, Acinetobacter/ Moraxella, Flavobacterium, Micrococcus, Streptococcus, Lilctobacillus and coliforms. Furthermore, it must be appreciated that infected udders are going to introduce potentially pathogenic bacteria into the milk. This problem is relatively widespread, it having been shown that some 30 % of British dairy herds suffer from mastitis. As milk is an ideal growth medium for bacteria it is essential to cool it as rapidly as possible. The introduction of refrigerated farm bulk milk tanks over the last 25 years, coupled with the bulk collection of milk in refrigerated tankers, has markediy influenced the bacteriological quality of raw milk supplies. The major effect of this change has been to reduce the quantity of milk spoiled by souring. Souring of milk at normal temperatures is caused by the lactic acid bacteria which grow mainly at temperatures above 100 e. These bacteria produce lactic acid from the milk sugar (lactose) which induces a sour flavour and later coagulation of the milk. Most lactic acid bacteria are killed by pasteurisation but a few (e.g. Streptococcus thermophilus) are thermoduric and can cause post-pasteurization problems. With rapid cooling and refrigerated storage of milk the problems are somewhat different. Nowadays it is the psychrotrophs, mainly pseudomonads, which are primarily responsible for spoilage problems. Psychrotrophic bacteria, originally derived from soil and water, are readily isolated from farm milking equipment, pipes and bulk milk tanks. Inefficient or delayed cooling of milk markedly increases the proportion of psychrotrophs but growth of this group continues more slowly at recommended storage temperatures for raw milk (3-7"C). Counts of psychrotrophic bacteria in bulk tanks vary from 104 to 106 per fF depending upon the extent and type of contamination and storage conditions. A large proportion of these psychrotrophs produce proteases and lipases, and many of these enzymes are unaffected by pasteurization; in fact temperatures of 150°C for 10 s may be required to inactivate them. Defects due to proteases include bitterness and the principal spoilage effect of lipases is rancidity.
7.3 PASTEURIZATION This process involves heating the milk to a temperature high enough to destroy all pathogenic bacteria such as Mycobacterium tuberculosis, Salmonella spp. and Brucella spp; in so doing the large
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MICROBIAL SPOILAGE OF FOODS
majority of other bacteria including spoilage bacteria are killed and the keeping quality of the milk is thus enhanced. Most of the milk produced is pasteurized by the high-temperature, short-time (HTST) method in which milk is held at 72°e for at least 15 s and then cooled rapidly to less than lOoe; the older low-temperature, long-time (LTLT) or 'holder' method (63°e for 30 min) is still occasionally employed but only on a small scale. The term 'thermoduric' is applied to those bacteria that survive pasteurization due to their innate heat resistance. Thermoduric bacteria consist mainly of a few species of Streptococcus (e.g. S. thermophilus), Micrococcus (e.g. M. luteus) and Corynebacterium (e.g. C. lacticum) together with the spores of certain Bacillus spp., particularly B. cereus. These bacteria are readily isolated from poorly cleaned dairy equipment and pipelines although numbers in refrigerated bulk milk tanks are usually small. Spoilage of pasteurized milk held at normal temperatures is caused principally by the thermoduric bacteria, the predominant organism at the time of spoilage usually being B. cereus. This organism causes the defect termed 'hitty cream' and is responsible for the 'sweet' curdling of pasteurized milk (i.e. coagulation of milk by rennin without acid formation). The psychrotrophic bacteria, so important in raw milk, are readily killed by pasteurization even though some of their enzymes are unaffected. However, psychrotrophs can be an important cause of spoilage in pasteurized milk if post-pasteurization contamination occurs. This contamination should be negligible but with inadequate cleaning of equipment it can be very significant. Such contamination should be minimized since pasteurized milk is usually stored at rc, a temperature at which psychrotrophs grow well. Pasteurized milk with minimal post-pasteurization contamination should have a shelf-life of at least 7-10 days at rc.
7.4 UHT MILK Ultra-high-temperature (UHT) milk is homogenized milk that is exposed to a temperature of not less than 132°e for at least Is, this
process rendering the milk practically sterile. The original form of sterilization of milk was to heat the milk at lOOoe for 30 min in hermetically sealed bottles; such milk was characterized by a cooked flavour and rich and creamy texture which, together with its darker appearance, rendered it a rather unattractive product. UHT milk does not suffer from these characteristics and has therefore largely superseded the older bottled sterilized milk. UHT milk is filled
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101
aseptically in special cartons (e.g. Tetra Pack) which are then heatsealed. This milk has the appearance, flavour and nutritional quality of pasteurized milk and should remain acceptable for several months without refrigeration. Spoilage of UHT milk can be caused occasionally by the growth of spore formers, mainly Bacillus stearothermophilus or B. subtilis, the spores of which have either survived the UHT process or are posttreatment contaminants. More commonly, spoilage can result from the continuing activities of heat-stable proteases and lipases produced by psychrotrophs in the raw milk. Gelation of stored UHT milk, which may also be caused by a chemical process, can also be attributed to proteases. 7.S BUTTER
Butter is a comparatively stable product microbiologically as it has a low moisture content (15 %) and a high fat content (80 %). The water is present in the form of a fine emulsion in the fat phase and the physical conditions in the water droplets probably exert an inhibitory effect on bacterial growth. Furthermore, many butters are salted at concentrations varying between 3 and 13 % NaCl and this helps in preservation. The main source of microorganisms in butter is the cream from which it is made; this is pasteurized in the case of 'sweet' butters. The churning of cream into butter increases the numbers of organisms as they become concentrated in the buttermilk but at the end of processing numbers are low and with the highly salted butters further reductions are likely during storage. Certain 'sweet' butters utilize a 'starter' culture. These cultures of known bacteria are inoculated into milk or cream to induce souring under controlled conditions thus giving predictable and desirable characteristics to the butter; with 'sweet' butters the cream, after inoculation, is held at low temperatures to keep the acidity from increasing prior to churning. 'Sour' butters are normally made from pasteurized cream inoculated with starter organisms (Streptococcus lactis and S. cremoris are commonly used); the cream is incubated at room temperature until a low pH (4.5-5) is attained. The cream is then churned but is not salted because salt and acid react together to give undesirable flavours. Large numbers (10 7 to 108 per gram) are required to produce the acid. An alternative process involves the natural souring of cream which is then pasteurized before churning.
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MICROBIAL SPOILAGE OF FOODS
Thus the microbial content of fresh butters varies considerably depending on the manufacturing process, 'sweet' butters containing far fewer microorganisms than 'sour' butters. Spoilage of butter can be of microbial, enzymatic or chemical origin; many undesirable flavours may stem from the cream itself but this aspect of spoilage is not considered here. Microbial spoilage is caused principally by psychrotrophic bacteria as butter is usually stored under refrigeration. Pseudomonads and related Gram-negative rods which enter the product postpasteurization are often responsible for rancidity caused by the hydrolysis of butter fat liberating fatty acids. Putrid flavours and surface taint result from the proteolytic activities of Alteromonas putrefaciens growing on the surface of the butter. Moulds also grow on the surface producing discolouration; commonly implicated are members of the genera Alternaria, Cladosporium, Aspergillus, Penicillium, Mucor and Rhizopus. Rancidity may be induced by lipases present in the cream and chemical reactions include the oxidation of unsaturated fats. 7.6 CHEESE There are over 400 known varieties of cheese grouped into about 20 general classes. Most of these can be made from the same milk by varying the microorganisms, enzymes and salt added, and by changes in the temperature during manufacture and curing. Cheeses are classified by their texture or the degree of hardness and two major groups of natural cheeses are recognized. The first group, the ripened cheeses, vary from the very hard, low moisture content grating cheeses (e.g. Parmesan), through the hard cheeses (e.g. cheddar) to the higher moisture content, semi-soft cheeses (e.g. Stilton) and soft cheeses (e.g. Camembert). The second group of cheeses are the unripened soft cheeses with a high moisture content such as cottage cheese. Most cheeses are manufactured using the same basic processes. Nowadays pasteurized milk is normally used but the ripening process occurs more slowly and, because the natural flora has been largely destroyed, 'starter' cultures of bacteria must be added to the milk. In the case of Cheddar cheese, starters may be mixed strains of Streptococcus lactis or a mixture of S. lactis and S. cremoris. These bacteria convert the sugar lactose into lactic acid thus inducing the first stage in cheese manufacture, i.e. souring or 'ripening' of the milk. When the
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milk has reached the required acidity rennet is added which helps in curd formation. Later stages in processing involve treatment of the curd which, after salting and pressing, is allowed to mature; with Cheddar cheese maturation takes about 4 months. Microbiologically the starter culture organisms reach a peak of 109 per g in cheese during ripening but after some 48 h the streptococci decline in numbers. They are replaced by lactobacilli which represent 99 % of the population in mature cheese. Lactobacilli slowly decompose protein during maturation and this helps to flavour the cheese; in fact lactobacilli (e.g. L. acidophilus) are often added deliberately to enhance flavour. When considering the spoilage of cheese it should be emphasized that the harder cheeses with lower moisture contents have longer shelf-lives than the softer cheeses. Microbiological spoilage of mature Cheddar cheese is caused primarily by the surface growth of moulds which produce discolouration effects although there is little penetration of or attack on the cheese. Many different moulds and yeasts have been implicated in such spoilage and include species of Penicillium (green discolouration), Cladosporium (green to black) and Candida (black). However, the harder cheeses have a wax coating or develop a rind and this minimizes the problem. In more recent years film wrapped and vacuum-packed cheeses have become popular and these forms of packaging prevent fungal growth by excluding air. Bacterial spoilage of cheeses is more common during their manufacture and ripening. If the pH is too high pseudomonads, which always contaminate the product to some extent, are able to grow rapidly and cause sliminess. 'Gassy' cheese is a common problem and is caused by coliforms such as Enterobacter spp. fermenting the lactose with the production of carbon dioxide; certain clostridia may also cause this effect. Control may be effected by the incorporation of nisin, an antibiotic produced by certain strains of Streptococcus lactis, in the cheese. This antibiotic is particularly active against clostridia in cheeses with a higher pH. Various flavour defects, of which bitterness and rancidity are the most important, can occur in cheese; many of these defects may be caused by microorganisms. Problems during manufacture should not occur if good starter cultures are used and a high standard of hygiene is maintained. 7.7 YOGHURT Yoghurt is a fermented milk product made by adding a mixed starter culture (Lactobacillus bulgaricus and Streptococcus thermophilus) to
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MICROBIAL SPOILAGE OF FOODS
milk that has been heated to destroy the indigenous flora. As with cheese, lactic acid is produced during incubation at 45°C and this reduces the pH to 4-0; traces of other products such as diacetyl and acetaldehyde contribute towards the flavour characteristics. After incubation the yoghurt is promptly cooled to 4°C to prevent further acid development and the low storage temperature coupled with the acidity of the product ensure that spoilage by proteolytic and other non-acid tolerant bacteria is prevented. Slow growth of the starter organisms continues at the storage temperature and this restricts the shelf-life to about 4 weeks as by that time the excess acid produced will impair flavour. Some microbial spoilage in dairy products are summarized in Table 7.1. Table 7.1 : Some Microbial Spoilage in Dairy Products Product
Spoilage
Microorganism
Milk Pasteurized, refrigerated Rancid
Canned Cream Butter Cheese
Pseudomonas, Alcaligenes, Staphylococcus Coliforms, Pseudomonas, Ropy or slimy Alcaligenes, Micrococcus, Bacillus subtilis Sour (acid, gas) Lactic acid bacteria Chromobacterium Discoloration Pseudomonas, Flavobacterium Bitter, fruity Alcaligenes, Proteus, Acinetobacter Swelling, gas Clostridium sporogenes Foamy Candida, Torulopsis Surface taint Pseudomonas, Alteromonas Gassy, butyric acid Clostridium tyrobutyricum Gassy, floating or Leuconostoc, S. lactis split curd subsp. diacetylactis Penicillium, Scopulariopsis Moldy Mucor, other molds contd ....
