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- Sprache: Englisch
Microbes in the Food Industry This newest volume in the groundbreaking new series, "Bioprocessing in Food Science," focuses on the latest processes, industrial applications, and leading research on microbes in the food industry, for engineers, scientists, students, and other industry professionals. Microbes in the Food Industry, the latest volume in the series, "Bioprocessing in Food Science," is focused on different aspects in food microbiology, food science and related subjects for individuals in the food industry, researchers, academics, and students. Microbes are key components of the food processing industry, and this book concentrates on topics that incorporate ideas and applications from various fields to address concerns relating to food safety, quality, and sensory attributes. Researchers around the globe will be able to use this information as a guide in establishing the direction of future research on food processing considering various aspects related to microbes. The main objective of this book is to disseminate knowledge about the recent technologies developed in the field of microbiology and their relation to the food industry. Written in an easy-to-understand style, the chapters gathered here are of interest to people in the industry with a great deal of experience and knowledge but also for students and newly hired professionals in the food industry. Whether for the veteran engineer or scientist, the student, or a manager or other technician working in the field, this volume is a must-have for any library.
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Contents
Cover
Series Page
Title Page
Copyright Page
Preface
1 Food Microbiology: Fundamentals and Techniques
1.1 Introduction
1.2 Food Microbiology: A Historical Perspective
1.3 Beneficial Microbes in Food
1.4 Harmful Microbes in Food
1.5 Classical Food Microbiological Techniques
1.6 Advances in Food Microbiological Techniques
1.7 Regulations Governing Food Microbiology
1.8 Conclusions
References
2 Fermented Foods in Health and Disease Prevention
2.1 Fermentation
2.2 Traditional Fermented Food
2.3 Application of Fermentation to Food
2.4 Effects of Fermentation on Nutrients
2.5 Health Benefits of Fermented Foods and Beverages
2.6 Food Safety and Quality Control
2.7 Conclusions and Future Perspectives
References
3 Probiotic Dairy Foods
3.1 Introduction
3.2 Classification and Phylogenetic Properties of Probiotic Microorganisms
3.3 Probiotics in the Dairy Matrix
3.4 Probiotic Dairy Products
References
4 Dairy Probiotic Products
4.1 Introduction
4.2 Fermented Milks
4.3 Conclusions and Perspectives
References
5 Design Schematics, Operational Characteristics and Process Applications of Bioreactors
5.1 Introduction
5.2 Fermenter Design and Operations
5.3 Fermenter Configuration
5.4 Types of Fermenter
5.5 Factors Influencing Operation of Fermenters
5.6 Conclusion
References
6 Enzymes in Food Industry and Their Regulatory Oversight
6.1 Introduction
6.2 Production of Enzymes
6.3 Applications of Enzymes in Food Industry
6.4 Safety Evaluation of Enzymes
6.5 Global Regulatory Frameworks
6.6 Regulatory Framework in India
References
7 Functional and Nutraceutical Potential of Fruits and Vegetables
7.1 Introduction
7.2 Biochemistry of Fruits and Vegetables
7.3 Nutritional Composition of Fruits and Vegetable By-Products
7.4 Extraction of Bioactives from Fruits and Vegetables
7.5 Processing Methods Used for Development of Functional Foods from Fruits and Vegetables
7.6 Fruits and Vegetable-Based Nutraceuticals
7.7 Influence of Processing Methods on Functional Ingredients
7.8 Influence of Storage on Functional Ingredients
7.9 Future of Functional Foods
Conclusion
References
8 Microbes as Bio-Factories for the Valorization of Fruit and Vegetable Processing Wastes
8.1 Introduction
8.2 Microbial Bio-Processing of Fruit and Vegetable Wastes
8.3 Valuable Commodities from Fruit and Vegetable Waste
8.4 Technical Challenges, Economics and Future Prospective
8.5 Conclusion
References
9 Solid-State Fermentation
9.1 Introduction
9.2 History of Solid-State Fermentation (SSF)
9.3 Factors Affecting SSF
9.4 Types of Solid-State Fermentation
9.5 Application of SSF Carried Out on Inert Support Materials
9.6 Modern Aspects of Solid-State Fermentation
9.7 Challenges to SSF
9.8 Conclusions
References
10 Pigments Produced by Fungi and Bacteria from Extreme Environments
10.1 Introduction
10.2 Extreme Environments
10.3 Extremophilic Microorganisms
Conclusion
Acknowledgments
References
11 Commercially Available Databases in Food Microbiology
11.1 Introduction
11.2 Functions of a Databases
11.3 Need for Databases
11.4 Predictive Microbiology in Foods
11.5 Predictive Microbiology and Its Models
11.6 Rapid Methods of Data Generation
11.7 Predictive Models
11.8 Guidelines for Modeling the Shelf Life of Foods
11.9 Databases in Foods
11.10 QMRA (Quantitative Microbial Risk Assessment)
11.11 Other Databases
11.12 Future Prospects
References
Index
Also of Interest
Wiley End User License Agreement
List of Figures
Chapter 1
Figure 1.1 Colonies of select microorganisms when grown on selective agar media.
Figure 1.2 Common plating techniques – (a) pour plate, (b) spread plate, (c) spiral plate.
Chapter 2
Figure 2.1 Classification of fermented milk products.
Chapter 4
Figure 4.1 Potential human health benefits of yogurts supplemented with probiotics. Adapted from [46–55].
Figure 4.2 Kefir grains composed of kefiran and microbial cultures.
Figure 4.3 Potential benefits of kefir for human health.
Figure 4.4 Potential human health benefits of ice creams supplemented with probiotics. Adapted from [226–232].
Chapter 5
Figure 5.1 Downstream process in fermenters.
Figure 5.2 (a) Continuous Stirred tank reactor (CSTR) (b) Plug flow reactor (PFR) [9].
