Food Microbiology: Principles into Practice

By Osman Erkmen and T. Faruk Bozoglu

This book covers application of food microbiology principles into food preservation and processing. Main aspects of the food preservation techniques, alternative food preservation techniques, role of microorganisms in food processing and their positive and negative features   are covered. Features subjects on mechanism of antimicrobial action of heat, thermal process, mechanisms for microbial control by low temperature, mechanism of food preservation, control of microorganisms and mycotoxin formation by reducing water activity, food preservation by additives and biocontrol, food preservation by modified atmosphere, alternative food processing techniques, and traditional fermented products processing. The book is designed for students in food engineering, health science, food science, agricultural engineering, food technology, nutrition and dietetic, biological sciences and biotechnology fields. It will also be valuable to researchers, teachers and practising food microbiologists as well as anyone interested in different branches of food.

• Language: English
• Publisher: Wiley
• Release date: Apr 13, 2016
• ISBN: 9781119237853

Osman Erkmen

Osman Erkmen is professor of food microbiology in the Department of Food Engineering, Gaziantep University. Gaziantep, Turkey. He started his career as a research assistant at the Department of Food Engineering in 1985 and later became an assistant professor in 1994 and associate professor of food microbiology in 1999. He has been working as a professor in this department since 2004, where he teaches courses in general microbiology, food microbiology, food sanitation, and food toxicology. His research focuses on the uses of nonthermal processes and natural antimicrobials in food preservation; the production of fermented foods; the microbial production of lycopene, thiamin, alcohol, and citric acid from industrial wastes; and microbial inactivation and modeling. He studies the combined effect of nonthermal processes, natural antimicrobials in the destruction of microbial cells and spores, and its application in food preservation, as well as characteristics of white and red wines production from Gaziantep grapes. Professor Erkmen has published over 150 research articles, reviews, book chapters, proceeding articles, and popular articles, edited two books, and authored three books in the ?elds of food microbiology, general microbiology, food toxicology and food sanitation, with more than 3,500 citations. He has more than 10 patents, organized more than 20 international scientific symposiums, and participated in more than 65 international symposiums.