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... contd. Product Soft
Cottage
Cheddar Swiss Yoghurt
Microorganism Mucor Torulopsis, Debaryomyces Pseudomonas Flavobacterium, yeasts, molds Pseudomonas, Alcaligenes, Flavobacterium, coliforms Fruity Yeasts Sweet, yeasty, fruity Yeasts Gassy, sweet Yeasts (Torulopsis) Off-odour C. sporogenes Yeasty Yeasts (Torulopsis)
Spoilage Black mold (eat's fur) Surface growth Slimy curd, putrid odour, Discoloration Slimy, gelatinous
DOD
SPOILAGE OF VEGETABLES, FRUITS AND THEIR PRODUCTS
8.1 PROLOGUE As soon as vegetables and fruits are harvested physiological changes occur and some of these lead to a loss in quality. Respiratory activity involving the breakdown of carbohydrates by the plant enzymes continues and the changes induced, whether advantageous or deleterious, are markedly influenced by the maturity of the plant when harvested; thus plants can usually be stored for lengthy periods with little change in quality if harvested at the right time. Many fleshy fruits such as bananas are harvested before maturation and ripening continues thereafter but citrus fruits only ripen satisfactorily on the tree. However, although spoilage can be induced by autolytic enzymes, it is caused more usually by the activities of microorganisms and this aspect is considered in this chapter. Microbial spoilage of various products of vegetables and fruits are also dealt in this chapter. 8.2 VEGETABLES Fresh vegetables contain microorganisms coming from soil, water, air, and other environmental sources, and can include some plant pathogens. They are 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 most common sp?ilage is caused
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by different types of molds; some of those are from the genera Penicillium, Phytopthora, 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. In addition to changes in colour, microbial rot causes loss of texture and off-odour. 8.3 FRUITS
Fresh fruits are high in carbohydrates (generally 10% or more), very low in proteins (~ 1.0%), but have pH 4.5 or below. Thus microbial spoilage of fruits and fruit products is confined to molds, yeasts, and aciduric bacteria (lactic acid bacteria, Acetobacter, Gluconobacter). Like fresh vegetables, fresh fruits are susceptible to rot by different types of molds from genera Penicillium, Aspergillus, Alternaria, Botrytis, Rhizopus and others. According to the changes in appearance, the mold spoilages are designated as black rot, gray rot, soft rot, brown rot, and others. Yeasts from genera Saccharomyces, Candida, Torulopsis and Hansenula have been associated with fermentation of some fruits such as apples, strawberries, citrus fruits, and dates. Bacterial spoilage associated with the souring of berries and figs has been attributed to the growth of lactic acid and acetic acid bacteria. 8.4 SOFT DRINKS, FRUIT JUICES AND PRESERVES AND VEGET ABLE JUICES
Carbonated and non-carbonated soft drinks, fruit juices, and preserved and concentrated fruit juices and drinks are low pH products (2.5 to 4.0). The carbohydrate (sucrose, glucose, and fructose) content ranges from 5 to 15% in juices and drinks, but 40 to 60% in concentrates and preserves. High sugar content reduces the Aw of these products, which in the concentrates and preserves can be about 0.9. Carbonated beverages also have low O-R potentials. Among the microorganisms that can be present in these products, only aciduric molds, yeasts, and bacteria (Lactobacillus, Leuconostoc, and Acetobacter) are able to cause spoilage if appropriate preservation methods are not used. In the carbonated beverages, some yeast species from genera Torulopsis, Candida, Pichia, Hansenula, and Saccharomyces can grow and make the products turbid. Some Lnctobacillus and Leuconostoc species can also grow to cause cloudiness
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MICROBIAL SPOILAGE OF FOODS
and ropiness (due to production of dextrans) of the products. Noncarbonated beverages can be similarly spoiled by the yeasts, Lactobacillus and Leuconostoc spp. In addition, if there is enough dissolved oxygen, molds (Penicillium, Aspergillus, Mucor, and Fusarium) and Acetobacter can grow; the latter will produce acetic acid to give vinegar-like flavour. Fruit juices are susceptible to spoilage by molds, yeasts, Lactobacillus, Leuconostoc, and Acetobacter spp. However, a particular type of juice may be susceptible to spoilage by one or another type of microorganism. Molds and Acetobacter can grow if enough dissolved oxygen is available. Yeasts can cause both oxidation (C0 2 and H 20) and fermentation (alcohol and CO2) of the products. Acetobacter can use alcohol to produce acetic acid. Heterofermentative Lactobacillus fermentum and Leuconostoc mesenteroides can ferment carbohydrates to lactate, ethanol, acetate, CO2, diacetyl, and acetoin. In addition, Leu. mesenteroides and some strains of Lilb. plantarum can produce slime due to dextran production. In fruit drinks, Lactobacillus and Leuconostoc spp. can also convert citric and malic acids (additives) to lactic and acetic acids and reduce the sour taste (flat flavour). In concentrated fruit drinks and fruit preserves, due to low Aw (0.9), only osmophilic yeasts can grow; molds can also grow if oxygen is available. To prevent growth of these potential spoilage microorganisms, several additional preservation methods are used for these products; these include heat treatment, freezing, refrigeration, and addition of specific chemical preservatives. Tomato juice has a pH of about 4.3. It is generally given high heat treatment to kill vegetative microorganisms. However, bacterial spores can survive. Flat sour spoilage of tomato juice due to germination and growth of Bacillus coagulans has been documented. Most other vegetable juices have pH values between 5.0 and 5.8 and many have growth factors for lactic acid bacteria. These products are susceptible to spoilage due to the growth of many types of microorganisms. Effective preservation methods are used to control their growth.
8.5 SAUERKRAUT Sauerkraut is another food produced by a fermentation process. The raw material is shredded cabbage to which salt (2-3 %) is added to control the fermentation and to release sugars required by the lactic acid bacteria for growth. Incubation is at around 20°C and the lactic acid produced reduces the pH to 3.5 thus giving a preservation effect. The principal organisms involved are leuconostocs and lactobacilli which thrive in the near anaerobic conditions prevailing.
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Spoilage of the sauerkraut occurs as a result of incorrect processing conditions. Thus 'slimy kraut' is due to the growth of the wrong strains of lactobacilli as a result of too high an incubation temperature. Poor salt distribution may allow the growth of a variety of pectolytic and proteolytic organisms causing "rotted kraut" or, with high salt densities, Rhodotorula spp. causing "pink kraut." 8.6 WINE Wine is produced by the fermentation of the juice of crushed grapes known as 'must'. The fermentation can proceed normally, induced principally by the activities of a sequence of natural yeast populations. It is customary, however, to treat the musts with sulphur dioxide to suppress the natural flora and then inoculate a starter yeast culture thus giving greater control over the fermentation process. At the end of active fermentation the wine is transferred to storage tanks for ageing after which it is filtered and bottled or otherwise stored. The microorganisms causing wine spoilage are principally wild yeasts and bacteria although, as with beer, defects are frequently non-microbial in origin. Important spoilage yeasts include Candida and Pichia spp. which produce growth films on the surface of wines. Some yeasts may be desirable in certain wines but may cause spoilage in others where residual sugar is desired. Wine spoilage bacteria are principally acetobacters and lactic acid bacteria. The former produce ropiness and sourness as with beer whilst the latter, represented by the genera Lactobacillus, Leuconostoc and Pediococcus, produce lactic and acetic acids from sugars; these acids are usually accompanied by turbidity, off-flavours and possibly the evolution of carbon dioxide. 8.7 CONTROL OF MICROBIAL SPOILAGE
Many microorganisms gain access or contaminate plant material during harvesting or subsequent handling. Therefore it is desirable to use equipment that is as clean as possible and to minimize mechanical damage of plant material; many external organisms can be removed from fruits and vegetables by being washed in water although washing can reduce the storage life of vegetables if inadequately drained. A suitable storage environment is essential so that physiological and microbiological deterioration are minimized. Storage is normally under chill conditions (O-S°C) but certain commodities such as potatoes and cucumbers are best stored at 7-1O°C. The optimum
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MICROBIAL SPOILAGE OF FOODS
relative humidity is in the 85-95% range and storage life can be enhanced with, for example, apples and pears by controlled atmospheric storage (reduced oxygen and increased carbon dioxide concentrations). Sealed plastic films encourage high humidity within the pack and increased microbial spoilage can result. Perforated plastic films largely overcome this problem but humidity can be higher than with the unwrapped product. Finally, various chemicals can be used which involve either pre-harvest or post-harvest treatment. In the latter case common control measures include dipping or spraying with fungicides/bactericides such as borax (sodium tetraborate), sorbic acid, phenylphenates, diphenyl and iodophors, and fumigation with sulphur-containing dusts or 502. Some microbial spoilage of vegetables and their products are summarized in Table 8.1; similarly microbial spoilage of fruits and their products are mentioned in Table 8.2. Table 8.1 : Some Microbial Spoilage of Vegetables and their Products
Food Fresh vegetables
Spoilage Soft rot, mushy
Soft black rot Black mold rot Blue mold rot Beans, snap
Anthracnose Blight
Cabbage
Leaf spot Gray mold
Carrots
Soft rot Fungal rot Decay, wet rot
Celery
Fungal rot
Organism
Envinia carotovora, Pseudomonas fluorescens Alternaria, Rhizopus nigricans Aspergillus niger Penicillium Colletotrichum Xanthomonas Alternaria B. cinerea E. carotovora Rhizopus stolonifer Fusarium Sclerotinia sclerotiorum Rhizoctonia carotae Mucor contd ....
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111
... contd. Food Onions
Spoilage Pink rot
Organism
Neck rot
Botrytis allii Pseudomonas cepacia P. aeruginosa Aspergillus niger Corynebacterium Fusarium Candida, Pichia, Hanseniaspora, Kloeckera Alternaria, Aspergillus, Botrytis, Colletotrichum. Monilia, Penicillium, Rhizopus Xanthomonas Byssochlamys fulva
Rot Brown rot Black mold Potatoes
Ring rot
Tomatoes
Dryrot Ferment Fungal rot
Bacterial spot Soft rot
s. sclerotiorum
Canned vegetables Com, green beans, peas
Tomato
Bacillus stearothermophilus Desulfotomaculum nigrificans Sulfide stinker Putrid swell Clostridium sporogenes Hard swell Clostridium thermosaccharolyticum Flat-sour Bacillus coagulans Butyric fermentation Clostridium pasteurianum, C. butyricum Flat-sour
Fermented vegetables Brine
Film forming
Yeasts (Candida,
Debaryomyces, Hansenula, Kloeckera, Pichia, Rhodotorula, contd ....
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MICROBIAL SPOILAGE OF FOODS
... contd. Food Pickles
Sauerkraut Vegetable juice
Spoilage
Organism Sacchnromyces)
Bacillus Bacillus Black Soft, mushy, slimy B. subtilis Reduced acidity Yeasts Pink Rhodotorula Lactobacillus, Acetobacter Sour
Soft
Table 8.2 : Some Microbial Spoilage of Fruits and their Products Food Fresh fruit, general
Apples Apricots Bananas
Berries, general Strawberries Citrus fruits Dates Figs Guava Olives
Organisms Penicillium expansum Aspergillus niger Streptococcus faecalis Byssochlamys fluva, Penicillium Fermentation Torulopsis, Candida, Pichia Gray mold rot Botrytis cinerea Storage rot Colletotrichum, Gloeosporium Botryodiplodia, Erwinia sp. Black rot Alternaria Crown rot Colletotrichum, Fusarium Verticillium Fungal rot Botrytis cinerea, Mucor mucedo B. cinerea Gray mold rot Fermentation Kloeckera Soft rot Penicillium Black rot Alternaria Fermentation Saccharomyces, Candida Hanseniasopra, Torulopsis Gluconobacter Souring Anthracnose rot Colletotrichum Softenins (stem-end Rhodotorula 8,zutinis, contd ....
Spoilase Blue mold rot Black mold rot Souring, bitter flavour Soft rot
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113
... contd. Food
Spoilage
Organism
shrivel)
R. minuta, R. rubra,
Saccharomyces, Hansenula Sloughing of skin Klebsiella, Enterobacter, Escherichia, Aeromonas liquefaciens Peaches Monilinia fructicola Brown rot Sclerotinia Rhizopus stolonifer Decay Canned fruit Clostridium Butyric acid Byssochlamys fulva, Soft rot B. nivea Rhizopus stolonifer, Softening Apricots R. arrhizus Banana puree Gas Bacillus licheniformis Grapefruit sections Gas (CO) Lactobacillus brevis Fruit juice Lactobacillus Souring, CO2 Acetification, vinegar Acetobacter Moldy surface Penicillium Cloudy Non-fermenting yeasts Cloudy, alcohol Fermenting yeasts Buttermilk flavour Lactobacillus, Leuconostoc Jelly, jam, preserves Fungal Xeromyces bisporus Fermentation Osmophilic yeasts Acetobaeter, Gluconobaeter Wine Acetification Mousy odour, cloudy, Lactic acid bacteria slimy Acetaldehyde Saccharomyces oviformis S. beticus Flowers Candida vini
000
SPOILAGE OF CEREAL PRODUCTS
9.1 PROLOGUE The cereals, which all belong to the Gramineae or t.1-te grass family, are one of the most important sources of carbohydrates in the human diet. Some of the products susceptible to microbial spoilage include high-moisture cereal grains, refrigerated dough, breads, soft pastas and pastries
9.2 CEREAL GRAINS Grains normally have 10 to 12% moisture, which lowers the Aw to .$.0.6 and thus inhibits microbial growth. However, during harvesting, processing, and storage, if the Aw increases above 0.6, some molds can grow. Some species of storage fungi from genera Aspergillus, Penicillium, and Rhizopus can cause spoilage of high-moisture grains. The microbial flora of harvested cereal grains such as com, wheat and oats contains up to many millions of bacteria and moulds per gram. However, the low Aw of grains effectively inhibits the growth of all microorganisms provided storage conditions are satisfactory but in moist conditions mould growth is likely. Certain steps involved in flour manufacture reduce the microbial load and of these bleaching has the greatest effect. Mould counts remain fairly constant, in the low thousands per gram, in properly stored flours and the most commonly isolated species are members of the genera Penicillium, Aspergillus and Rhizopus. Bacteria decrease in numbers during storage and counts of < 1000 per g are usual, with Bacillus spp. the predominant group. Where the moisture
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115
content is above normal mould growth is likely and at still higher Aw levels growth of Bacillus spp. will occur. 9.3 BREAD AND CAKES Commercially produced bread should be of sufficiently low moisture content to inhibit growth of most microorganisms except moulds which are the principal spoilage agents; in fact, moulds are said to be responsible for the loss of 1% of annual bread production. Amongst the most common are Rhizopus nigricans, the 'bread mould' which produces characteristic black dots of sporangia, Penicillium and Aspergillus spp., which produce green conidia in abundance, and Neurospora sitophila, the 'red bread' mould. Mould spoilage is encouraged by slicing, wrapping the bread when too warm and storage in a warm, moist environment. Ropiness of bread, caused by Bacillus spp., is now rarely seen in commercially produced bread. It is initially characterized by brownish spots accompanied by an unpleasant odour and, later, disintegration of the crumb or slices follows; the spoilage is caused by hydrolysis of flour protein and starch which produces stickiness and stringiness in the bread. Control is best achieved by low temperature storage, the addition of preservative (e.g. calcium propionate or sorbic acid) and the use of good-quality flour. Moulds are responsible for most spoilage problems in cakes although the situation is complicated by the wide variety of ingredients that may be incorporated some of which, such a:; dairy and imitation creams, custard and chocolate, have been implicated in bacterial food poisoning incidents. Generally, mould growth is again controlled by low temperature storage and low Aw levels together with the use of preservatives as in bread. However, some materials used as fillings may have high Aw' which allows for bacterial growth. 9.4 REFRIGERATED DOUGH Refrigerated dough (for biscuits, roles, and pizza) are susceptible to spoilage (gas formation) from the growth of psychrotrophic heterolactic species of Lactobacillus and Leuconostoc. Rapid CO2 production can blow the containers, especially when the storage temperature increases to lOoC and above. 9.5
PASTAS
Pastas can be spoiled by microorganisms prior to drying due to improper manufacturing practices. Dry pastas do not favour
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MICROBIAL SPOILAGE OF FOODS
microbial growth. However, soft pastas can be spoiled by bacteria, yeasts, and molds. Anaerobic packing and refrigeration storage can prevent mold growth and slow down the growth of yeasts and anaerobic and facultative anaerobic psychrotrophic bacteria. Suitable preservatives can be used to prevent their growth.
9.6 LIQUID SWEETENERS AND CONFECTIONERIES Liquid sweeteners include honey, sugar syrups, maple syrups, corn syrups, and molasses. Confectionery products include softcentered fondant, cream, jellies, chocolate, and Turkish delight. Most of these products have an Aw of 0.9 or below and are normally not susceptible to bacterial spoilage. Under aerobic conditions, some xerophilic molds can produce visible spoilage. However, osmophilic yeasts from genera Zygosaccharomyces (Zyg. rouxil), Saccharomyces (Sac. cerevisiae), Torulopsis (Tor. holmil), and Candida (Can. valida) can ferment these products. To prevent growth of yeasts in some of these products with slightly higher Aw (such as in maple syrup), chemical preservatives are added.
9.7 BEER The main ingredients of beer are malted barley, hops and water. The malt is ground up with water to form a mash and the enzymes naturally present convert the starch into an easily fermentable sugar, maltose. This 'wort' is then boiled together with hops which are added principally for flavouring. After cooling, yeasts (Saccharomyces spp.) are added to the cooled wort to convert maltose to alcohol and carbon dioxide. The beer is subsequently matured in storage tanks at ODC for several weeks after which finishing processes are performed. Poor-quality beer is most commonly caused by non-microbial effects but only microbiological factors will be considered here. One of the most important is the need to maintain active, healthy yeasts with the required characteristics as the quality of the final product is markedly influenced by the yeast used. Microbial beer contamination, which occurs during or after the cooling of the wort, produces hazes, pellicles, acidification and undesirable flavours or consistency. Beer can be affected by different types of 'wild' yeast (often contaminants of the 'starter' yeast culture) which can cause cloudiness, flavour defects and pellicle formation. 'Ropiness' in beer, in which the liquid becomes viscous and pours as an 'oily' stream, is caused by Acetobacter, Pediococcus or Iilctobacillus spp. Acetobacters also cause sourness in beers
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SPOILAGE OF CEREAL PRODUCTS
by oxidizing ethyl alcohol to acetic acid. Qiacetyl (CH3COCOCHJ is produced by a pediococcus and this flavour defect of lager beers is characterized by honey-like odour and taste. Some microbial spoilage of cereals, cereal products and other carbohydrate foods are summarized in Table 9.1. Table 9.1 : Some Microbial Spoilage of Cereal Products and Carbohydrate Foods Food Beer
Spoilage Ropy Gas, slime Off-flavour
Turbid Fruity Haze, diacetyl Bread
Candy (chocolate cream) Cereals, grains Wheat Com Chocolate sauce Coconut Dough "refrigerated"
Ropy, slime Black mold Blue mold Pink mold Sour
Organism
Gluconobader Streptococcus lactis Lactobacillus, Pediococcus, Brettanomyces, Candida, Pichia Candida, Brettanomyces Candida Lactic acid bacteria, pediococci
Bacillus subtilis Rhizopus nigricans Penicillium Neurospora Lactic acid or coliform bacteria
Red Fermentation
Serratia marcescens
Moldy Discolora tion Pink Blue-eye Cloudy Rancidity
Aspergillus, Penicillium Rhizopus nigricans Erwinia rhapontici Penicillium martensii Xeromyces bisporus Micrococcus luteus, Bacillus subtilis Lactabacillus, Leuconostoc, Streptococcus
Gas, slime, sour
Yeasts
contd ....
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MICROBIAL SPOILAGE OF FOODS
... contd.
Food
Spoilage
Honey Molasses Peanuts
Fermented, yeasty Torulopsis, osmophilic yeasts Clostridium, osmophilic yeasts Gas, frothy Moldy Fusarium, Penicillium,
Soft drinks Sugar solutions
Turbidity Slime Ferment, yeasty
Organism
Aspergillus Yeasts
Leuconostoc mesenteroides Osmophilic yeasts
000
SPOILAGE OF CANNED FOODS
10.1 PROLOGUE
Traditionally, canning is a method of food preservation in which food is closed within hermetically sealed containers. Heat is applied to the containers in such a way as to destroy or inactivate microorganisms, their toxins and enzymes, thereby rendering the food free from possible spoilage or harmful effects. From a biological viewpoint the process may fail in one or both of two ways. The first is a post-process infection involving leakage of microorganisms inwards through faulty seams and the second is the survival of organisms as a result of inadequate heat treatment. A more recently introduced canning process, particularly for fluid foods, involves a high-temperature, short-time (HTST) heat treatment (e.g. 130°C for 30 s) combined with aseptic filling of pasteurized containers but the problems here are still fundamentally the same as in the more traditional method. Before making a more detailed review of the microbiological spoilage of canned foods it should be mentioned that spoilage may also be caused by chemical changes, the most important of which is the 'hydrogen swell'. This results from the reaction of the can metal (iron) with acidic foods when the hydrogen liberated causes the can to swell. The higher the acidity of the food, the greater the likelihood of this problem developing although suitable intemallacquers should largely eliminate this fault.
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MICROBIAL SPOILAGE OF FOODS
10.2 LEAKER SPOILAGE
A wide variety of microorganisms may be associated with spoilage following post-process seam leakage and the main source of these organisms is the water used for cooling the cans after processing. The microbiological quality of this water thus markedly influences the frequency of reinfection and total bacterial counts of the water must be less than 100 per ml. An interesting facet of this problem is that motile bacteria gain entry through seams more readily than non-motile forms. Adequate chlorination of cooling water is the means by which numbers of bacteria are reduced to an acceptable level.
+
(d)~~ "..
(e)
--
--+(f)
Fig. 10.1 : Steps in the manufacture of the conventional can: (a) formation and notching of metal sheet; (b) bending of notched ends; (c) formation of body by (d) interlocking and' compressing notched ends; (e) treatment of body with lining compound and solder; (f) flanging of body.
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121
Many other factors contribute to post-process reinfection and defective cans are amongst the most important. The basic steps in the construction of a conventional can are illustrated in Figs 10.1 and 10.2. The most likely point of entry for microorganisms is at the junction of the side seam with the double seams of the can lid or base; control of the seaming operation at these points is therefore very important. A further leakage point is the double seam itself (Fig. lO.3) and it is essential to ensure optimum seam thickness and overlap of cover and body hooks; the quality and amount of lining compound in the double seam area is also of great importance. A third possible although least likely entry point is directly through a small hole or cut in the metal from which the can was formed.
Fig. 10.2 : Steps in can closure after filling: (a) positioning of lid, (b) to (e) progressive operations in the formation of double seam.
In a detailed review of microbiologicalleaker spoilage Put et al. list certain guiding principles which, if adhered to, will ensure that canned foods remain sterile, wholesome and safe for the consumer. These principles are: (1) ensure that the construction of the double seam, the lap and the side seam are in accordance with accepted quality standards; (2) avoid rough handling of cans; (3) avoid excessive deformation of can
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MICROBIAL SPOILAGE OF FOODS
ends during sterilization and cooling due to sudden pressure changes; (4) correctly chlorinate cooling water to a residual level of 1-2 mg/litre of free chlorine measured in the water drained from the seam after the cooling operation. Also ensure that the cooling water conforms to the chemical and bacteriological standards laid down for drinking water; (5) wash and disinfect at frequent intervals all surfaces of mechanical handling equipment which might corne in contact with the double seam; (6) dry cans immediately after cooling and transport them on clean dry surfaces; (7) check cannery hygiene by regular microbiological surveys; (8) insist on and supervise high hygienic standards amongst employees. seam -'
t-thi kn I cess I J
I
I I
'J ,,~__-
body hook
can body
Fig. 10.3 : The finished double seam .
. In fact it has now been agreed that lead-soldered side seams should be phased out by 1985. In consequence there is an everincreasing proportion of cans with welded side seams which have a minimum overlap only. As a result, the junctions of the side seam of the can with the base and lid are far less bulky so that the chances of ba~teria gaining entry at these points are substantially reduced. Another change in can construction which is rightly gaining in popularity is that the base and side of the can are now being formed continuously from the same sheet of metal; this eliminates the need for side seams as well as the base-side junction so that seam leakage in such cans is only possible at the junction between the body and lid unless, of course, there are blemishes in the tin or aluminium plate. Where spoilage results from seam leakage the bacteria implicated usually have relatively low optimum growth temperatures
SPOILAGE OF CANNED FOODS
123
(25-35°C) and would be readily killed by the heat processing. In addition, more than one type of bacterium is usually isolated; these types commonly include members of the genera PseudonumilS, Alcaligenes and Flavobacterium, together with coliforms and micrococci. The above types are common contaminants where chlorination levels in the cooling water are minimal or non-existent; where chlorination is only slightly sub-standard, Bacillus and Clostridium spp. may be involved since their spores are highly resistant to chlorination. When it is remembered that nearly two-thirds of all forms of spoilage in canned foods are due to reinfection after heating, the importance of control measures, especially adequate chlorination of cooling water, cannot be overstressed.
10.3 SPOILAGE DUE TO INADEQUATE HEAT TREATMENT The aim of the heat processing is to destroy or inactivate microorganisms and their products, that is, to ensure canned foods are 'commercially sterile'. Such foods may not be sterile in the absolute sense of the term but any surviving spores or microorganisms are no longer capable of growth. The heat process necessary for commercial sterilization is determined to a considerable extent by the pH of the food. Above pH 4.5 Clostridium botulinum spores, which are markedly heat-resistant, can germinate and growth of vegetative cells with consequent toxin production is thus a distinct possibility. Foods with pH values above 4.5 are therefore given severe heat treatments at temperatures in the 110121°C range. Conversely foods with pH values of below 4.5 are given relatively mild heat treatments, temperatures in excess of lOO°C being usually unnecessary. The rate of killing bacterial spores (or vegetative cells for that matter, although they are much less heat-resistant) is a function of temperature and time; the higher the temperature, the greater the rate of destruction for any given time. Bacterial death is said to be logarithmic which means that equal percentages of surviving cells are killed in each successive unit of time. As can be seen in Fig. 10.4, the D value, represented by the slope of the survival curve is defined as the time at any given temperature for a 90 % reduction in viability to be effected (thus for the heat-resistant spore the D value is 10 min whilst for the heat-sensitive spore the value is less than I min). This means that the greater the number of spores that are present in any given food, the longer the time necessary for their destruction. From a practical standpoint it is therefore important to ensure that foods being canned
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MIawBIAL SPOILAGE OF FOODS
have a predictable number of spores in them which can be destroyed by the normal heat treatment applied; thus the microbiological quality of raw materials and process line sanitation standards must be carefully monitored.
5 10 15 20 Heating time at 110°C (min)
25 - -..30
Fig. 10.4 : Hypothetical survivor curves showing: (a) curves for heatresistant spore, and (b) curve for heat-sensitive spore.