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CONTENTS

Volume 1: Microorganisms Related to Foods, Foodborne Diseases, and Food Spoilage

Cover

Title Page

Copyright

About the Authors

Preface

Section I: Microbiology and Microbial Behavior in Foods

Chapter 1: History and Development of Food Microbiology

1.1 Introduction

1.2 History of Microorganisms in Foods

1.3 Fields of Food Microbiology

Chapter 2: Microbial Growth in Foods

2.1 Introduction

2.2 General Principles of Microbial Growth

Chapter 3: Types of Microorganisms in Foods

3.1 Introduction

3.2 Nomenclature of Microorganisms

3.3 Microorganisms in Foods

3.4 Microbial Genetics

3.5 Significance of Microorganisms in Foods

Section II: Microbial Sources and Factors Affecting Microorganisms

Chapter 4: Presources of Microorganisms in Foods

4.1 Introduction

4.2 Primary Sources of Microorganisms Present in Foods

Chapter 5: Factors Affecting Microbial Growth in Foods

5.1 Introduction

5.2 Intrinsic Factors

5.3 Extrinsic Factors

Section III: Foodborne Diseases

Chapter 6: Important Factors in Foodborne Diseases

6.1 Introduction

6.2 Important Facts in Foodborne Diseases

6.3 Immune Responses

Chapter 7: Bacterial Pathogenicity and Microbial Toxins

7.1 Introduction

7.2 Bacterial Pathogenicity

7.3 Bacterial Toxins

Chapter 8: Foodborne Invasive Infections

8.1 Introduction

8.2 Types of Foodborne Invasive Infection

Chapter 9: Foodborne Toxicoinfections

9.1 Introduction

9.2 Types of Foodborne Toxicoinfection

Chapter 10: Foodborne Intoxications

10.1 Introduction

10.2 Bacterial Foodborne Intoxication

10.3 Mycotoxins

10.4 Mushroom Toxins

10.5 Biogenic Amines

Chapter 11: Parasites, Marine Toxins, and Virus Food Poisonings

11.1 Introduction

11.2 Parasites

11.3 Marine Toxins

11.4 Chemical Poisoning

11.5 Foodborne Viruses and Prion

11.6 Food Allergy

Chapter 12: Indicators of Foodborne Pathogens

12.1 Introduction

12.2 Establishment of Microbiological Criteria

12.3 Indicators of Pathogens in Foods

Section IV: Detection of Microorganisms

Chapter 13: Conventional Techniques in Food Microbiology

13.1 Introduction

13.2 Sampling Plan and Sample Preparation

13.3 Conventional Microbial Counting Methods

Chapter 14: Advanced Techniques in Food Microbiology

14.1 Introduction

14.2 Developing Rapid Methods

14.3 Physical Method

14.4 Chemical Methods

14.5 Immunoassay Methods

14.6 Other Methods

14.7 Limitation of Rapid Methods

14.8 Future Developments in Rapid Methods

Section V: Microbial Food Spoilage

Chapter 15: Principles of Food Spoilage

15.1 Introduction

15.2 Food Spoilage

Chapter 16: Spoilage of Meat and Meat Products

16.1 Introduction

16.2 Meat and Meat Products

16.3 Poultry

Chapter 17: Spoilage of Eggs and Egg Products

17.1 Introduction

17.2 Microbial Contamination

17.3 Spoilage

17.4 Preservation of Eggs and Egg Products

Chapter 18: Spoilage of Fish and Other Seafoods

18.1 Introduction

18.2 Microbial Contamination

18.3 Spoilage

18.4 Preservation of Fish and Other Seafoods

Chapter 19: Spoilage of Milk and Milk Products

19.1 Introduction

19.2 Milk Composition and Microbial Contamination

19.3 Spoilage

19.4 Preservation of Milk and Milk Products

Chapter 20: Spoilage of Vegetables and Fruits

20.1 Introduction

20.2 Vegetables and Fruits Spoilage

20.3 Fruit Juice and Beverage Spoilage

20.4 Fermented Vegetables and Fruits Spoilage

Chapter 21: Spoilage of Cereals and Cereal Products

21.1 Introduction

21.2 Contamination

21.3 Spoilage

21.4 Control of Mold and Mycotoxin Contamination

Chapter 22: Spoilage of Canned Foods

22.1 Introduction

22.2 Canned Foods

22.3 Canned Food Spoilage

Chapter 23: Spoilage of Miscellaneous Foods

23.1 Introduction

23.2 Spoilage

Chapter 24: Enzymatic and Nonenzymatic Food Spoilage

24.1 Introduction

24.2 Spoilage

Chapter 25: Indicators of Food Spoilage

25.1 Introduction

25.2 Indicators of Food Spoilage

Chapter 26: Psychrotrophs, Thermophiles, and Radiation-Resistant Microorganisms

26.1 Introduction

26.2 Psychrotrophic Microorganisms

26.3 Thermophilic Microorganisms

26.4 Radiation-Resistant Microorganisms

References

Index

Volume 2: Microorganisms in Food Preservation and Processing

Cover

Title Page

Copyright

About the Authors

Preface

Section I: Food Preservation Techniques

Chapter 1: Principles in Food Preservation Techniques

1.1 Introduction

1.2 Food Preservation Principles

Chapter 2: Food Preservation by High Temperatures

2.1 Introduction

2.2 Mechanism of Antimicrobial Action of Heat

2.3 Factors Affecting Heat Resistance

2.4 Heat Treatment in Food Processing

2.5 Thermal Process

Chapter 3: Food Preservation by Low Temperatures

3.1 Introduction

3.2 Effects of Low Temperatures on Microorganisms

3.3 Mechanisms of Microbial Control

3.4 Factors Affecting Storage of Foods at Low Temperatures

3.5 Methods Used in Food Preservation

Chapter 4: Food Preservation by Reducing Water Activity

4.1 Introduction

4.2 Principles of Dehydration Process

4.3 Mechanism of Food Preservation

4.4 Factors Affecting Food Preservation by Reducing Water Activity

4.5 Methods to Reduce Water Activity of Foods

4.6 Control of Microorganisms and Mycotoxin Formation

4.7 Characteristics of Low-Moisture Foods

Chapter 5: Food Preservation by Additives and Biocontrol

5.1 Introduction

5.2 Properties of Preservatives

5.3 Factors Affecting Activity of Antimicrobial Agents

5.4 Types of Antimicrobial Agents Used in Foods

5.5 Naturally Occurring Antimicrobial Agents

5.6 Biocontrol (Biologically Based Preservation Systems)

Chapter 6: Food Preservation by Irradiation

6.1 Introduction

6.2 Characteristics of Radiations

6.3 Mechanisms of Microbial Inactivation by Irradiation

6.4 Factors Affecting Inactivation of Microorganisms by Irradiation

6.5 Application of Irradiation on Foods

6.6 Regulatory Status of Irradiation

Chapter 7: Food Preservation by Removal Methods

7.1 Introduction

7.2 Removal Methods

Chapter 8: Food Preservation by Modified Atmosphere

8.1 Introduction

8.2 Packaging

8.3 Packaging Methods in Food Preservation

8.4 Effects of Modified Atmosphere on Microorganisms and Foods

8.5 Factors Affecting Efficiency of Modified Atmosphere

8.6 Packaging Materials

8.7 Application of Modified Atmosphere in Food Preservation

8.8 Food Safety and Future Outlook

Chapter 9: Food Preservation by Combination of Techniques (Hurdle Technology)

9.1 Introduction

9.2 Hurdle Technology

9.3 Principles of Combined Preservation Methods

9.4 Mechanisms of Antimicrobial Effects of Hurdles

9.5 Application of Hurdle Technology in Food Preservation

9.6 Limitations and Requirements of Hurdle Technology

Section II: Alternative Food Preservation Techniques

Chapter 10: Kinetic Parameters in the Inactivation of Microorganisms

10.1 Kinetic Parameters

Chapter 11: Alternative Food Processing Techniques

11.1 Microwave Processing

11.2 Ohmic Heating

11.3 High-Pressure Processing

11.4 Pulsed Electric Fields

11.5 High-Voltage Arc Discharge

11.6 Pulsed Light Technology

11.7 Magnetic Fields

11.8 Ultrasound

11.9 Pulsed X-Rays

11.10 Ozone

11.11 Antimicrobial Edible Films

Section III: Role of Microorganisms in Food Processing

Chapter 12: Microbial Metabolism of Food Components

12.1 Introduction

12.2 Microbial Physiology and Metabolism

Chapter 13: Basic Principles of Food Fermentation

13.1 Introduction

13.2 Fermentation and Fermenting Microorganisms

13.3 Factors Affecting Fermentation

13.4 Benefits of Fermented Foods

Chapter 14: Fermented Dairy Products

14.1 Introduction

14.2 Nutritional Significance of Milk

14.3 Types of Microorganisms Used in Dairy Fermentation

14.4 Types of Fermented Dairy Products

Chapter 15: Fermented Meat Products

15.1 Introduction

15.2 Sausages

Chapter 16: Fermented Vegetables and Fruits

16.1 Introduction

16.2 Fermented Alcoholic Drinks

16.3 Products of Mixed Fermentations

Chapter 17: Fermented Cereal and Grain Products

17.1 Introduction

17.2 Fermented Products

Chapter 18: Starter Culture and Bacteriophage Problems

18.1 Introduction

18.2 Starter Culture

18.3 Bacteriophage Problems of Starter Cultures

Chapter 19: Probiotics and Prebiotics

19.1 Introduction

19.2 Abiotics, Prebiotics, and Probiotics

19.3 Probiotic Microorganisms

19.4 Probiotic Product Development

19.5 Functional Foods

Chapter 20: Microbial Food Ingredients and Enzyme Applications in Food Processing

20.1 Introduction

20.2 Microbial Food Ingredients

20.3 Microbial Enzymes in Food Processing

Appendix

Bibliography

Index

End User License Agreement

List of Tables

Table 2.1

Table 2.2

Table 3.1

Table 5.1

Table 5.2

Table 5.3

Table 5.4

Table 6.1

Table 6.2

Table 6.3

Table 7.1

Table 9.1

Table 10.1

Table 12.1

Table 16.1

Table 19.1

Table 19.2

Table 19.3

Table 19.4

Table 20.1

Table 20.2

Table 20.3

Table 20.4

Table 21.1

Table 21.2

Table 21.3

Table 22.1

Table 22.2

Table 22.3

Table 24.1

Table 25.1

Table 25.2

Table 1.1

Table 1.2

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 2.5

Table 4.1

Table 4.2

Table 5.1

Table 5.2

Table 5.3

Table 6.1

Table 6.2

Table 6.3

Table 8.1

Table 9.1

Table 9.2

Table 11.1

Table 11.2

Table 12.1

Table 12.2

Table 13.1

Table 13.2

Table 13.3

Table 14.1

Table 14.2

Table 14.3

Table 14.4

Table 14.5

Table 15.1

Table 15.2

Table 15.3

Table 16.1

Table 16.2

Table 16.3

Table 16.4

Table 17.1

Table 18.1

Table 18.2

Table 19.1

Table 19.2

Table 19.3

Table 19.4

Table 19.5

Table 20.1

Table 20.2

Table 20.3

Table A.1

List of Illustrations

Figure 2.1

Figure 2.2

Figure 3.1

Figure 4.1

Figure 5.1

Figure 5.2

Figure 5.3

Figure 10.1

Figure 13.1

Figure 13.2

Figure 16.1

Figure 1.1

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 4.1

Figure 4.2

Figure 6.1

Figure 6.2

Figure 6.3

Figure 8.1

Figure 9.1

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5

Figure 11.6

Figure 12.1

Figure 12.2

Figure 12.3

Figure 12.4

Figure 12.5

Figure 12.6

Figure 12.7

Figure 14.1

Figure 14.2

Figure 14.3

Figure 14.4

Figure 14.5

Figure 14.6

Figure 15.1

Figure 16.1

Figure 16.2

Figure 16.3

Figure 16.4

Figure 16.5

Figure 16.6

Figure 16.7

Figure 17.1

Figure 17.2

Figure 17.3

Figure 17.4

Figure 17.5

Figure 17.6

Figure 17.7

Figure 17.8

Figure 20.1

Figure 20.2

Figure 20.3

Figure 20.4

Food Microbiology

Principles into Practice

Volume 1: Microorganisms Related to Foods, Foodborne Diseases, and Food Spoilage

Osman Erkmen

Department of Food Engineering, University of Gaziantep, Turkey

T. Faruk Bozoglu

Department of Food Engineering, Middle East Technical University, Turkey

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Library of Congress Cataloging-in-Publication Data

Names: Erkmen, Osman, 1955-, author. | Bozoglu, T. Faruk, 1950- , author.