10.3.1 Spoilage of Low Acid Foods (pH> 4.5) Foods in this category include canned meats, poultry, fish, vegetables, soups, baked beans, spaghetti and milk puddings, and spoilage of these foods is caused mainly by Bacillus and Clostridium spp. due to the heat resistance of their spores. B. stearothermophilus is the only species of Bacillus of commercial importance and is responsible for 'flat sour spoilage', i.e. it produces acids from carbohydrates to sour the food but no gas is formed so the ends of the can retain their normal shape. B. stearothermophilus is an obligate thermophile, all strains grow at 65°C and none below about 35°C. Extremely heat-resistant spores are produced which are some ten times more resistant than the spores of C. botulinum. Thus foods that are processed to destroy C. botulinum spores may well contain viable B. stearothermophilus spores. However, because of its high minimum growth temperature, cans held at temperatures below 35°C will not spoil even though the spores may be present. This is a good example of the
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125
concept of a commercially sterile pack. Canned foods susceptible to this type of spoilage include peas and similar vegetables which receive a fairly lengthy heat process. Although B. coagulans has also been implicated in flat sour spoilage, the spores of this organism are not very heat-resistant and it is of little importance nowadays. C. thermosaccharolyticum is another important thermophilic organism producing spores that are not as heat-resistant as those of B. stearothermophilus; it produces 'swell' or 'TA' (thermophilic anaerobe) spoilage in canned foods. Such spoilage is caused by the fermentation of carbohydrates to form acids and large quantities of gas, carbon dioxide and hydrogen; if sufficient pressure builds up, the ends of the can become distended and eventually the side 3eam may split and the can contents burst out. C. nigrificans, or more correctly Desulfotomaculum nigrificans, produces 'sulphur stinker' spoilage. D. nigrificans is essentially a Gramnegative obligate anaerobe producing spores with a heat resistance intermediate between B. stearothermophilus and C. thermosaccharolyticum spores. Hydrogen sulphide, produced by the breakdown of protein, is soluble in the product and reacts with the iron of the container to produce iron sulphide causing blackening of the food. As excess hydrogen sulphide is formed, the aroma of the characteristic spoilage is very unpleasant. Fortunately this type of spoilage is rare due to the extremely low incidence of the spores of the organism and to its high minimum growth temperature. Sulphur stinker spoilage has been , associated with both canned mushrooms and milk puddings in recent years. Control of thermophilic spore formers which survive the heat treatment is best achieved by ensuring that cans are cooled as rapidly as possible after processing. In particular, 'flat sour' thermophiles multiply very rapidly in the 50-70°C range and therefore failure to cool cans immediately after processing to a temperature of 35°C could allow considerable multiplication and lead to serious spoilage. Control is also aided by: (Ci) ingredient (sugar, starch, dried milk, etc.) selection to assure freedom from or only low numbers of thermophilic spores; (b) thorough cleaning of raw materials and (c) sound line sanitation. 10.3.2 Spoilage of High Acid Foods (pH < 4.5) Foods with pH values of <4.5 include tomatoes, pears, peaches, pineapples and other fruits and their juices, together with pickles and sauces. Microorganisms causing spoilage of these products are much
126
MICROBIAL SPOILAGE OF FOODS
more varied and a number of the more important ones and the spoilage effects they induce are outlined below. The thermophilic 'flat sour' organism B. coagulans can grow in pH values in the 4.0-4.5 range and is sometimes involved in the spoilage of tomatoes and tomato juice, usually as a result of seam leakage. B. coagulans spores are heat-sensitive and should be destroyed by the mild heat treatments used, although as an additional safety measure processing temperatures in excess of lOO°C have been recommended. Certain clostridia, particularly C. pasteurianum and C. butyricium, also grow well in this pH range a.'1d cause spoilage, with gas production, of canned tomatoes, pears and other fruits. The spores produced by these clostridia are even more heat labile and since they are destroyed in 15 min at lOO°C it is unlikely that processing is inadequate. Amongst the non-spore forming bacteria the lactic acid bacteria are most frequently incriminated in the spoilage of these high acid foods. Lactobacillus brevis commonly causes fermentation of tomato ketchup, Worcester sauce, pickles and salad dressings. Other lactobacilli and leuconostocs occasionally cause spoilage of a range of canned fruits and fruit juices. These lactic acid bacteria (Le. some Lactobacillus spp. and all Leuconostoc spp.) produce gas as well as acid from the syrup sugar so that spoilage is accompanied by can distension; other end products include acetic acid and ethyl alcohol so that with these diverse products such lactic acid bacteria are termed 'heterofermentative'. With 'homofermentative' lactic acid bacteria (Le. the remaining lactobacilli and all Streptococcus spp.) only lactic acid is produced by the fermentation of sugar. Yeasts are extremely heat-sensitive and are therefore rarely involved in spoilage of canned foods. Certain Torulopsis spp. may occasionally cause gaseous spoilage of sweetened condensed milk, which relies upon the high sugar content rather than upon a substantial heat treatment as the method of preservation. Other yeasts, Saccharomyces spp., have produced spoilage in citrus juices and pickles. Like yeasts, moulds rarely cause spoilage but there are two notable exceptions in Byssochlamys fulva and B. nivea the ascospores of which are unusually heat-resistant tolerating 85°C for 30 min: The foods attacked are mainly strawberries and raspberries which may totally disintegrate in spoilage due to the action of the pectolytic enzymes produced by the organisms. Control of this type of spoilage is best achieved by pre-treating the infected fruit with gaseous methyl bromide or peracetic acid and by careful cleaning of both the raw material and the processing equipment.
DOD
SPOILAGE OF FROZEN FOODS
11.1 PROLOGUE Freezing normally commences in foods at -1 to -3°C and as the temperature is further reduced more of the food becomes frozen. For meat and fish the water may only be completely frozen at -50 to 70°C although for fruit and vegetables the corresponding figures are 16 to -200C. Thus at temperatures slightly below O°C unfrozen water is available for the growth of microorganisms and growth down to -~C is possible for a few specialized bacteria and even down to -lOoC for certain moulds. As the temperature falls from O°C a series of eutectics (i.e. ice: solute mixtures) is formed which is accompanied by an increasing concentration of dissolved solids in the unfrozen water. These increasing solute concentrations progressively lower the a w and this has an increaSingly deleterious effect on the microbial population, thus organisms capable of growth in foods at sub-zero temperatures must also tolerate lowered a w values.
11.2 FACTORS AFFECTING VIABILITY OF MICROORGANISMS DURING FREEZING Although some microrganisms are killed by freezing, approximately 50% may survive although this figure is influenced by a number of factors including the type of organism, the rate of freezing and the composition of substrate being frozen. Bacterial spores are unaffected by freezing and, in general, Gram-positive rods and cocci are more resistant than Gram-negative bacteria. At conventional freezing rates varying from the 'slow' process
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MICROBIAL SPOILAGE OF FOODS
used in the home freezer unit to the' quick' processes used by the food industry it has long been established that viability of organisms is enhanced as the freezing rate increases [Fig. ILl, curve (a)]. This increase of survival is probably mainly due to the diminishing contact time of the susceptible organisms with harmful high solute concentrations in the unfrozen water. When freezing is more rapid, viability decreases probably due to the formation of internal ice crystals causing destruction of the cell membranes [curve (b)]. With extremely fast freezing rates, as, for example, when liquid nitrogen is used, ice crystal formation is reduced and is replaced by 'vitrification' [curve (c)]. When foods are frozen commercially the bacterial viabilities obtained will be predominantly as in curve (a). There are certain substances such as glucose, milk solids, fats and sodium glutamate which are known to be 'protective' and improved viabilities are obtained in their presence; the protective mechanism has not been elucidated. 80 (a)
(b)
(c)
°l~--------~l~O~--------l~OO~--------~l~~~------~l~O~OOO
Cooling rate ("C / min)
Fig. 11.1 : Effect of freezing on viability of typical gram negative rod.
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129
11.3 EFFECT OF COLD STORAGE Whilst the main losses in viability occur during initial freezing, further kill-off of bacteria occurs during frozen storage. Provided the storage temperature is low enough, death rates are minimal but at normal frozen food storage temperatures (-20°C) some loss in viability is evident, particularly in the early days of storage. Foods show a far greater reduction in viable counts when held at -5 to -lOoC than at20°C but whilst the higher storage temperatures may be an effective method of reducing counts they contribute to an increased rate of deterioration of the food resulting from other causes. Even when microbial growth is completely inhibited the product quality can still deteriorate due to the continued activity of released microbial enzymes or to the indigenous enzymes present in the food; in the case of vegetables these enzymes must be destroyed by blanching. Other harmful physico-chemical and biochemical changes can occur during freezing and cold storage. 11.4 FREEZING INJURY TO CELLS When bacteria are frozen and subsequently thawed three categories of cells can be distinguished: uninjured, injured and killed. Uninjured cells are capable of growth on minimal nutritive media or on the selective media normally used in their isolation; conversely, killed cells would be unable to grow on any medium. Injured cells are more demanding nutritionally whilst repair of the freezing-induced injury is being effected; they only grow on media which provide certain energy-requiring factors necessary for the repair of the injury. This repair is rapid being completed in under 2 h; it can also be performed by cells in the thawed-out food provided the required nutrients are available. This finding has important applications when using selective media for the enumeration of bacteria from frozen foods; recoveries may be substantially reduced, giving a false picture, if injury repair is precluded. Repair is best effected either by pre-incubating the samples in a complex medium or by allowing the food to thaw out for 2 h before enumerating bacteria on selective media. 11.5 THAWED FOODS AND THEIR SPOILAGE For reasons which are not clear, the rate of thawing influences the number of microorganisms surviving the freeze-thaw cycle, somewhat higher recoveries being obtained with faster thawing. The survivors start multiplying, as in the normal growth cycle, after a lag period but this period is extended by the inherently low temperature of
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the food so that the log phase of growth may take 3-6 h to become established. When frozen foods are allowed to defrost over a long period at, say, 3-lOoC psychrotrophs may dominate the flora and subsequently cause spoilage. In other cases the types of organisms growing will depend on the temperature at which the thawed food is held but with most foods the organisms predominating would be similar to those in the equivalent unfrozen product. Particular problems arise with larger packs, e.g. frozen turkeys, where a temperature gradient is established between the warm surface; and the cold interior. If such packs are defrosted at too high a temperature growth of bacteria on the surface could become quite rapid. In general, however, if thawing is reasonably fast and the food is used within a few hours no problems should arise. Within this time it is perfectly safe to refreeze thawed food, although to maintain textural, flavour and nutritional qualities it may be unwise. There are a number of cases where the spoilage flora of the thawed frozen food is different from that of the fresh counterpart and of these the spoilage of defrosted peas is a good example. During processing, leuconostocs and streptococci build up on the production lines and it is these organisms which dominate the spoilage flora; they attack the sugars (mainly sucrose) present in the peas with a consequent drop in pH accompanied by a yellow appearance. Other changes induced include the production of copious amounts of slime on the surface of the peas together with typical 'vinegary' or 'buttery' odours. The spoilage profiles in other frozen green vegetables are essentially similar.
DOD
INDICATORS OF MICROBIAL FOOD SPOILAGE
12.1 PROLOGUE Microorganisms are capable of causing food spoilage in two ways. The most important one is through the growth and active metabolism of food components by the live cells. The other one is produced, in the absence of live cells, by their extracelllular and intracellular enzymes that react with the food components and change their functional properties, leading to spoilage. The loss of food due to microbial spoilage has economic consequences for the producers, processors, and consumers. With the increase in population in the world, loss of food due to microbial (and non-microbial) spoilage means less food is available for the hungry mouth. To fight against world hunger, efforts should be directed not only to increase food production, but also to minimize spoilage so that enough food is available for consumption. Many preservation methods have been devised to reduce microbial spoilage. Under certain methods of preservation, both raw and partially processed (semi-preserved, perishable, and non-sterile) foods are susceptible to microbial spoilage. This is more evident in foods that are expected to have a long shelf life. To reduce loss of raw and partially processed foods due to microbial spoilage, two things are important. One is to predict how long a food, following production, will stay acceptable under the condition(s) of storage normally used for that food; i.e., what is its expected shelf life under normal conditions
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MICROBIAL SPOILAGE OF FOODS
of handling and storage? The other is to determine the current status, with respect to spoilage, of a food that has been stored for some t1.J.TIe. This information needs to be available well before a food has developed obvious detectable spoilage and thus is unacceptable. Many criteria have been evaluated to determine their efficiency as indicators to predict expected shelf life, as well as to estimate stages of microbial food spoilage. These criteria or indicators can be grouped as sensory, microbiological, and chemical (microbial metabolites). The sensory criteria (e.g., changes in colour, odour, flavour, texture, and general appearance) have several drawbacks as indicators, especially if used alone. Changes in texture and flavour generally appear at the advanced stages of spoilage. Odour changes can be masked by the spices used in many products. Odour changes from volatile metabolites may not be detected in a product that is exposed to air, as compared to the same product in a package. Colour changes, such as in meat exposed to air, may not be associated with microbial growth. Finally, individuals differ greatly in their perception for organoleptic criteria. However, sensory criteria can be used advantageously along with microbiological and/ or chemical criteria. Studies by a large number of researchers have clearly revealed that a single microbiological or chemical test is not effective in predicting either the shelf life of a product or its spoilage status. The contributing factors in microbial spoilage of a food include the type of product, its composition, methods used during processing, contamination during processing, nature of packaging, temperature and time of storage, and possible temperature abuse. As these factors differ with products, it may be rational to select indicator(s) on the basis of a product or a group of similar products. Some of the factors to be considered in selecting a microbial or chemical indicator(s) for a product (or several similar types of products) are: 1. In a good fresh product, it (or they) can be present in low numbers (microbial) or absent (chemical). 2. Under normal conditions of storage (temperature, time, packaging), it (or they) should increase (microbial or chemical) in quantity to reach a very high level. 3. When spoilage occurs under normal storage conditions, it (or they) should be the predominant causative agent(s) (microbial or chemical). 4. It (or they) can be detected rapidly (microbial or chemical).
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It (or they) can be used reliably to predict shelf life and spoilage status (microbial or chemical). 6. It (or they) should have a good relationship with the sensory criteria of spoilage of the particular product (microbial or chemical). Different microbial groups and their metabolites (chemicals) were evaluated for their suitability as indicators of food spoilage. As bacteria are the most predominant microbial group in food spoilage, the effectiveness of some bacteria and metabolites as indicators is briefly discussed. In addition, the effectiveness of testing microbial heat-stable enzymes in predicting the shelf life of products susceptible to spoilage by them is also discussed.
5.