Title: Food microbiology : principles into practice / Osman Erkmen, T. Faruk Bozoglu.

Description: Chichester, West Sussex ; Hoboken, NJ : John Wiley & Sons, Inc., 2016. | Includes bibliographical references and index.

Identifiers: LCCN 2016005530 | ISBN 9781119237761 (cloth)

Subjects: | MESH: Food Microbiology | Foodborne Diseases

Classification: LCC RA1258 | NLM QW 85 | DDC 615.9/54—dc23 LC record available at http://lccn.loc.gov/2016005530

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Cover image: Getty/BlackJack3D

About the Authors

The photograph representing Osman Erkman.

Osman Erkmen

Born in 1955 in Konya, Turkey, Osman Erkmen is professor of food microbiology in the Department of Food Engineering under the University of Gaziantep (Gaziantep, Turkey) since 2004. He received his BS degree in Biology (1985) and MS degree in Food Microbiology (1987) from the Middle East Technical University (Ankara, Turkey). He did his PhD in General Microbiology from the Department of Microbiology under the University of Gaziantep in 1994. He started his career as a research assistant at the Department of Food Engineering in 1985 and later became assistant professor in 1994 and associate professor of Food Microbiology in 1999. Since 2004 he is working as professor in this department. At the Department of Food Engineering, he expanded his research to the use of nonthermal processes and natural antimicrobials in food preservation; in the production of fermented foods; in the microbial production of thiamin, alcohol, and citric acid from industrial wastes; and in the microbial inactivation kinetics and modeling. He received funding for research from the University of Gaziantep Foundation, the Scientific and Technological Research Council, and the Republic of Turkey State Planning Organization. He has been studying the combined effect of nonthermal processes and natural antimicrobials in the destruction of microbial cells and spores, its application in food preservation, and in the microbial production of lycopene from industrial wastes. He teaches courses in Food Microbiology, General Microbiology, Food Sanitation, and Food Toxicology.

Professor Erkmen has published over 100 research articles, reviews, book chapters, proceeding articles, and popular articles in the fields of Food Microbiology, Food Toxicology, Food Sanitation, and General Microbiology with more than 1500 citations. He is the editor of the book Gıda Mikrobiyolojisi (Food Microbiology) in Turkish language and is author of two books: A Laboratory Manual in General Microbiology and Basic Methods for the Microbiological Analysis of Foods.

T. Faruk Bozoglu

The photograph representing T. Faruk Bozoglu.

Born in 1950 at Ankara, Turkey, Professor Dr. T. Faruk Bozoglu received his BS degree in Chemistry (1973) and MS degree in Organic Chemistry (1977) from the Middle East Technical University (METU), Ankara, Turkey. He did his PhD in Food Microbiology from the Department of Food Science under the North Carolina State University, Raleigh, NC (1982). He joined the Department of Food Engineering at METU and is working as full-time Professor since 1992. He has carried out many collaborative researches with American and European Universities, especially on nonthermal processes. He has to his credit more than 60 SCI publications (BOZOGLU F* and BOZOGLU TF*) and more than 1100 citations. He is the advisor of 21 PhDs and more than 30 MS graduates. He has conducted two NATO ASI and participated in more than 70 international symposiums. He is also the chairman of METU Sport Club and Vice President of Turkish Dance Sports Federation.

Preface

This book deals with microorganisms affecting foods, foodborne diseases, and food safety, and it is intended as a reference source for academic institutions and food industry. A main characteristic of this book is that it is fundamental and comprehensive, not requiring any background knowledge of microbiology. Therefore, its usage is not bound to a particular time. It is hoped that the book will serve varied departments such as Food Engineering, Faculty of Health Science, Agricultural Engineering, Food Technology, and Nutrition and Dietetic Department, as well as anyone interested in different fields of food study. An enormous food industry exists, producing different food products ranging from milk, meat, eggs, and poultry to cereals. Therefore, many communities, including engineers, food producers, and people from other fields, deal with the relationships between microorganisms and food. Food safety and application of food standards greatly depend on the awareness of microorganisms in foods. Actually, this book aims to give food producers and other related people valuable information on this field and help them to gain new perspectives. Thus, it will be a valuable source informing the reader about the importance of microorganisms in food industry, protection of foods against microbial hazards, and solutions to problems such as foodborne diseases, food spoilage, and toxin formation. In addition, its readily comprehensible language and the concise explanation of concepts make this book all the more appropriate and useful for the people who have an interest in the field.

Due to the diverse relations between food materials and microorganisms, the authors have designed this volume primarily for students who lack in knowledge of microorganisms. Sections I and II concentrate on organism's habitats, their activities, and the factors that affect their growth and death. Section III focuses on foodborne diseases, the topic that is believed to be the most important as well as troublesome. Section IV presents the principles for the detection of unwanted microorganisms in food and their toxins. Finally, Section V covers food spoilage that occurs as a consequence of either microbial growth in food or the release of enzymes during their growth in the food environment. Numerous references have been recommended in this volume for those who are interested in having an in-depth knowledge of microbiology.Osman Erkmen and T. Faruk BozogluGaziantep, 2016

Section I

Microbiology and Microbial Behavior in Foods

There are microbiological, chemical, and physical hazards in foods. Microorganisms are living microscopic sized organisms and include bacteria, viruses, yeasts and molds (named together as fungi), algae, and protozoa. They play important roles in other living organisms and in ecosystems. Microorganisms have both desirable and undesirable roles in foods. The use of microorganisms in foods and their isolation involve use of specific methods. Some of the simplest techniques in use today in food microbiology have been developed over the last 300 years. Food microbiologists must understand the basic principles of microbiology, have knowledge of food systems, and be able to solve the microbiological problems that occur in complex food ecosystems. Different types and numbers of microorganisms in raw and processed foods are important with respect to foodborne diseases, food spoilage, and food bioprocesses. Microorganisms metabolize some food components to provide needed energy and cellular materials. This section presents discovery of microorganisms, food microbiology subjects, and microbial growth characteristics in foods.

History and Development of Food Microbiology

Microbial Growth in Foods

Types of Microorganisms in Foods

Chapter 1

History and Development of Food Microbiology

1.1 Introduction

Microbiology is the branch of biological science that deals with microorganisms and agents (prions, viroid, etc.) that are invisible to the naked eye. It helps to understand the smallest of all biological life. With time, the importance of microorganisms in human and animal diseases, soil fertility, plant diseases, fermentation, food spoilages, and foodborne diseases was recognized, and microbiology was developed as a specific discipline. Later, microbiology was divided into several subdisciplines, such as medical microbiology, mycology, soil microbiology, plant pathology, and food microbiology. Except for a few sterile foods, all foods contain one or more types of microorganisms. Some of them have desirable roles in food, such as in the production of fermented food, whereas others cause food spoilage and foodborne diseases. To study the role of microorganisms in food and to control them when necessary, it is important to isolate them in pure culture and indicate their morphological, physiological, biochemical, and genetic characteristics. Some of the simplest techniques in use today for these studies have been developed over the last 300 years.