12.2 MICROBIOLOGICAL CRITERIA Previous discussions indicated that spoilage microorganisms differ with the products or, more accurately, with the intrinsic and extrinsic environments of the products. It is rational to select the microorganism(s) predominantly involved in spoilage of a food (or a food group) as the indicator(s) of spoilage for that food. As an example, refrigerated ground meat during aerobic storage will normally be spoiled by the Gram-negative psychrotrophic aerobic rods, most importantly by the Pseudomonas spp. Thus the population level of the psychrotrophic Gram-negative rods should be the most appropriate indicator of spoilage for this product (or for raw meats stored under the same conditions), both for predicting the shelf life of this product and to estimate the status of spoilage during storage. Aerobic plate count (APC), which measures the mesophilic population, may not be a good indicator for this product since many mesophiles do not multiply at psychrotrophic temperature and, conversely, there are some psychrotrophic bacteria that do not multiply at 35°C in the 2 d used to enumerate APe in meats. However, APe (also standard plate count, Spc, for dairy products) has special importance in food microbiology. In fresh products, it indicates the effectiveness of sanitary procedures used during processing and handling of the product. A high APC or SPC in a food product such as hot dogs and pasteurized milk is viewed with suspicion, both for stability and safety. Thus it is a good idea to include APe or SPC along with the method suitable to detect the load of an appropriate indicator group for a food. Some of the specific microbial groups that can be used as indicators in different foorl" (or food types) are listed here.
MICROBIAL SPOILAGE OF FOODS
Refrigerated raw (fresh) meats stored aerobically: Enumeration of colony-forming units (cfu/g or cm2) of psychrotrophic aerobes, especially Gram-negative aerobes. The data may be available in 2 to 7 d depending upon the incubation temperature (10 to 25°C) and plating methods (pour or surface) used. 2. Refrigerated raw (fresh) meats stored anaerobically (vacuum-packaged): Enumeration of cfu/g or cm2 of psychrotrophic lactic acid bacteria (by plating in a suitable agar medium adjusted to pH 5.0) as well as psychrotrophic Enterobacteriaceae (in violet-red bile glucose agar medium). Depending upon incubation temperature, the data can be available in 2 to 7 d. The plates may be incubated in a CO2 environment. The products can also be tested for psychrotrophic Clostridium spp., such as Cia. laramie, using specific methods. 3. Refrigerated low heat-processed, vacuum-packaged meat products: Enumeration of cfu/ g or cm2 of psychrotrophic lactic acid bacteria (by plating in an agar medium adjusted to pH 5.0) as well as psychrotrophic Enterobacteriaceae (in violet-red bile glucose agar medium). Depending upon incubation temperature, the results can be available in 2 to 7 d. The plates may be incubated in a CO2 environment. The products can be tested for psychrotrophic Clostridium spp., such as Clo. laramie, using specific methods. 4. Raw milk: SPC, psychrotrophic Gram-negative rods, thermoduric bacteria. 5. Pasteurized milk: SPC, psychrotrophic bacteria (Gramnegative and Gram-positive). 6. Butter: Lipolytic microorganisms. 7. Cottage cheese: Psychrotrophic, especially Gram-negative rods. 8. Fishery products (raw): Psychrotrophic Gram-negative rods. 9. Beverages: Aciduric bacteria, yeasts, and molds. 10. Salad dressing and mayonnaise: Lactobacillus spp. (especially Lab. jructivorans) and yeasts. It is quite evident that several days are necessary before the population levels of the indicator microorganisms from enumeration of colony-forming units become-available. This is the major 1.
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disadvantage of the microbiological methods. To overcome this problem, several indirect methods that indicate the probable population of microorganisms in foods have been devised. One such method is the determination of lipopolysaccharides (LPS) present in a food. LPS is specifically found in the Gram-negative bacteria. Thus, by measuring LPS concentration, an estimate of the level of Gram-negativc bacteria in a food can be obtained. However, this method is not applicable for spoilage by Gram-positive bacteria. Several other indirect methods are measurement of ATP (ATP concentrations increase with high numbers of viable cells), impedance/conductivity (electric conductivity decreases with increase in cell numbers), and dye reduction time (the higher the population, the faster the reduction). However, each method has specific advantages and disadvantages. 12.3 CHEMICAL CRITERIA As microorganisms (particularly bacteria) grow in foods, they
produce many types of metabolic by-products associated with the spoilage characteristics. If a method is developed that is sensitive enough to measure a specific metabolite in very low concentrations and long before the spoilage becomes obvious, then the results can be used to determine the spoilage status of a food. Methods studied thus far to measure microbial metabolites include I-IzS production, NH3 production by colorimetric or titration methods, production of volatile reducing substances, CO2 production, diacetyl and acetoin production, indole production, and others. However, different metabolites are produced by different species-strains of bacteria and the results are not consistent; thus they cannot be used for different types of products. Change in food pH, especially in meat and meat products, due to microbial growth has also been used to determine the spoilage status of a food. In normal meats, with a pH of about 5.5, metabolism of amino acids by some spoilage bacteria generates NH3' amines, and other basic compounds. They will shift the pH to the basic side (as high as pH 8.0). In contrast, the metabolism of carbohydrates (present or added) by some bacteria produces acids and reduces the pH further to the acidic side. Thus measurement of pH of a stored meat product can also give some indication of its spoilage status. As the pH increases, the proteins become more hydrated, i.e., its water-holding capacity (WHC) increases and, when pressed, this meat will have less extract-release volume (ERV). In contrast, when the pH shifts toward the acid side, the WHC will be lower and ERV will be higher. However, many low-
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MICROBIAL SPOILAGE OF FOODS
fat products are formulated with high phosphate and generally have a pH close to 7.0 {to increase WHC).:me buffering action of phosphate may not allow the pH to shift to the basic or acid side from the microbial metabolism of amino acid and carbohydrates, respectively. In these products, pH measurement (or the measurement of WHC or ERV) may not be good indicators of their spoilage status. None of the microbiological and chemical criteria studied fulfil all the factors necessary for a good indicator that will indicate expected shelf life of a fresh product as well as its spoilage status during storage. More emphasis needs to be given to develop suitable indicators that reduce the loss of food due to microbial spoilage. 12.4 ASSAY OF HEAT-STABLE ENZYMES 12.4.1 Heat-Stable Proteinases in Milk Proteinases of some psychrotrophic bacteria, such as
Pseudomonas fluorescens strain B52, when present as low as 1 ng/ml raw milk, can reduce the acceptance quality of UHT-treated milk during normal storage. Because of this, it is very important that sensitive assay method{s) be used in their estimation to predict the shelf life of dairy products. Some of the earlier methods, such as UV absorbance, Folinciocalteu reagent reaction, and gel diffusion assay, are probably not sensitive enough for this purpose. Several new methods, such as the use of TNBS (trinitrobenzene sulfonic acid) and fluorescamine reagents are quite sensitive and being tested to assay proteinases in milk. In the TNBS method, the reagent reacts with free amino groups and, under the experimental conditions, develops colour that can be colourimetrically measured to determine the extent of free amino acids present due to proteolysis. Fluorescamine reacts with amino acids to form fluorescent compounds at pH 9.0 and thus can be fluorimetrically measured to determine protein hydrolysis. Other methods, such as enzyme-linked immunosorbent assay (ELISA) and luciferase inactivation assay are extremely sensitive methods and need further development before they can be used reliably. 12.4.2 Heat-Stable Lipases in Milk . Due to the presence of naturallipases in milk, the measurement of lipases produced specifically by the psychrotrophic bacteria creates some difficulties. However, it can be overcome by heating the milk, which destroys milk lipases but not the bacterial heat-stable lipases. Assay methods that measure release of free fatty acids (FFA) due to
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137
hydrolysis of milk fat by the lipases can be titrated to determine the potential of lipolysis of the lipases. As milk contains FF A naturally, this method may not be accurate. Methods in which esterases of chromogenic and fluorogenic compounds react with lipases to produce colour or fluorescent products have also been developed, but they have limitation. Recently, a rapid and sensitive sandwich ELISA method was tested to determine lipases of Pseudomonas spp. An antibody produced against a Psuedomonas fluorescens strain and linked to horseradish peroxidases reacted with lipases from many strains of Pseudomonas spp.
000
MICROBIOLOGICAL TESTING OF FOOD
13.1 PROLOGUE There may be a variety of reasons why it is necessary to examine foods qualitatively and/or quantitatively for microorganisms. The principal objectives of microbiological testing are to ensure: (1) that the food meets certain statutory standards; (2) that the food meets internal standards set by the processing company or external standards required by the purchaser; (3) that food materials entering the factory for processing are of the required standard and/or meet a standard agreed with a supplier; (4) that process control and line sanitation are being maintained. The microbiological test methods used to monitor food quality are themselves also varied and are largely dependent on the food being analysed. It is convenient to list tests under the following general headings: 1.
Estimations of total numbers of microorganisms.
,2.
Estimations of numbers of indicator organisms.
3.
Examining for, or estimating numbers of, food spoilage organisms or other selected groups.
4.
Examining for, or estimating numbers of, food poisoning ur foodborne pathogenic microorganisms.
MIOWBIOLOGICAL TESTING OF FOOD
5.
139
Examinations of the metabolic products of microorganisms; this often requires more sophisticated te.-:hniques only available in the larger laboratories.
13.2 SAMPLING
13.2.1. Sampling Rate One of the biggest problems associated with the microbiological testing of foods is the question of the number of samples that should be analysed from a batch or a day's production to ensure that the product or food material is of the required standard. Obviously the greater the number of samples taken, the greater the confidence placed in the results obtained but, since food used for analytical purposes represents a non-recoverable expense, a compromise must be achieved between the accuracy and the economics of testing. Sampling schemes in current use include testing five or ten samples per batch, or the square root of the number of packs per batch; however, it could be argued that such a high level of sampling is totally unjustified where consistently good results are obtained. Thus the available microbiological resources should be used prudently and sampling rates should be highest with foods that are known to be hazardous or which give erratic results; sampling rates should also be high for foods which have been subjected to a process change or after a process line failure. It should also be appreciated that the level of testing is affected by the test rationale; thus fewer tests are necessary where confirmation of a satisfactory standard is the object than where elimination of an unsatisfactory product is the aim. Any worthwhile sampling scheme should be statistically based and readers requiring information on such schemes are strongly recommended to consult Microorganisms in Foods, Vol. 2 [International Commission on Microbiological Specifications for Foods (lCMSF, 1974)] where much other pertinent information is also available.
13.2.2 The Representative Sample As defined by the aforementioned international commission (ICMSF, 1974), 'a representative sample is one whose condition is as similar as possible to that of the lot (or batch) from which it is drawn'. It is therefore necessary to avoid any form of bias and to ensure that
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MICROBIAL SPOILAGE OF FOODS
.. sufficient samples are taken. This is best achieved by some form of random sampling by the use of random number tables as recommended by the Commission (lCMSF, 1974). However, account should be taken of the possible build up of microorganisms on ,equipment, and hence in food, during a production run; a product may contain far fewer microorganisms at the start of a shift than towards the end and this should be allowed for in any sampling scheme. Alternatively, if equipment has been inadequately cleaned foods may remove large numbers of bacteria at the start of a production run. It may be advantageous, therefore, to remove products on a regular time basis; this method does not undermine random sampling and is frequently employed. Other difficulties in sampling can arise if the product has a heterogeneous consistency and more frequent sampling is required if the food is not homogeneous. At one extreme only a small sample of well-mixed milk would be required to give a representative sample whereas with some of today's multi-component products it may prove impossible to get the required quantity of each component into a single representative sample and therefore a separate analysis of each component may have to be made. Sampling must sometimes be deliberately biased when microorganisms are confined to specific zones of the food, e.g. foods such as meats, fish and fruits carry the majority of their organisms on the outer surfaces and these must therefore constitute the principal sampling regions.
13.2.3 Sampling Techniques Liquid foods present no problem and are normally sampled by means of sterile pipettes after thorough mixing. Solid foods often present problems for the sampler who uses a variety of techniques to overcome the difficulties. With larger food units such as poultry and fish the whole carcass may be rinsed by agitation in a sampling medium although it is recognized that only a part of the flora is recovered by such a treatment; higher counts are obtained if abrasives such as sterile sand are included in the rinse solution. Moistened cottonwool swabs have been widely used for many years for surface sampling and have the advantage of being easy to handle and, when used in conjunction with a metal template, of presenting a known surface area
MICROBIOLOGICAL TESTING OF FOOD
'141
for analysis; after swabbing, the head of the swab is broken off into diluent which is then shaken to release the organisms. Recoveries obtained by these swab techniques are poor due to the adherence of microorganisms to the food surface and to their retention in the swab but recoveries can be improved if the sampled area is re-swabbed with a second dry swab. Agar contact methods, in which a sterile agar-based medium is pressed against the surface to be sampled, give even lower counts than swabbing techniques but they are the simplest sampling methods to use since the medium can be incubated directly. . With meats, fish and poultry the highest counts are obtained with so-called 'destructive sampling'. Samples can be taken with presterilized cork borers which have the advantage of analysing a known surface ar~a in conjunction with a core sample. Scalpels and knives are also popular but with these instruments the surface area: weight ratio is impossible to control. Known areas can also be sampled by using a skin scraper together with a sterile metal cylinder into which the diluent is poured. Comminuted meats and particulate foods such as peas and flour are easier to handle as a known weight can be taken. Certain of the microbiological sampling techniques discussed above are also used for the examination of food processing equipment: viz. rinsing with a known volume of water or diluent, swabbing, possibly with template, agar contact methods.
13.2.4 Treatment of Sample A known weight of food sample (e.g. 10 or 25 g) is taken except where swabs or rinses have been used. The food is added to a suitable sterile diluent such as 1/4 strength Ringer's solution or 0.1% peptone water and then treated so as to release into the diluent microorganisms on or in the food. The treatment typically involves mechanical blending or 'stomaching'. In the latter technique, which is now very popular, the sample together with a known volume of diluent is put in a sterile plastic bag which is pounded by paddles inside the 'stomacher'. The volu:ne of diluent used is usually nine times the weight of sample (e.g. 25 g peas in 225 ml diluent) so that a 10-1 homogenate is prepared. From this, suitable dilutions can be prepared (10-2, 10-3, 10-4, etc.) depending upon the microbiological quality of the food or surface under test (Fig. 13.1).
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MICROBIAL SPOILAGE OF FOODS
1 ml Hr' d,lulIon transferred
I mIlO' dIlution
1 mllO~ dllulJon
stOlllilchcr llag
(and so on as reqUJred)
1
homogtmate (10- dllutlon) of (ood (25g) stomached In 225 ml diluent
Y mI dIluent + 1 mIlO" d,lulJon mIxed = leT' dIlutIon
9 tnl dIluent + 1 milO" dIlutIon mIxed:; Hr' dlluhon
1rnl + nutrIent ag'1r~
9 ml dIluent + 1 mllO-' dIlulJon mIxed I ()' dIlulJon
=
11 t 6/ ;;,.",' ""
ml + nutrient agar
1 ml + nutrient agar
dIlulJon + 10-15 ml molten nutnent agar mIxed by gently rotalIng plate ag.r then allowed to solIdIfy .nd plates Incubated at required temperature
Figure 13.1 : Schematic representation of 'pour plate' method used in the enumeration of bacteria.
13.3. MICROBIOLOGICAL TEST PROCEDURES IN COMMON USAGE 13.3.1 Total Viable Count One of the most common microbiological tests carried out on foods is the total viable count which is also known as the standard plate count or aerobic plate count. In this, suitable dilutions of the food sample are plated on or in agar-based media containing complex nutrients which support the growth of as wide a range of microorganisms as possible. Nutrients included in, for example, Nutrient Agar (Oxoid) are beef extract, yeast extract and peptone (a proteolytic enzyme digest of fresh meat containing a variety of inorganic salts, growth factors and peptides). The pH of the medium is usually adjusted to 7.0-7.4 so that bacteria rather than yeasts or moulds are recovered. The individual bacterial cells transferred to the plate in the diluent divide in the normal way during incubation. Thus an estimate of the total number of viable cells (i.e. cells capable of growth in the recovery medium) in the dilution plated out can be calculated by counting the total number of bacterial colonies which develop following incubation; clearly, under normal aerobic conditions obligate aerobes and facultative anaerobes will grow.
MICROBIOLOGICAL TESTING OF FOOD
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The incubation temperatures selected depend on the food being examined. Commonly used temperatures are 55°C for thermophiles, 3537°C for mesophiles and 20°C for many spoilage bacteria. Whilst the latter temperature is suitable for psychrotrophic bacteria as well as for many mesophiles, lower temperatures (e.g. 1-rC) are sometimes used for more accurate estimates of psychrotrophs; it must always be borne in mind that no one incubation temperature completely excludes all organisms from another group. Many plating techniques are employed for enumerating total numbers of viable bacteria and five are described briefly. Enumeration of colonies is traditionally performed manually using an illuminated colony counter with the operator counting each individual colony. This can be a tedious operation and, unless a suitable number of colonies has developed in the growth medium (ideally below 300, although for statistical reasons a minimum of 30 colonies is also required), it can also be inaccurate. In recent years automatic colony counting devices have been developed which enable accurate counts to be obtained in a few seconds.
13.3.1.1 The 'Pour Plate' Method In this method a set (or preferably duplicate sets) of Petri dishes is inoculated with 1 ml aliquots from appropriate dilutions of the food. Some 10-15 ml of melted nutrient agar, cooled to 45°C, is then added to each of the Petri dishes and mixed carefully with its aliquot. After the agar has solidified, the plates are incubated at the required temperature for a period of time depending upon the incubation conditions (e.g. 12 days at 3rC, 3-4 days at 20°C and 7-10 days at 5°C). After incubation, plates containing 30-300 colonies should be counted from which the number of viable cells per gram (or per cm2) of food can be readily calculated: if an average count of 112 colonies is obtained for the 10-4 dilution the total count per gram of food = 112 x 104 = 1.1 x 106.
13.3.1.2 The 'Spread Plate' Method Here the medium is pre-poured and allowed to solidify in the Petri dishes; 0.1 ml quantities of the dilutions are spread evenly over the whole surface of the medium using sterile L-shaped glass rods. Plates are incubated as above. Advantages of this technique are, first, that heat-sensitive cells (i.e. psychrotrophs) are not killed by the molten agar which may occur to an extent in the 'pour plate' method if the temperature of the agar is too high; second, all the colonies develop on the surface of the agar and can be easily observed and picked off if
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MICROBIAL SPOILAGE OF FOODS
necessary, whereas many colonies develop embedded in the agar in the 'pour plate' method, and these may be restricted in size and may be more difficult to subculture.
13.3.1.3 The 'Drop Plate' Method Solidified medium is again used with this technique. Specially calibrated pipettes, delivering 0.02 ml per drop are used and five separate drops (i.e. 0.1 ml) are delivered onto the surface of the plate, the drops being dried before incubation. Dilutions giving under 20 colonies per drop should be counted.
13.3.1.4 The 'Agar Droplet' Method Since the above methods are both time-consuming and expensive in materials, a more rapid technique using far smaller quantities of materials has been developed; this is the 'agar droplet' method. In this technique the dilutions are prepared in molten agar , and colonies develop in the solidified droplets (0.1 ml) during incubation. Counting of the colonies is facilitated by using a projection viewer which magnifies the droplets approximately ten-fold.
13.3.1.5 The 'Spiral Plate' Method Another semi-automatic method becoming more popular is the 'spiral plate' in which a machine continuously plates a known volume of sample on the surface of a rotating agar plate. The amount of sample deposited decreases as the dispensing stylus is moved from the centre to the perimeter of the rotating plate; thus the microbial colonies develop along a spiral track during incubation. Counting can be performed manually using a counting grid but laser-based automatic colony counters have been developed specifically for use with this technique. Comparisons with the more traditional methods have shown no significant differences in counts obtained. 13.3.2 Counting Using Electrical Impedance Measurements As microorganisms multiply in a growth medium minute changes in impedance occur which can be measured by passing a small electric current through the medium. At a particular concentration of microorganisms there is a marked change in impedance and clearly with higher initial numbers this threshold concentration is reached more rapidly. Thus estimates can be made of the numbers of microorganisms initially present in a food by recording the time taken (detection time) to reach the threshold using samples of the food diluted
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MICROBIOLOGICAL TESTING OF FOOD
in a growth medium. It has been shown that there is a good agreement between conventional viable COlmts and detection times. Furthermore, the times required for analysis are normally only about 5 h so that rapid quality assessments are made possible.
13.3.3 Counting by Measurement of Adenosine Triphosphate (ATP) A bioluminescent technique has been developed based on action involving ATP and the luciferase enzyme derived from fireflies. The reaction in much simplified form can be expressed as AT P + luciferase
~
light
The total amount of light produced is directly proportional to the quantity of ATP present and since all bacteria contain roughly the same amount of ATP per cell (approximately 10-15 g) it should be possible to measure the number of bacterial cells in any sample. Unforhmately, foods (e.g. meats) contain large amounts of non-microbial ATP and it is necessary to remove this ATP or separate the bacteria from the remainder of the food before bacterial ATP can be estimated. Simple separation techniques have been reported (Stannard & Wood, 1983) which enable results to be obtained in 20-25 min, the results correlating with the bacterial counts obtained using more conventional counting methods.
13.3.4 Counting Using the Direct Epifluorescent Filter Technique This technique, commonly abbreviated to DEFT, was originally developed for the rapid enumeration of bacteria in raw-milk but it has found wider use more recently. In this method, pre-filtered suspensions of the food (the pre-filtering is necessary with foods to remove debris after 'stomaching') are subsequently passed through a fine polycarbonate membrane filter. Bacteria retained on the surface of the filter are then stained with a fluorescent material, acridine orange, and enumerated by means of an epifluorescence microscope. The acridine orange absorbs energy from a special source of illumination provided on the microscope but the absorbed energy is lost immediately in the form of fluorescent light which is transmitted towards the eyepiece; thus individual cells fluorescing in the preparation can be counted readily by the viewer.
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MICROBIAL SPOILAGE OF FOODS
13.3.5 Direct Microscopic Count It may sometimes be necessary to obtain the total numbers of microorganisms (i.e. viable and non-viable cells) present in food samples as in, for example, canned foods or pasteurized milk. Smears of the food sample, possibly diluted, are prepared on glass microscope slides and stained with a suitable dye. The total number of microbial cells in a given number of microscopic fields is counted and from this the total number of organisms per gram of food can be roughly calculated.
13.3.6 Indicator Organisms The routine examination of foods for a wide range of pathogenic bacteria is impracticable in most laboratories either because they are inadequately equipped or because the sample size would be impractical to handle. Thus it has become normal practice to examine foods for bacteria whose presence indicates the possibility of food poisoning or other pathogenic bacteria being present. These bacteria are thus termed 'indicator organisms' and they are often regarded as being of great significance when assessing the microbiological safety and quality of foods. The principal bacteria employed as indicators are the coliforms, enterococci and, more recently, Enterobacteriaceae. Total viable counts at 37"C are sometimes a useful rough guide although their relationship with the safety of foods is a rather more tenuous one. 13.3.6.1 Coliforms
The principal coliform bacteria are Escherichia coli and Enterobacter aerogenes. The former is normally found in the gastrointestinal tract of man and other animals and is rarely found elsewhere whereas E. aerogenes is normally associated with vegetation and is only occasionally found in the intestine. In water testing E. coli is the classical indicator for the possible presence of enteric pathogens. Here there is a direct relationship between the numbers of E. coli present and the extent of faecal pollution, the higher the nU!Il.bers the greater the pollution; this is because the organism cannot multiply in water and, in fact, numbers slowly decline unless renewed pollution occurs. In food, the presence and concentration of E. coli is of less significance and the occurrence of this organism, even in large numbers, does not necessarily imply recent heavy faecal pollution. The numbers can be influenced by many factors such as actual growth in the food, poorly cleaned equipment and
MIrn.OBIOLOGlCAL TESllNG OF FOOD
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contamination from personnel. Thus all that can be concluded with many foods is that faecal contamination, indirect or direct, tested for the presence of indole. E. coli is virtually alone in being able to produce gas from lactose and indole from peptone at this elevated temperature so that positive reactions confirm E. coli. As an alternative to BGBB a solid medium (Eosin Methylene Blue agar) can be employed. Presumptive or confirmed coliforms from stages 1 and 2 are inoculated onto the surface of pre-poured plates of this medium which are then incubated at 37"C for 24 h; E. coli is distinguished by the metallic sheen of the blue-black colonies.
13.3.6.2 Enterococci As stated earlier, the enterococci comprise two species found in human and animal intestines, namely Streptococcus faecalis and S. faecium. The former is associated principally with the human intestinal canal whereas the latter is found in both man and animals. The enterococci are sometimes used as indicators of faecal pollution in water testing, one of the advantages being that they die out less rapidly than E. coli. However, a disadvantage of this group is that they are found more frequently than E. coli in non-faecal environments and hence their isolation is less conclusive evidence of faecal contamination. In foods it has often been argued that enterococci are a better indication of sanitary quality than E. coli; they are generally more resilient than coliforms, particularly in frozen and dried foods and in foods given a moderate heat treatment. However, this resilience further undermines their value as indicator organisms as their presence in, for example, heat-treated foods may be of little value if less-stable pathogens such as salmonellas have been killed by the process. Many techniques are available for the isolation and enumeration of enterococci but they usually rely on sodium azide as the selective agent and often employ a high incubation temperature (45°C). An example of a commonly used medium is KF Streptococcus agar (Difco) which, as well as the usual nutrient sources, also contains tetrazolium chloride, an ingredient which imparts a red colour to the colonies; incubation is at 37°C for 48 h. Alternatively, glucose azide broth incubated at 45°C may be used with assessments of numbers being made by the MPN technique in conjunction with probability tables. Tubes showing acid production are regarded as positive (N.B. enterococci do not produce gas from glucose.) Further identification to species or strain level is normally not required.
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MICROBIAL SPOILAGE OF FOODS
13.3.6.3 Enterobacteriaceae The family Enterobacteriaceae comprises many genera including those characterized by lactose fermentation (e.g. Escherichia and Enterobacter) and those not fermenting lactose (e.g. the nonenteropathogenic Proteus and Serratia as well as Salmonella and Shigella). Mossel et al. (1963) claimed that a close correlation existed between counts obtained for total numbers of the family Enterobacteriaceae and the extent of faecal pollution, particularly in relation to contamination by salmonellas; because many discrepancies were found when the more conventional tests for coliforms were employed, they suggested that a test for total Enterobacteriaceae would be more reliable. The discrepancies noted when coliform tests were used were: (1) the product may contain only Salmonella spp. and therefore falsely reassuring results may be obtained; (2) non-lactose fermenting strains of E. coli may occasionally predominate; (3) occasionally strains may fail to produce gas even though they dissimilate lactose; (4) coliforms are poorly defined anyway and on these grounds the new test was justified. The test procedure suggested by Mossel et al. (1963) involves enriching occurred at some stage and that the safety of the food is possibly in question. The detection and enumeration of E. coli are performed in three stages. The first stage, the presumptive coliform count, involves the enumeration of coliforms, both faecal and non-faecal, by use of selective media; these can be described briefly as nutrient media containing selective agents such as antibiotics, dyes or chemicals which suppress the growth of many organisms but allow the selected organisms, resistant to the agents, to grow. With coliforms bile salts are commonly incorporated in the culture media as the selective agent. Examples of selective media used for this purpose are MacConkey agar and Violet Red Bile agar. The former additionally contains a nutrient source (peptone), lactose, which is dissimilated by coliforms with the production of acid (and gas) and a pH indicator (neutral red); the latter medium contains crystal violet as an additional selective agent but is otherwise based on similar principles. Enumeration techniques include direct plating of dilutions of homogenized food samples on the above or similar media employing the 'pour' or 'spread' plate method; incubation is for 24 h at 37°C, coliforms then appearing as red colonies. Alternatively, dilutions can be added to MacConkey or similar broths such as lauryl sulphate tryptose broth which should contain inverted fermentation (Durham) tubes to collect gas produced by the breakdown of lactose by coliforms.