The Earth is about 4.6 billion years old. The surface area of Earth was cooled, and oceans and atmosphere were formed about 3.8 billion years ago. The first living simplest cells from simple molecules evolved in the Earth's vest oceans between 3.8 and 3.5 billion years ago. This primitive life form on the Earth is known as the universal ancestor. The oldest known fossils from sedimentary rocks are prokaryotic cells, 3.5 billion years in age. They were found in Western Australia and South Africa. The nature of these fossils and the chemical composition of the rocks indicate that they have lithotrophic and fermentative modes of metabolism and they first evolved prokaryotic Archaea cells. Photosynthetic microorganisms known as cyanobacteria evolved about 3 billion years ago. Photosynthesis arose and oxygen was accumulated by the atmosphere. They were prokaryotic cells and lack from membrane-bound organelles (such as mitochondria, nucleus, and golgi apparatus). For approximately 2 billion years ago, prokaryotic cells were the only form of life on the Earth. The larger, more complicated eukaryotic cells (fungi) appeared much later, between 1.5 and 2.1 billion years ago. Sexual reproduction evolved about 1.2 billion years ago and this initiated a rapid increase in the evolution of organisms. Sexual reproduction from two parent organisms resulted in increasing of genetic variations and biological evolution.

1.2 History of Microorganisms in Foods

1.2.1 Early Development on Foods

During the last ice age, 10 000–20 000 BC, nomadic populations of humans used crops beside wild animals. The barley was flourished in Nile from around 18 000 BC. Around 8000 BC, as agriculture and animal husbandry, they were adopted by the early civilizations and food supply, especially agricultural products became available during the growing seasons. Preservation of foods became important for uniform supply of food around the year. The first animals to be domesticated were goats and sheep in Near East in about 9000 BC. The first evidence of beer manufacture has been traced to ancient Babylonian in 7000 BC. The first fermented milk has been used in diet between 6100 and 5800 BC in Anatolia after the cow was domesticated. Wines have been prepared by Assyrians in 3500 BC. Milk, butter, and cheese were used by the Egyptians as early as 3000 BC. Fermented sausages were prepared by the ancient Babylonians and Chine as far back as 1500 BC. By 3000 BC, the people of summer (now Iraq) had developed an agricultural economy and livestock breeding. They constructed irrigation canals. They could move their livestock during their migration and slaughtered when needed. Between 8000 and 1000 BC, many food preservation methods, such as drying, cooking, baking, smoking, salting, sugaring (with honey), low-temperature storage (in ice), storage without air (in pits), fermentation (with fruits, grains, and milk), pickling, and spicing, were used, probably mainly to reduce spoilage.

1.2.2 Discovery of Microorganisms

From the time of Renaissance period until the late nineteenth century, it was generally accepted that some life forms arose spontaneously from nonliving matter. Such spontaneous generation appeared to occur primarily in decaying matter. The spontaneous generation theory argued that animalcules (an older term for a microscopic life) could not generate by themselves (biogenesis), but they were present in different matters only through abiogenesis (spontaneous generation). Some scientific minds were curious to determine where do animalcules come from, they observed them in many different matters that were emanating. The earliest attempt in spontaneous generation from air and matter was proved by Francesco Redi. In 1668, he placed meat in several dishes, half of these were covered with gauze and an empty dish was served as controls. After several days, the uncovered meat dishes were covered with maggots, but neither the covered meat, nor the empty dishes had similar infestations. Thus, the spontaneous generation of maggots in spoiled meat resulted from the presence of flies in air (nonliving matter). John Turberville Needham (1745) boiled broth and then tightly sealed to exclude exterior air. When the containers were opened, they were found to be full of animalcule. After repeating the experiment with several other broths, Needham concluded that spontaneous generation actually did occur from nonliving matter.

In 1768, Lazzaro Spallanzani repeated the experiments of Needham and Redi, but removed air from the flask by vacuum. Days later, the unsealed bottle seemed with small living things. The sealed bottle showed no signs of life. He proved that spontaneous generation could not occur without air and the air was a source of contaminants but nonliving matter was not generating life. Thereby, he disproved Needham's theory. Anthonie van Leeuwenhoak (1676–1683) observed different types of animalcules under microscope up to 300x magnification. He observed them in saliva, rainwater, vinegar, and other materials. He sketched three morphological groups (cocci, bacilli, and spiral) and also described some to be motile. Francois Nicholoas Appert, in 1804, developed methods to preserve foods in sealed glass bottles (canning) by heat in boiling water. He credited to Spallanzani's research. Schulze (1830), Theodor Schwann (1838), and Schroeder (1854) passed air through a filter and they showed that bacteria failed to appear in boiled meat infusion even in the presence of air. They also credited to Spallanzani's research. In 1859, Louis Pasteur placed nutrient solutions in flasks that had necks bent into S-shaped curves. He then boiled the solution for a few minutes and allowed them to cool. Growth was not taking place in the contents of the flasks because dust and living things had been trapped on the walls of the curved necks. To prove his assumptions were correct, he simply broke the necks of the flask and then solutions became cloudy with the growth of organisms. He demonstrated that bacteria could grow only in the infusion that was contaminated from dust particles in air. He proved that bacteria were able to reproduce (biogenesis), the contamination come from life forms in the air and life could not originate by spontaneous generation (abiogenesis). John Tyndall, in 1870, also showed that boiled infusion could be stored in dust-free air in a box without microbial growth.

1.2.3 Development of Food Microbiology

In 1664, Robert Hook described the structure of molds. Theodor Schwann (in 1837) proved that yeast cells were responsible for the conversion of sugars to alcohol, a process they called alcoholic fermentation. In 1838, Ehrenberg introduced the term bacteria and has reported at least 16 bacterial species in four genera. In 1875, Ferdinand Cohn developed the preliminary classification system of bacteria. He also discovered that some bacteria produced spores. Louis Pasteur studied on milk souring (1857), causes of diseases (1862), and defects in wine (1866). He showed how to keep solutions sterile. Pasteur's discoveries led to the development of aseptic techniques to prevent contamination of microorganisms. He found that yeast ferments sugars to alcohol and bacteria can oxidize the alcohol to acetic acid. He demonstrated that all fermentations were due to the activities of specific yeasts and bacteria (1857). He reported that some fermentative microorganisms were anaerobic and could live only in the absence of oxygen, whereas others were able to grow either aerobically or anaerobically. In 1870, Pasteur placed heat preservation methods of foods on a scientific basis. He heated the wine (at 60 °C for 30 min) to destroy undesirable microorganisms, known as pasteurization. He developed an anthrax vaccine by using heat-treated (inactivated) bacterial cells. He later used vaccination to fowl cholera and anthrax, both diseases caused by bacteria. He also made many discoveries including food spoilage, food preservation, diseases, and immunity. Microbiology and food microbiology become sciences by the studies of Pasteur.

John Tyndall (1877) realized that some bacteria had the ability to form resistant structures known as spores. Through a series of boiling and cooling steps, he inactivated these structures. He first allowed spores to germinate (by incubation) and then killed the new cells that arose from spores. He repeated this experiment on three successive days. He produced sterile broths. This technique was given the name tyndallization in his honor.

Robert Koch (1890) isolated bacteria in pure cultures from diseased cattle with anthrax. He developed techniques of agar plating methods to isolate bacteria in pure cultures and staining methods for better microscopic observation of bacteria. He introduced germ theories (Koch's postulates) from his research including for criteria to identify the causative agent of disease.