MIOWBIOLOGlCAL TESTING OF FOOD
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When broths are used in the enumeration of coliforms; etc. the Most Probable Number (MPN) technique is usually employed, in this, five tubes of broth are inoculated with each dilution. 'i'he presumptive MPN is based on the number of broths showing acid and gas production after 24-48 h incubation and is calculated using statistical tables. The second stage is to confirm the presence of coliform organisms although, in practice, this step is frequently missed out as the majority of presumptive coliforms are confirmed. This confirmation is made by subculturing all the broths showing acid and gas production or suspected colonies from agar into tubes of Brilliant Green Bile Broth (BGBB) which is then incubated at 37°C for 48 h. The inclusion of brilliant green renders BGBB more selective than the two media employed earlier and the production of gas in this medium confirms the presence of coliforms. The final stage is to confirm the presence of E. coli. This can be done directly from stage 1 using BGBB as above but with an incubation temperature of 440C for a 24 h period. At the same time a tube of peptone water is inoculated, again incubated at 44°C for 24 h and subsequently samples of homogenized food in BGBB (the rationale of the enriching technique is given in the next section); the BGBB used in this test is modified from that used in the confirmation of coliforms by the substitution of glucose for lactose since all Enterobacteriaceae members, by definition, dissimilate glucose with acid production. Incubation is for 24 h at 37°C and this is normally followed by plating out broths showing growth,(i.e. turbid) onto modified MacConkey or Violet Red Bile agar; again modification is by sugar substitution. All colonies developing with a characteristic red or purple appearance can be regarded as Enterobacteriaceae. It is important to reiterate that the presence of even substantial numbers of indicator organisms does not, in itself, indicate with certainty that direct faecal contamination of processed foods has occurred; it may equally well suggest inadequate processing, post-process contamination or insanitary processing condItions especially where foods have subsequently been stored at temperatures permitting microbial growth.
13.3.7 Food Poisoning Organisms Only the more important food poisoning bacteria will be considered in this section and test procedures for salm.onellas,
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MICROBIAL SPOILAGE OF FOODS
Clostridium perfringens and C. botulinum, Staphylococcus aureus, Bacillus cereus and Vibrio parahaemolyticus are briefly discussed in tum. 13.3.7.1 Salmonellas The isolation and identification of Salmonella spp. is a complex process involving a number of stages and enumeration is not normally attempted. After the sample of food (25 or 50g) has been taken it is blended as described previously (see Section 13.2.4) except that the process is normally performed in a pre-enrichment medium such as nutrient broth. The broth is then incubated at 37"C for a few hours in order to facilitate the recovery of injured salmonellas. The second stage involves the inoculation of a sample of the broth into an enrichment medium which encourages the growth of salmonellas but restricts or completely iru'Libits growth of competitive organisms such as coliforms. Selective enrichment is a general term used to indicate that one or a group of organisms is being allowed to grow ~hilst competitors are being inhibited. Two commonly used enrichment media for salmonellas are selenite broth and tetrathionate broth. The former contains sodium selenite as the inhibitory agent although the dissimilation of lactose, which is incorporated in the medium, by many of the enteric bacteria present causes a pH drop which favours salmonella growth. Tetrathionate broth contains a number of inhibitory agents including bile salts, brilliant green and tetrathionate which, in combination, permit the salmonellas to grow whilst effectively inhibiting most competitors. Incubation of these broths can be at either 37 or 43°C for 24 h. After enrichment the broths are usually plated out on selective media and both MacConkey agar and Violet Red Bile agar have been used for this purpose. However, media such as Brilliant Green MacConkey agar and Desoxycholate Citrate agar, both having more marked selective properties, are now g~nerally used; both these media contain a variety of selective agents, the usual nutrients and lactose plus a pH indicator. Salmonellas as non-lactose fermenters form green and colourless colonies, respectively, on these media after incubation at 37"C for 24 h and are thus readily distinguished from the red colonies produced by the lactose fermenters. Unfortunately many other organisms produce colonies indistinguishable from those of Salmonella spp. on these media and therefore further tests are necessary before isolation can be confirmed. Confirmation is made by picking off suspect colonies and, after checking for purity, reinoculating them into media to test for the dissimilation of further carbohydrates, production of
MICROBIOLOGICAL TEsTING OF FOOD
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hydrogen sulphide, decarboxylation of lysine and motility. Provided these screening tests give the correct pattern of results, the isolate may be regarded as a salmonella and a final confirmation is made on the basis of serological tests. Characterization to specific serotype is possible using appropriate H and 0 antisera although, in general, confirmation using polyvalent H and 0 antisera (representative of all the serotypes likely to be encountered) is sufficient.
13.3.7.2 Clostridium perfringens and C. botulinum Unlike salmonellas, the isolation of low numbers of C.
perfringens from food does not necessarily mean that a food poisoning danger exists. Only when large numbers are present is there a definite hazard and therefore enumeration techniques are essential with this organism. Pour or spread plate techniques are used with dilutions of food homogenates in conjunction with selective media. Many such media have been devised for C. perfringens but the rr.a.jority are agarbased, and contain suitable nutrients, indicator systems and selective agents. The ICMSF has carried out a comparative study on methods for the enumeration of C. perfringens in foods and concluded that Sulphite Cycloserine agar gives highest recoveries of this organism together with the lowest number of false positives. TItis medium contains the antibiotic cycloserine and, in addition, there is an indicator system that utilizes the fact that C. perfringens, like a number of other clostridia, reduces sulphite to sulphide giving black colonies in the presence of an iron salt. After anaerobic incubation of pour plates at 37°C for 24 h, suspect colonies are inoculated into a confirmatory medium checking for motility (c. perfringens is non-motile) and the ability to reduce nitrate to nitrite. An alternative confirmatory test is to plate out suspect colonies on an egg yolk agar to one half of which has been added C. perfringens antitoxin. After incubation, colonies which have developed in the half containing no antitoxin are surrounded by a zone of opacity (the Nagler reaction) whereas colonies in the other part show no change due to the specific neutralization of the reaction with the antitoxin. C. botulinum is not normally enumerated in foods and tests generally involve an examination of the food for botulinal toxins and the isolation of C. botulinum followed by toxin assays. Extreme care should be taken when examining suspect foods and it is essential to seek expert advice before contemplating such analyses. Where facilities are suitable, food homogenates can be examined directly for the presence of intact cells and spores using fluorescent staining methods.
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MICROBIAL SPOILAGE OF FOODS
Food samples, unheated and heated, should also be streaked out on Blood agar, preferably containing egg yolk so that the typical C. botulinum colony reaction (i.e. a zone of opacity around the colony and a 'pearly layer' on the surface) can be obtained after anaerobic incubation at 30°C for 3 days. Suspect colonies are then cultured in a cooked meat broth and the supernatant liquid is then tested for botulinum toxin after a suitable incubation period (N.B. toxin assay is sometimes performed on meat broths inoculated directly with suspect food samples). Finally, a toxin assay can be performed directly on extracts of the original food. Assays essentially involve the inoculation of the food extracts or culture supernatants into mice some of which have been protected with A, B or E antitoxin whilst the others remain unprotected. The inoculated mice are observed, over a number of days and if the unprotected mice die with typical symptoms but the mice protected with a specific antitoxin do not then the test is positive for that C. botulinum type.
13.3.7.3 Staphylococcus aureus Examining methods for S. aureus can be grouped into two distinct areas: first, the enumeration of staphylococci and, second, testing for the presence of the enterotoxin in the food. As only about 50 % of S. aureus strains are enterotoxin producers it will be appreciated that the latter test is more conclusive when investigating food poisoning outbreaks; furthermore, small numbers of S. aureus are commonly isolated from certain foods and this cannot be regarded as a danger. The range of agar-based selective media which have been used for the enumeration of S. aureus from foods is a particularly extensive one and, as with C. perfringens, the ICMSF has carried out a comparative study of some of those in common use. It was concluded that Baird-Parker agar performed most satisfactorily. This medium contains potassium tellurite and lithium chloride as inhibi tory agents and egg yolk emulsion as an indicator system. After 24-48 h incubation at 37°C S. au reus produces black colonies surrounded by a clear zone (2-5 mm wide) in the medium; within this zone there is a smaller opaque region which only develops in the later stages of incubation. These reactions are highly specific for S. aureus but a confirmatory test (coagulase reaction) must be made. The detection of enterotoxin from food extracts or from filtrates of cultures isolated from suspect foods normally involves serological procedures which are more sensitive and less expensive than the animal
MICROBIOLOGICAL TESTING OF FOOD
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tests previously used. As levels of enterotoxin in foods are extremely low, extraction and concentration methods have had to be employed; since such techniques are rather time-consuming, more rapid techniques have been introduced in recent years. Haemagglutination and radioimmunoassay methods give results within a few hours and, furthermore, require no concentration of the food extracts.
13.3.7.4 Bacillus cereus Only quantitative determination of B. cereus in suspect foods is necessary and large numbers must be isolated to be of any significance. The selective media used for enumerating this organism (e.g. Holbrook & Anderson, 1980) often include polymyxin since Bacillus spp, are largely unaffected by this antibiotic. An indicator system involving egg yolk and possibly mannitol in conjunction with a pH indicator is often incorporated; B. cereus can be presumptively identified by the opaque zone surrounding the colony after incubation at 37°C for 24 h, the zone being similar to that seen with S. aureus in egg yolk containing media. B. cereus does not dissimilate mannitol so that the colonies are pale with a purple colouration of the surrounding agar whilst mannitol fermenters produce yellow colonies.
13.3.7.5 Vibrio parahaemolyticus
V. parahaemolyticus is halophilic (tolerates 8.5 % NaCl) and thrives at a high pH (8.6) and these characteristics are utilized in selective media devised for the isolation and enumeration of this organism. One commonly used medium is Thiosulphate Citrate Bile Salt Sucrose agar which contains bile salts as additional selective agents. Two indicator systems are incorporated in the medium, one involving thiosulphate and ferric citrate (see Sulphite Cycloserine agar) and the other sucrose with pH indicator. Vibrios, unlike many enteric bacteria, fail to produce hydrogen sulphide whilst most V. parahaemolyticus strains are unusual amongst vibrios in not diSSimilating sucrose. Thus, after overnight incubation at 3~C, V. parahaemolyticus produces large dark green colonies whilst sucrosefermenting organisms form yellow colonies in the presence of bromothymol blue indicator. Further biochemical tests are necessary to confirm the identity of the isolates and, in particular, the Kanagawa test for potential pathogenicity must be performed. This involves plating out suspect cultures on an agar-based medium containing a 20% suspension of washed human blood cells which are haemolysed after
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MICROBIAL SPOILAGE OF FOODS
overnight incubation (Kanagawa +ve); this confirms the potential virulence of the culhlre. . 13.3.8 Food Spoilage Organisms Using standard nutrient media devoid of any selective ingredients it is possible to encourage the growth of particular groups of organisms by varying the incubation conditions. Incubation of such media at elevated tempera hIres (e.g. 55°C) will enable thermophiles to grow and such organisms may cause problems where foods are processed at high temperatures. Canned foods and foods processed at lower temperatures (50-90°C) are examples of foods with potential thermophile problems. By incubating plates anaerobically a further selective element can be introduced as only the obligately anaerobic and facultatively anaerobic thermophiles can now grow. Conversely, by incubating non-selective nutrient media at low temperatures psychrotrophs can be recovered. An incubation temperature of 5°C is recommended for most foods but a temperature of 7"C is used by the dairy industry as refrigerated milk is frequently exposed to temperatures of up to 7'c. Unfortunately because of the low incubation temperature, colonies take longer to develop and counting is delayed for 7 or more days. For this reason media are often incubated at 20°C which is ideal for most psychrotrophs, although the growth of many mesophiles at this temperature means that only a rough, although more rapid, indication of psychrotroph density is obtained. Obviously selective media could also be based on extremes of pH and salt level, and examples will be presented in the following sections of media commonly used for the detection and/or enumeration of the principal groups of spoilage bacteria.
13.3.8.1. Pseudomonas Masurovsky et al. (1963) described a medium specifically designed to detect and enumerate pseudomonads in foods. The medium contained a number of inorganic salts, arginine and yeast extract as nutrients and two antibiotics, erythromycin and chloramphenicol. The authors found that the Gram-positive bacteria tested were completely inhibited and only a few Gram-negative forms survived and these in low numbers. This medium has become popular but substitution of the original antibiotics by penicillin and streptomycin has increased its selectivity. Solberg et ai. (1972) developed a medium containing cetyl trimethyl ammonium bromide (Cetrimide) and hydroxytrichlorodiphenyloxide as selective agents which was equally effective
MICROBIOLOGICAL TESTING OF FOOD
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but allows the growth of certain coliforms as do the media described above. Grant & Holt (1977) described a new medium containing a number of selective agents to allow the almost exclusive growth of pseudomonads; the agents consist of the antibiotic nalidixic acid (suppresses enteric Gram- negative bacteria), nitrofurantoin and triphenyl tetrazolium crloride (suppress aerobic Gram- negative rods other than pseudomonads), the dye basic fuchsin (suppresses Grampositive bacteria) and cyclohexamide (suppresses fungi). With all the above media incubation should be for 2-3 days in the 20-30°C range; further biochemical tests may be necessary on selected colonies to confirm" them as pseudomonads and additional screening is necessary where specific identification is required. Alternatively, use of a non-selective medium incubated at 5°C in parallel with one of the selective media outlined above could be considered; high counts on both media are strongly indicative of an overwhelming predominance of Pseudomonas spp.