The pathogen must be present in all diseased animals.

The pathogen can be isolated from diseased animal and grown in pure culture.

The pathogen from the pure culture must cause the disease when it is injected into a healthy animal.

The pathogen must be reisolated from the new diseased animal and shown to be the same symptoms as the originally inoculated pathogen.

Sergei N. Winogradsky (1907) and Martinus W. Beijerinck prepared the enrichment culture technique. Paul Ehrlich (1915) found that some chemical agents have the ability to inhibit or kill microorganisms without damaging the animals. Alexander Fleming (1928) recognized that some microorganisms exhibit antibiosis; they are able to produce natural compounds that inhibit the growth of competitors. He showed that the bacterium (Staphylococcus aureus) was inhibited by the mold (Penicillium notatum). Later, Howard Florey and Ernst Chain (1940) cultivated Penicillium and purified the first widely available antibiotic, penicillin G.

1.2.4 Modern Microbiology

The use of lenses and lens systems to increase the apparent size of an object is the most important fact in the development of microbiology as a true science. The Italian astronomer Galilei (1564–1642) was the first scientist to use a lens to magnify the image of a small object. The first microscope was constructed by a Dutch scientist Anthonie van Leeuwenhoek (1676) to examine different matters using microscope. He drew three bacterial shapes (rods, cocci, and spirals). These shapes are very good approximations of actual forms known today.

In 1838, Matthias Schleiden proposed that all plants are composed of cells. One year later, Theodor Schwann (1837) would extend this concept to animals and vegetables. He also proposed that tissues originate from cells. Rudolf Virchow (1843) indicated the idea of self-replication. This leads Virchow to purpose every cell from a cell. In time, the combined works of Schleiden, Schwann, and Virchow purposed the cell theory that says (1) all living things are composed of cells and (2) all cells arise from other cells. This theory is universally accepted today.

Since the 1940s, knowledge of microbiology has expanded with increasing advances in microscopy, biochemistry, and genetic research. In 1953, James D. Watson and Francis H.C. Crick defined the structure of the DNA molecule. In 1956, F. Jacob and E.L. Wollman discovered the circular structure of the bacterial chromosome. Two years later, M. Meselson and F. W. Stahl described the DNA replication. In 1970s, discoveries in microbiology led to the development of recombinant DNA technology and genetic engineering. In 1980s, phylogenetic tree of life (three domain system; Bacteria, Archae, and Eukaryote) was proposed from similarities and dissimilarities of nucleotides sequenced rRNA.

1.3 Fields of Food Microbiology

1.3.1 Importance of Microorganisms in Foods

In the early twentieth century, studies continued to understand the association and importance of microorganisms in foods. Sanitation was used in the food handling to reduce contamination by microorganisms. Specific methods were studied to prevent microbial growth as well as to destroy the spoilage and pathogenic microorganisms. Specific methods were developed for the isolation and identification of microorganisms. Beneficial bacteria used in food fermentation, especially dairy fermentation, were isolated and characterized. However, after the 1950s, food microbiology entered a new era. Basic information on the physiological, biochemical, and biological characteristics of microorganisms in foods (such as microbial interactions in food environments and microbial physiology, biochemistry, genetics, and immunology) has helped open new frontiers in food microbiology. Among these are food fermentation/probiotics, food spoilage, foodborne diseases, and food safety.

1.3.1.1 Foodborne Diseases

Many pathogenic microorganisms can contaminate foods during various stages of their handling, production, storage, serving, and consumption. Foodborne illness may result from consumption of water and foods in raw or cooked when they contain the pathogenic microorganisms or their toxins in sufficient quantity. Foodborne diseases cannot only be fatal, but they can also cause large economic losses. Foods of animal origin associate more with foodborne diseases than foods of plant origin. Mass production of foods, new processing technologies, storage of foods, changes in food consumption patterns, and the increase in imports of food from other countries have been increased the chances of higher number of outbreaks as well as the introduction of new pathogens. On the other hand, effective methods are developed to ensure the safety of consumers against foodborne diseases.

Foodborne diseases are attributed primarily to pathogenic bacteria, toxigenic molds, and enteric viruses and protozoa. Some of bacteria responsible for foodborne diseases are Aeromonas hydrophila, pathogenic Escherichia coli, Listeria monocytogenes, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, S. aureus, Yersinia enterocolitica, Salmonella, Shigella, and Vibrio. Some of toxigenic mold species present in the genera are Penicillium, Aspergillus, and Byssochlamys. Some of the viruses of concern in foods are hepatitis A virus, Norwalk virus, Norwalk-like virus, and rotavirus. Cryptosporidium parvum, Cyclospora cayetanensis, Giardia lamblia, and Toxoplasma gondii are some pathogenic parasites. Beside microorganisms, chemicals and natural toxins in foods can also cause foodborne diseases.

1.3.1.2 Food Spoilage

Spoilage is the unfitness of food for human consumption. Food may be spoiled by chemical and biological agents. Biological spoilage can result from the action of inherent enzymes, growth of microorganisms, invasion of insects, contamination with parasites, and presence of worms and the like. About one-fourth of the world's food supply is lost through action of microorganisms alone. Chemical spoilage results from purely chemical reactions, such as browning and oxidation reactions. The chance of food spoilage and association of new types of microorganisms have greatly increased due to new marketing trends, new processing techniques, extending shelf-life, and changes of temperature between production and consumption of foods. Many food materials are processed to destroy enzymes and microorganisms, thus prolong the keeping quality of foods for hours, days, months, or even years.

1.3.1.3 Food Bioprocessing

Microorganisms can play some positive role in food. They can be consumed in themselves as the edible fungi and algae. Many microorganisms are used to produce different kinds of fermented foods using raw materials from animal and plant sources. The main desirable microorganisms used in the production of fermented foods are lactic acid bacteria (LAB). LAB produce new product in milk, brined vegetables, many cereal products, and meats with added carbohydrate. Examples to such fermented foods are cheeses, yogurt, wine, beer, pickles, sauerkraut, and sausages. In addition to being more shelf stable, all fermented foods have aroma and flavor characteristics. In some instance, the vitamin content of the fermented food is increased along with increasing digestibility of the raw foods. Consumption of these foods has increased greatly over the last 10–15 years and is expected to increase still more in the future. Genetic recombination techniques are being used to obtain better fermentative microorganisms for new products and to improve quality of foods.

1.3.1.4 Food Biopreservation

Biopreservation refers to extending storage life and enhancing safety of foods using natural microflora, starter culture, and antimicrobials. In fermented foods, beneficial microorganisms can reduce pH and produce antimicrobial agents, such as H2O2, organic acids, and bacteriocins. These produce are shelf-stable foods. Many food ingredients including enzymes, pigments, aromatic and flavoring compounds, and so on, may be produced by natural or engineered microorganisms. Antimicrobial metabolites of microorganisms are being used in foods to control undesirable microorganisms. LAB have a major potential for use in biopreservation because they are safe to consume and produce desirable products.

1.3.1.5 Probiotic

Probiotic means for life and is the live microbial cell preparation with survival in the colon. Microorganisms contributing the health and balance of the intestinal tract are referred to as the friendly, beneficial, or good microorganisms. When they are ingested, they maintain a healthy of intestinal tract, and help fight illness and disease. Many beneficial bacteria survive in the gastrointestinal tract of humans. Probiotic microorganisms are usually of the genus Lactobacillus and Bifidobacterium.

1.3.1.6 Food Safety

Total quality management can be applied from farm to fork to control microorganisms, to prevent microbial growth, and to protect foods against contamination of spoilage and pathogenic microorganisms. Food safety can be provided by application of hazard analysis and critical control points (HACCP) in food production, processing, and preservation. Microbiological characteristics of foods, such as unprocessed and low-heat-processed ready-eat foods, can be indicated for product safety. Food safety legislation provides production of foods according to the standards. It is impossible to conduct microbiological studies for each food product to ensure safety and stability of food products. Mathematical models can be used to determine the influence of combinations of several parameters on microorganisms. Although they may not be accurate, they can provide first-hand information very rapidly, and be helpful to eliminate many of hazards. Information from mathematical models can then be used to conduct a traditional study that is feasible both experimentally and economically. They can be used to predict growth and inactivation of pathogenic and spoilage microoganisms in food products by studying microbial growth rate at different pH, aw, temperature, preservatives, and the other factors.

1.3.1.7 Microbial Physiology and Food Preservation

Microbial physiology is cell structure, growth factors, metabolism, and genetic composition of microorganisms. Physiological characteristics of microorganisms are studied through analysis of the cellular response to different environmental conditions. Microbial physiology performs a qualitative and/or quantitative characterization of certain microbial species, such as growth on different carbon, nitrogen, and energy sources. Clearly, microbial physiology is an important research field on microbial species and in all applied aspects of microbiology, such as food microbiology, industrial microbiology, environmental microbiology, and medical microbiology.

All food preservation techniques exert their effect by manipulating one or more intrinsic and extrinsic factors with slowing or stopping microbial growth and inactivating (killing) microorganisms. Where microbial growth is slowed, shelf life of food is extended and different microorganisms may predominate with changing the character of the spoilage. Similarly, where microorganisms are inactivated or killed, the shelf life will depend on types of microorganisms surviving in the inactivation treatment whether the product is subjected to any posttreatment contamination. Though, modification of one intrinsic or extrinsic factor can often achieve an acceptable degree of preservation, this often means that the product's qualities are changed in a dramatic way. For example, to preserve a food by acidification, it may be necessary to produce a very acidic product of possibly limited acceptability. More frequently though a number of factors are adjusted less severely to achieve the overall antimicrobial effect in what is known as the hurdle concept or multiple-barrier concept of food preservation. Each factor modifies the food's sensory and other properties. For example, the hurdles of low pH, ethanol content, dissolved CO2, and hop resins combine to restrict the range of microorganisms that can grow in spoil beer.

1.3.1.8 Microbiological Analysis of Foods

In food, microorganisms are present as mixed population. Studying the behavior of microorganisms in foods involves their isolation and enumeration. In the case of enumerating microorganisms, a food sample is generally diluted in a relatively inert liquid diluent that will not subject the microorganisms to osmotic and pH stress, and the dilutions are inoculated on to an appropriate solid or liquid medium and incubated. Several dilutions are usually inoculated in this way so that a detectable result or countable number of colonies is obtained. A reasonable count and the dilution can be related to the microbial number in the analyzed food. Identification of microorganisms can also involves isolating individual colonies (pure culture). There is no universal culture medium for counting and isolation of all microorganisms; whether a microbial growth will depend on the components of the medium and the incubation conditions (such as temperature and gaseous atmosphere). A microorganism can be counted or isolated using selective and/or differential media that favor the growth of a particular target microorganism and/or allow the presence of target microorganisms to be clearly distinguished from other microflora. Cultural techniques are simple and complex in the isolation and enumeration of microorganisms. But they can be labor-intensive, have high recurrent costs, and have long incubation time before a result is obtained. Waiting for a microorganism to grow and produce turbidity or a colony can typically take longer time such as up to 18 h.

1.3.1.9 Food Safety Management Systems

The International Organization for Standardization (ISO) 22000 contains a number of standards each focusing on different aspects of food safety management. ISO's food safety management standards help organizations to identify and control food safety hazards. ISO 22000 as an international standard specifies the requirements for a food safety management system that involves the following elements: interactive communication, system management, prerequisite programs, and HACCP in organizations, production, processing, transport, and distribution of food products. This type of certification is suited to businesses that require international recognition of their food safety management system. Business can apply a systematic way in order to produce and serve food with the minimum risk of hazards (such as microbiological risks, chemical risks, and others). The testing foods to indicate pathogens or other microorganisms of concern are necessary for controlling quality. Application of good practices during production of food is a more effective way of controlling quality. The introduction of Good Manufacturing Practices (GMP) provides a framework for the hygienic production of food rather than identifying problems by accepting or rejecting batches based on microbiological risks. GMP involves aspects, such as plant layout and design, and the control of operating procedures.

Microbiological risk analysis (MRA) on foods comprises three interrelated activities: risk assessment, risk communication, and risk management. MRA involves estimation of the level of risk. Microbiological risk assessment consists of the following four stages:

Hazard identification is the identification of pathogens that may be present in a particular food.

Hazard characterization is a qualitative/quantitative evaluation of the adverse effects of a risk including if possible the relationship between pathogen dose and effect (dose/response).

Exposure assessment is an estimation of the particular hazard based on food consumption patterns and incidence of the hazard.

Risk characterization is a qualitative/quantitative estimation of the probability and consequences of illness causing by the hazard.

MRA should contribute to food safety objective. It is a statement of the maximum frequency of occurrence or level of a hazard in a food considered acceptable for consumer protection and something that should be deliverable through the application of good hygiene practices and HACCP.

1.3.2 Food Microbiology Course

Food microbiology is a course to study the relationship of habitat; the food preservation; the occurrence of microorganisms in foods; the effect of environment on growth of microorganisms in foods; the food bioprocessing and biopreservation; the microbiology of food spoilage; the food manufacture; the foodborne diseases; the physical, chemical, and biological destruction of microorganisms in foods; the metabolic, physiological, and genetic characteristics of microorganisms; and the microbiological examination of foods and food safety.

New technologies used in the food production, processing, distribution, storage, and consumption introduce new problems. Food microbiology deals with these problems and tries to effectively solve them. Information from these researches helps to develop methods for rapid and effective detection of spoilage and pathogenic microorganisms, to improve microbiological quality of foods, to develop desirable microbial strains by recombinant DNA technology, to produce fermented foods of better quality, to develop thermostable enzymes for food processing, to develop methods to remove bacteria from food and equipment surfaces, and to develop methods for effective control of spoilage and pathogenic microorganisms in food.

An individual who has completed courses in food microbiology should gain knowledge in the following areas:

Determination of microbiological quality of foods.

Uses of food ingredients with appropriate techniques.

Knowing sources of microorganisms contaminating foods.

Determination of microbial types involved in food spoilage and foodborne disease.

Design correct procedures to control the spoilage and pathogenic microorganisms in food and food processing area.

Design methods to overcome the new problems.

Identify how new technologies adapt in food processing.

Effective use of desirable microorganisms in bioprocessing and biopreservation.

Understand microbiological problems of food products.

Application of food safety principles in food production.