13.3.8.2 Micrococci The medium most commonly used for the isolation and enumeration of micrococci from foods is Mannitol Salt agar (Chapman, 1945). This medium contains 7-5% NaCl as a selective agent and suppresses the growth of all organisms with the exception of micrococci and staphylococci. To differentiate between them mannitol is incorporated in the mediwn with a pH indicator (phenol red), it being assumed that only staphylococci will attack this carbohydrate with acid production. Unfortunately this assumption is incorrect as there are a number of mannitol positive Micrococcus strains and mannitol negative staphylococci. Curry & Borovian (1976) devised a selective medium totally inhibiting the growth of staphylococci by the incorporation of furazolidone and this medium has been successfully used with foods for the detection of micrococci.
13.3.8.3 Lactobacilli and Leuconostocs As lactobacilli and leuconostocs have complex growth requirements, any medium used for their enumeration must contain a wide variety of nutrients. A suitable and widely used medium is Rogosa's Acetate agar which in addition to the nutrients contains Tween '80' (polyoxyethylene sorbitan mono-oleate) as a specific growth stimulant. Selectivity is achieved by the low pH (5.4) together with a 0.2m concentration of sodium acetate. There is no indicator system incorporated so that organisms capable of growing on the medium are presumptively identified as lactic acid bacteria. As lactobacilli, in
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particular, are microaerophilic, best growth is obtained by incubating inoculated plates in an atmosphere of 95% hydrogen and 5% carbon dioxide (5 days at 30°C). Preliminary screening of isolates involves Gram staining, catalase reaction (should be negative) and fermentation of carbohydrates (homofermentative or heterofermentative). The latter test is used for further identification to species level.
13.3.8.4 Streptococci Methods for the specific enumeration of enterococci have already been discussed (see Section 13.3.62) but streptococci have many different characteristics, and can be isolated from a variety of environments by various means. A medium that is frequently used for enumerating streptococci in foods is the Thallous Acetate Tetrazolium Glucose agar of Barnes (1956). Thallous acetate is included as a selective agent against various Gram-positive and Gram-negative bacteria whilst the triphenyl tetrazolium chloride LC; included as an indicator; this latter salt is reduced by certain streptococci giving characteristic red colonies. The lactic streptococci, so important in the dairy industry, can be enumerated on the ~-glycerophosphate medium of Terzaghi & Sandine (1975). This medium is again nutritionally complex to satisfy the requirements of the streptococci and the glycerophosphate is incorporated as a buffer to minimize the pH drop resulting from the formation of lactic acid by the dissimilation of lactose present in the medium. Incubation of the above media is at 30-37°C for 1 or 2 days.
13.3.8.5 Spore Formers Methods for the isolation of Clostridium botulinum, C. perJringens and Bacillus cereus have been described earlier but it may be necessary to enumerate the total numbers of spore formers present in foods. As a preliminary, samples or dilutions containing them are heat treated at 80°C for 10 min to destroy all vegetative cells and then cooled and plated. The heating encourages spores to germinate (heat shocking), a process that is often difficult to initiate without a suitable stimulus. Standard nutrient media are employed when enumerating Bacillus spp., the plates being incubated aerobically. Obviously members of this group present as vegetative forms in food do not figure in any enumeration of this type. Anaerobic incubation in a sealed jar is necessary for clostridia and this involves evacuation of the air and its replacement by hydrogen. The more extreme obligately anaerobic clostridia require the inclusion
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of a catalyst such as palladium in the jar; this converts an'f residual oxygen to water by combination with the hydrogen. Preliminary enrichment of the diluted sample in a cooked meat broth medium is sometimes practised but direct plating onto nutritionally complex solid media is preferable where enumeration is required. Differential Reinforced Clostridial medium is frequently employed and, although a liquid medium, has many advantages. Recoveries are higher than on solid media and anaerobic jars are unnecessary. Accurate counts are not possible but if decimal dilutions are inoculated figures with a range of a times ten factor are available (e.g. > 1000 but < 10000 per g). The medium contains a sulphur source/iron salt indicator system so that blackening of the medium indicates growtl~ of clostridia almost all of which produce hydrogen sulphide.
13.3.8.6 Yeasts and Moulds Yeasts and moulds have generally been cultivated on media with a low pH (3.5-5.5) and at a temperature of 20-30°C but many bacteria are able to grow under these conditions. To inhibit bacteria, 'broad-spectrum' antibiotics are now incorporated which suppress their growth. A typical medium is Oxytetracycline Glucose Yeast Extract agar which utilizes the antibiotic oxytetracycline and nutrients including glucose, and which is adjusted to a relatively high pH of 6.5. Assay methods for aflatoxins differ somewhat depending on the food. Generally the food is comminuted to reduce particle size and then extracted with a suitable solvent such as chloroform. The extract is purified and the next stage of analysis and detection involves some form of chromatography, usually thin layer chromatography. Using suitable solvents which clearly separate the toxins it is possible to compare any suspicious fluorescent spots under UV with appropriate controls. 13.3.9 Canned Foods Because the heat treatment of canned foods should destroy or inactivate all microorganisms, their presence in the food indicates a process failure. Thus microbiological examining techniques are restricted only to the isolation and identification of organisms. Media used fall into two main groups depending on the pH of the food. For low acid foods (pH> 4.5) the principal media are Dextrose Tryptone
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agar and Reinforced Clostridial medium while for high acid foods (pH < 4.5) Tomato Juice agar and Malt Extract agar are commonly used.
13.3.9.1 Dextrose Tryptone Agar This medium is used for the cultivation of 'flat-sour' thermophiles, particularly Bacillus stearothermophilus, and incubation is therefore at 55°C and aerobic. The organism produces acid from dextrose and a colour change from purple to yellow ensues in the presence of bromocresol purple indicator. This medium can also be used for the isolation of mesophiles and in this case incubation will he at 37"C.
13.3.9.2 Reinforced Clostridial Medium This medium is similar to the Differential Reinforced Clostriclial medium used in the enumeration of spore formers except that it is a solid medium (i.e. contains agar) and does not include the sulphur /iron salt indicator system.
13.3.9.3 Tomato Juice Agar This medium is used for the cultivation of lactobacilli, Bacillus coagulans and clostridia growing in low pH canned foods. The pH of the medium can be adjusted by the addition of lactic acid to correspond with that of the food under investigation.
13.3.9.4 Malt Extract Agar An example of a low pH medium (ca 4.5) used for the cultivation of yeasts and moulds from canned foods is Malt Extract agar. It is beyond the scope of this book to describe other essential procedures in the examination of canned foods. Readers requiring such information and further details of the media just described should consult appropriate literahlre in the Bibliography section. 13.3.10 Frozen and Dehydrated Foods When examining frozen or dehydrated foods the standard microbiological methods described in this chapter are employed. However, many of the cells may be metabolically injured and thus require an adequate thawing out or rehydration period during which repair may be effected; an alternative procedure is to preincubate food samples in nutritionally complex media. Failure to carry out one of these steps will result in reduced counts on selective media.
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13.3.11 Miscellaneous Tests 13.3.11.1 Methylene Blue Reduction Test [MBRT] This test, used in the examination of milk, is an example of a dye reduction test, the time taken for decolorization to occur being related to the number of organisms in the sample. This relationship is very approximate, however, as milk heavily contaminated with inert bacteria may give an extended reduction time whilst short reduction times may be due to natural reducing systems present in the milk. In the test 1 ml of a standard methylene blue solution is added to 10 ml of milk in it test-tube, mixed and then incubated in a water bath at 37°C. The milk and suitable controls are observed at 30 min intervals until decolorization of the milk is complete; the longer the time, the better the quality of the milk. Raw milk is considered satisfactory if it fails to decolorize methylene blue in 30 min. 13.3.11.2 The Limulus Lysate Test Gram-negative rods are important in the spoilage of proteinaceous foods and they produce lipopolysaccharide endotoxins which are actually a layer of their cell walls. Only minute (picogram) quantities of these endotoxins are required to gel a lysate protein obtained from the blood of the horseshoe crab (Limulus) and this reaction forms the basis of the test. Dilutions of the food sample are reacted with the lysate for 1 h at 3~C after which the end of a capillary tube is placed in the reaction mixture; the height to which the mixture is drawn up the tube determines whether or not gelling has occurred. This test has been developed particularly as an indicator of the microbiological quality of raw minced meats but it has also been found useful for the assessment of raw milk quality. With the former food it has been fOlmd that a close correlation exists between a positive test result and the numbers of bacteria present, the titre (i.e. highest dilution of food sample gelling lysate) increasing 1000-fold as the food spoils.
13.3.11.3 Microcalorimetry When microorganisms degrade food materials heat is produced, the amount depending on the numbers and types of organisms involved. By using temperature-sensitive instruments (mic>:ocalorimeters), the amount of heat evolved can be accurately measured. This technique has been used to identify canned foods undergoing spoilage during storage.
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13.3.12 Compilation of Specifications Employing the microbiological test methods described in this chapter it should be possible to maintain a measure of control of food processing operations. The application of these methods in food processing is discussed in later chapters but one aspect that can best be reviewed here is that of the compilation of specifications. Specifications are, in essence, microbiological standards that are agreed within or between companies and generally have no direct legal implications. Thus specifications can be prepared for raw materials supplied to a food processor, for foods at various stages of preparation and for final products. In the last case the microbiological standards may be those agreed as reasonable and attainable by the company or they may be standards imposed by or jointly agreed with an external agency. From a microbiological viewpoint and depending on the food, specifications may include standards for total numbers of microorganisms and or for food poisoning, indicator or spoilage organisms. When compiling specifications for raw materials and final products it is desirable to start with as wide a range of relevant test methods as is practicable so that comprehensive data on the background microbiology can be built up. However, from the outset it is essential to obtain agreement with the other companies involved on the best methods of sampling and analysis. With the plethora of techniques available it is no wonder that different laboratories have their preferred methods but the aim should be to use methods that give the highest recovery of organisms, that give reproducible results and that are easy to use. Ideally test samples should be exchanged between laboratories and analysed by the agreed methods to ensure that similar results are being obtained. As was mentioned at the beginning of this chapter, greater attention should be paid to raw materials and foods where erratic or unexpected results are obtained than to foods giving a consistent picture; attempts should be made to ascertain the reasons for these variations so that they may be eliminated. Where a clear pattern of results is difficult to establish, microbiological standards must be regarded as tentative in the short term although with food poisoning organisms additional constraints clearly apply. Specifications should reflect what is attainable under good manufacturing practice but should include tolerances to allow for sampling inaccuracies and for foods that are marginally outside the agreed standards. For example, a specification could include 'total viable count (30°C incubation)-not more than 5000 per g', but what about the occasional sample with a
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count of 5500, 6000 or even 8000 per g? To cover for these contingencies the specification might be better written thus: 'total viable count ... 5000 per g but counts of < 10 000 per g accepted in no more than 10 % of samples examined'. Such tolerances would not be permissible for salmonellas in, for example, frozen liquid whole egg; here the specification would include 'salmonella-absent from 50 g sample' and that requirement would be absolute. Perhaps before getting too enthusiastic about the need for a massive microbiological test regime for foods it would be as well to remind ourselves of the often quoted remark of the eminent bacteriologist, Sir Graham Wilson, who said "Bacteriologists are better employed in devising means to prevent or overcome contamination than in examining more and more samples."
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INFORMATION SOURCES ON INTERNET The Internet is a huge and valuable resource with many sites of interest to food microbiologists. Here we have listed a few, many of which will lead you on to other related sites. Food Safety Resources on the Internet: http://bc-ciphi.cnx.net/ food%205afety .html Centre for Food Safety & Applied Nutrition, USA: http://vm.cfsan.fda.gov I -particularly useful is the 'Bad Bug Book': http://vm.cfsan.fda.gov I -mow lintro .html Mycotoxicology Newsletter: an international forum for mycotoxins, especially useful for summaries of recent symposia: http://www.mycotoxicology.orgl mtn12c.html Ministry of Agriculture, Fisheries and Food, UK: http://www.maff.gov.uk/maffhome.htm Department of Health, UK: http://www.open.gov.uk/ dohl dhhome/htm Institute of Food Science and Technology, UK:
http://www.ifst.org/ -particularly the hot topics section: http://www.ifst.org/hottop.htm World Health Organisation (WHO), Geneva:
http://www.who.ch/
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-particularly the food safety programme: http://www. who.int/fsf/ Canadian Food Inspection Agency:
http://www.cfia-acia.agr.ca/ Foodnet, Canada:
http://foodnet.fic.ca/ National Centre for Food Safety and Technology (NCFST) is a consortium of industry, academia and government organized to address the complex issues raised by emerging food technologies: http://www.iit.edu/ -ncfsl CSIRO, Australia: http://www.dfst.csiro.au/ Australian Office of Food Safety:
http://www.dpie.gov.au/ocvo/ ofs.html
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