Chapter 2

Microbial Growth in Foods

2.1 Introduction

Microbial growth (cell multiplication), except viruses, in raw and processed foods is important with respect to foodborne diseases, food spoilage, and food bioprocesses. Microorganisms utilize nutrients in foods to obtain energy, cellular components, and many end products. There are different types of factors influencing the microbial growth in foods. The factors influencing the microbial growth are helpful in designing methods to control spoilage or hazard and stimulate their growth (as in bioprocessing and detection). The log phase of microbial growth and many types of microbial lethality follow first-order kinetics. The doubling time, D value, and z value are used in kinetic constants base on the first-order kinetics. Microbial growth characteristics are presented in this chapter.

2.2 General Principles of Microbial Growth

2.2.1 Importance Being Small Size

Bacteria are very small, most approximately 0.2–4.0 μm in diameter. Surface area/volume (s/v) ratio for spherical bacteria (4πr²/(4/3πr³) = 3/r) is high compared to large microbial cells of similar shape. High s/v ratio accounts high rate of metabolism and growth. Because, the cell substances (such as genetic material, ribosome, enzymes, and others) are very close to the surface; therefore, no circulatory mechanism is needed to distribute the nutrients that are absorbed, and there is little or no cytoplasmic movement within cell. Small cells can also easily exchange nutrients and remove end-products out compared to the large cells (low s/v ratio). Despite these advantages, a high s/v ratio limits the size of bacteria to microscopic dimensions.

A newly divided cell has a higher surface s/v ratio. So, a young cell easily uses nutrients to obtain energy and synthesize cellular components. As the cell size increases, s/v ratio decreases, which adversely affects the transport of nutrients into and end-products out of the cell. Therefore, growth rate of large cells is low than small cells.

2.2.2 Microbial Reproduction

2.2.2.1 Reproduction of Fungi

Reproduction of Molds

The wide variety of fungi demonstrates many reproductive ways. In general, most molds reproduce by producing spores (about 7 trillion spores). Fungi typically follow a reproductive cycle that involves the production of sexual and asexual spores. Asexual spores are the products of mitotic division of a single parent cell. Asexual reproduction is accomplished through the formation and spreading of asexual spores. Asexual spores germinate into a hyphae structure under favorable conditions. Sexual spores are formed through a process involving fusion of the two parental nuclei followed by meiosis, a type of nuclear cell division that produces offspring with half the genetic material as the parents. This develops into hyphae and a mycelium that produce enormous numbers of sexual spores that repeat the reproductive cycle. Spore types provide important basis for classification of fungi. Molds grow on foods with cottony appearance, filamentation, and branching. During the life cycle of mold, the dispersed mold spores settle on a suitable substrate and send out germ tubes that elongate to hyphae. Through continuous growth and branching, an extensive mycelium (mass of hyphae) is produced.

An increase in the number (or mass) of vegetative cells of bacteria, yeasts, and molds is used to reflect growth for microorganisms. Most bacteria reproduce by binary division: one (parent) cell asexually divides into two new cells of equal size. In binary division, the cell initiates division by forming constriction on the cell surface, followed by formation of transverse-wall formation, and separating the cellular materials equally between two sides of cell. The division can occur in one or more planes depending on the species of bacteria.

Reproduction of Yeasts

A yeast (mother) cell can reproduce by binary fission or budding (asexually). It produces a bud on its cell surface. Bud separates out from the original cell and daughter cells are born. In binary division, yeast cells split into two equal cells. They can also reproduce sexually. Most yeasts reproduce asexually by budding and a few species by combination of fission and budding. True yeasts (Ascomycetes) reproduce sexually by ascospores. False yeasts, fungi imperfecti (Deuteromycetes), do not produce sexual spores, reproduce asexually, such as Candida, Rhodotorula, and Cryptococcus.

2.2.2.2 Reproduction of Viruses

Viruses cannot reproduce by themselves. Viruses are composed of nucleic acid (DNA or RNA) and protein (capsid). Bacterial virus (bacteriophage) attaches to specific receptors on the surface of specific bacterial cell. The DNA in the bacteriophage head passes through the cell wall and enters into the cytoplasm of the bacterium. Viral DNA follows two cycles in the bacterial cells: the lytic cycle and the lysogenic cycle. In the lysogenic cycle, the virus DNA combines with the bacterial chromosome. Once it has inserted itself, virus DNA is known as a prophage and the cell is called a lysogenic cell. The phage DNA is replicated when host DNA is replicated and after cell division, each daughter cells gets a copy of virus DNA. This may go on for many generations of cell division without causing any damage on lysogenic cell. Many of the bacterial toxins (such as diphtheria, botulism, and toxic shock syndrome) are coded by genes of prophage in the cell DNA. Bacterial cell that carries a prophage has the potential to release viral DNA at any time and enter to lytic cycle, this is called induction.

In lytic cycle, free phage DNA controls bacterial DNA and converts bacterial cell to bacteriophage-producing cell; the cell starts to synthesize viral particles and produces phage-specific nucleic acid and proteins. Phage-specific nucleic acids, enzymes, and proteins are synthesized. Assembly of phage particles results with formation of phages in bacterial cell. The enzyme endolysin is synthesized by bacterial DNA, this enzyme lyses the bacterial cell and cell releases new bacteriophages.

2.2.2.3 Reproduction of Bacteria

The growth rate and growth characteristics of a microbial population can be studied by counting cell numbers (plating methods) or indirectly detecting cell numbers (turbidity, dry weight, and the other methods). From microbial population at different times of growth, a growth curve is plotted using log10 cell number versus time. A growth curve in batch (fixed volume) culture has five growth phases (Figure 2.1): (1) lag phase, (2) logarithmic (exponential) phase, (3), (4) death phase, and (5) cryptic phase.

A graphical representation where long number of cell is plotted on the y-axis and time on the x-axis. A growth curve in batch ( fixed volume) culture has five growth phases: (1) lag phase, (2) logarithmic (exponential) phase, (3), (4) death phase, and (5) cryptic phase.

Figure 2.1 Microbial growth curve.

During lag phase, the population does not change. The cells try to adapt to their new environment by inducing enzyme synthesis for new substrate and initiating chromosome and plasmid replication. The cells assimilate nutrients. The length of the lag phase depends on the temperature, number of cells (high number of cells usually have shorter lag phase), and the physiological history of the microorganisms. If actively growing cells are inoculated into an identical fresh medium at the same temperature, the lag phase may not appear. Growth factors can be manipulated to extend the lag phase.

Following lag phase, the cell number starts to increase, first slowly (phase of positive acceleration). The cells in the population differ in metabolic rate, initially only some multiply and then almost all cells multiply. The cells divide very rapidly and are called log (exponential) phase. During the log phase of growth, microorganisms reproduce by binary division. In binary division, one cell divides into two cells, which divide into four cells, which divide into eight cells, and so on.

At the end of the log phase, the growth rate slows down (phase of negative acceleration) and then the cell numbers would be constant (stationary) due to nutrient shortage (nutrient depletion), accumulation of toxic products, and others; a few cells die and a few cells multiply. This keeps the living population stable and is called stationary phase.

At the end of the stationary phase, the number of death cell would be higher than dividing cells (accelerated death phase) due to effect of toxic compounds and exhaust of available nutrients. Then the growth phase is characterized with a rapid decrease in the population, this phase is called death phase. Speed of death depends on relative resistance of cells to toxic and unfavorable conditions.

After a long period of time, some cells may still remain viable due to use of death cell components as a nutrient. This survival is called cryptic growth. This is important for the determination of some microbiological criteria in food, especially in controlling spoilage and pathogenic microorganisms. In cryptic growth, some cells undergo involution and assume a variety of shapes, become long filamentous rods or branching, or some cells loose structures, such as cell wall (protoplast).

2.2.3 Growth and Death

2.2.3.1 Growth Kinetics

Food microbiology deals with four phases of microbial growth. Growth curve of a culture is obtained by plotting the number of cells on a log scale (log10 cell number) versus time. This plot represents the state of microbial populations rather than individual microbes. For the log (exponential) phase of growth, first-order reaction kinetics can be used to describe the change in cell numbers. This involves the use of doubling time to describe the rate of logarithmic growth. Doubling time or generation time (g) is related to classic kinetic constants, as shown in Table 2.1.

Table 2.1 First-order kinetics to describe exponential growth and inactivation of microbial population.

First-order microbial growth kinetic can be represented mathematically by the expression:

(2.1) equation

where dx/dt is the rate of change of biomass or numbers x with time t, and μ is the specific growth rate.

Integration of Equation 2.1 gives

(2.2) equation

or taking natural logarithms and rearranging:

(2.3) equation

where N0 (or x0) is the initial number of microbial population (colony forming unit (cfu) ml−1) at time t0 and N (or x) is the final number of microbial population (cfu ml−1) at time t.

The doubling or generation time (g) of a microorganism can be obtained by substituting x = 2x0 (or N = 2N0) in Equation 2.3:

(2.4) equation

An alternative way of representing exponential growth (N) in terms of the doubling time is

(2.5) equation

where duration time t/g is the number of generation (n).

Each microorganism divides at constant intervals during exponential phase. The time needed to divide a single cell is called generation time (or doubling time). All microbial species cannot divide at the same time or at the same rate. The generation time of a microbial species under different conditions provides valuable information for developing methods to preserve foods. In general, under optimum conditions of growth, bacteria have the shortest generation time, followed by yeasts and molds. Generation time of microorganisms in food systems is usually much longer than in a microbiological medium. The value of generation time for the same microbial species will change by changing the growth conditions. The generation time can be detected from the number of generations, n, that occurs in a particular time interval, t. The generation time can be calculated for changing growth conditions by the following formula:

(2.6) equation

where g is generation time (min), 0.3 is a constant (value of log10 2 and indicates doubling), t is the duration of study in minute.

During exponential growth, the growth rate, μ, is the reciprocal of the generation time. It is also the slope of the straight line obtained when the log number of cells is plotted against time:

(2.7) equation

The growth rate slows down at either side of optimum (to minimum and maximum) growth temperature until the growth stops. The growth temperature ranges and optimum growth temperature of a microorganism at the specific condition provide valuable information for its inhibition, reduction, or stimulation of growth in a food. The influence of different parameters on a food's microbial load can be illustrated by manipulating the equations in Table 2.1. For example, Equation 1a states that the number of organisms (N) at any time is directly proportional to the initial number of microorganisms (N0). Equation 3a can be used to determine how long initial number will take a microbial population to reach a level. Consider the case of ground meat manufactured with an N0 = 1.2 × 10⁴ cfu g−1. How long can it be held at 7 °C before reaching a level of 10⁸ cfu g−1 with μ = 0.025. According to Equation 3a, t = [2.3(log 10⁸/10⁴)]/0.025 = 165.6 h.

The relationship between g and μ is more obvious if Equation 2a is written using natural logs (such as ln (N/N0) = μΔt) and solve for the condition where t = g and N = 2N0. The solution for Equation 2a is g = 0.693/μ (Equation 4a). The growth rate constant, μ, is related to k by the equation μ = 0.693 k (Equation 5a). Both rate constants characterize populations in the exponential phase of microbial growth.

2.2.3.2 Death Kinetics

Inactivation of microorganisms is the main progress for the safety and high quality food production. The success of a food preservation technique depends on the mechanism of microbial inactivation. More information about the effects of factors on microorganisms would help to develop better food preservation processes. Microbial cell death mainly occurs with changing two facts: structural damage (such as disruption of cell membrane, DNA damages, ribosome alterations, and protein aggregations and denaturation) and physiological disorders (such as membrane-selective permeability disorder and lack of function of key enzymes that contribute vital reactions in and out of the microbial cell).

Death rate kinetics of microorganisms explain effects and optimization of process factors. Different microorganisms have different resistances to process conditions (such as high temperature), in that vegetative cells are generally the most susceptible while endospores are much more resistant, with viruses between these two extremes. The food matrix that surrounds the microorganisms also has an extremely large influence, especially its pH, water activity (aw), and concentration and type of components. Kinetic parameters are used for different food processing conditions in order to ensure safety and quality of foods. Basics of kinetics in the microbial inactivation depend on survival of microorganisms after treatment in foods. Microorganisms are more susceptible to processing conditions at the log and death phases. Microbial cell death is caused by heat, pressure, radiation, acid, chemicals, bacteriocins, and others. But some processes can also be used to support growth of microbial cells, such as in the case of fermented food products.

Kinetic models can be used for describing changes in microbial population based on linear reduction of microbial cells or spore numbers. For example, an initial number (N0) of population is reduced to final number (N) of population after a process time t at a constant process temperature. The plot of log (N) to times gives linear survival curve (Figure 2.2). Survival curve follows first-order decrease in the number of microorganisms or spores and provides data on the rate of destruction in specific conditions. Inactivation model assumes that all of the cells or spores in a population have identical resistance to processing conditions. Shoulders and tails that are deviations from the linear declines in the log numbers can also frequently occur in survival curve. Shoulders can be defined as a lag period, during which microbial population remains constant.

A graphical representation where log(N/N0) is plotted on the y-axis on a scale of 0–9 and time(h) on the x-axis on a scale of 0–9. A straight declined line depicting survival of microorganisms at a constant temperature.

Figure 2.2 Survival of microorganisms at a constant temperature.

The rate of death of population in the first-order decrease is directly proportional to the number of microorganisms in process. The result of first-order kinetics means that there is a definite time during which the number of microorganisms falls to one-tenth. After one time interval, the number of microorganisms will have fallen to one-tenth of the original number, after a second time interval, which is one-hundredth of the original value, and so on (Figure 2.2). Thermal death of populations depending on time of exposure at a constant temperature can be expressed mathematically in terms of number of viable microorganisms or spores by the equation:

(2.8) equation

where dN/dt is the rate of death of microorganisms or spores, N is the number of viable cells, k is the inactivation rate constant, t is the time, and the minus sign (−) signifies that N is decreasing. Integrating this equation between limits, numbers of N1 at time t1 and numbers of N2 at time t2, gives:

(2.9) equation

At time t (a processing time), this equation may be written as follows:

(2.10) equation

This equation can be changed in a logarithmic expression as follows:

(2.11)

equation

where N is the final number of viable cells or spores as colony forming unit (cfu ml−1 or g−1) at time t (min), N0 is the initial number of viable cells or spores (cfu ml−1 or g−1) at time 0, k is the inactivation rate constant (min−1), and log10 (N/N0) is the number of log cycle reduction in microorganism or spore numbers in time interval. If N/N0 ratio is taken as survival ratios (S), the equation