Figure 5.3 Fed-batch fermenter [11].
Figure 5.4 Stirred tank [1].
Figure 5.5 Airlift reactor [28].
Figure 5.6 Bubble column reactor [28].
Figure 5.7 Fluidized bed reactor [28].
Figure 5.8 Packed bed reactor [28].
Figure 5.9 (a) Sidestream membrane bioreactor (b) immersed or submerged membrane bioreactor [49].
Figure 5.10 Heat transfer configuration for fermenter are (a) jacketed vessel (b) external coils (c) internal helical coils (d) internal baffle type coils (e) external heat exchanger [62].
Chapter 6
Figure 6.1 Production of enzymes.
Chapter 7
Figure 7.1 Chemical structures of common polyphenols and flavonoids in fruits and vegetables.
Figure 7.2 Chemical structures of major carotenoids in fruits and vegetables.
Figure 7.3 Chemical structures of common vitamins in fruits and vegetables.
Figure 7.4 Chemical structures of common glucosinolates in fruits and vegetables.
Figure 7.5 Chemical structures of common phytoestrogens observed in fruits and vegetables.
Figure 7.6 Extraction approach for utilization of functional potential of fruits and vegetables.
Figure 7.7 Bioactive compounds extracted from fruits and vegetables using novel extraction techniques.
Chapter 8
Figure 8.1 Valorization of fruits and vegetables waste into potential biocommodities via microbial processing.
Chapter 9
Figure 9.1 The phases within a solid-state fermentation system.
Figure 9.2 Process of solid-state fermentation and its applications.
Figure 9.3 Factors influencing solid-state fermentation.
Figure 9.4 Comparison of anaerobic solid-state fermentation and aerobic solid-state fermentation.
Figure 9.5 Illustration shows the conversion of glucose into various fermented products.
Figure 9.6 Schematic diagram of a tray bioreactor and for an individual tray [67].
Figure 9.7 Schematic diagram of a traditional packed bed bioreactor.
Figure 9.8 Schematic diagram of stir type rotating drum bioreactor [71].
Figure 9.9 Schematic representation of (a) PLAFRACTOR bioreactor with multiple modules, (b) plate schematic and (c) compile up of plate and frames form vertical stacks [73, 74].
Figure 9.10 Schematic representation of Novozymes Bio A/G based modular bioreactor [75].
Figure 9.11 Schematic diagram of air-solid fluidized bed bioreactor [71].
Chapter 10
Figure 10.1 Extreme environments and their biotic factors.
Figure 10.2 Chemical structure of the main natural pigments detected in microorganisms.
List of Tables
Chapter 1
Table 1.1 Different types of food poisoning caused by food pathogen infections.
Table 1.2 Incubation conditions and most commonly used growth media for select microorganisms.
Table 1.3 Typical appearance of select microorganism colonies.
Chapter 2
Table 2.1 Fermented foods and their role in health.
Chapter 3
Table 3.1 Proposed reclassification of genus of
Lactobacillus
[15].
Table 3.2 Some examples of proposed genus and species of
Lactobacillus
genus (compiled from [9]).
Table 3.3 Most recent studies on probiotic yogurt.
Table 3.4 Summary of some current studies on probiotic cheeses.
Table 3.5 Some dairy-based probiotic beverages commercially marketed on a global scale.
Table 3.6 Some recent studies on probiotic whey-based drinks.
Chapter 4
Table 4.1 Types of fermented milks.
Table 4.2 The influence of supplementation with probiotics on the properties of yogurt.
Table 4.3 Probiotic or potentially probiotic microorganisms identified in spontaneous fermentation or intentionally added in kefir.
Table 4.4 Probiotic strains added to cheeses and their effects
in vitro
or
in vivo
. Adapted from [103].
Table 4.5 Studies on the incorporation of probiotics in butter.
Table 4.6 The influence of supplementation with probiotics on the properties of ice cream.
Table 4.7 Probiotic and/or symbiotic dairy desserts.
Chapter 5
Table 5.1 Parts for construction of Fermenter and their functions.
Table 5.2 Fermenter systems and their application.
Chapter 6
Table 6.1 Application of enzymes in food processing industry.
Chapter 7
Table 7.1 Nutritional composition of various fruits and vegetables. (
Continued
)
Table 7.2 Nutraceuticals compounds observed in fruits and vegetables.
Table 7.3 Bioactive compounds extracted from various fruits and vegetables.
Table 7.4 Fermented products obtained from different fruits and vegetables.
Chapter 8
Table 8.1 Summary of yield of bioproducts by solid state fermentation of food wastes and the process conditions.
Table 8.2 Summary of bioproducts from fruit and vegetables wastes via microbial processing.
Chapter 9
Table 9.1 Several definitions of solid-state fermentation prescribed by various authors.
Table 9.2 Applications of solid-state fermentation (SSF).
Table 9.3 Bioreactors used for the production of end products using solid-state fermentation.
Chapter 10
Table 10.1 Main pigments produced by polar, alpine/altitude, desert and saline fungi.
Table 10.2 Main pigments produced by polar and alpine bacteria.
Table 10.3 Main pigments produced by desert, saline, and volcanic bacteria.
Guide
Cover Page
Series Page
Title Page
Copyright Page
Table of Contents
Preface
Begin Reading
Index
Also of Interest
Wiley End User License Agreement
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Scrivener Publishing100 Cummings Center, Suite 541JBeverly, MA 01915-6106
Bioprocessing in Food Science
Series Editor: Anil Panghal, PhD
Scope: Bioprocessing in Food Science will comprise a series of volumes covering the entirety of food science, unit operations in food processing, nutrition, food chemistry, microbiology, biotechnology, physics and engineering during harvesting, processing, packaging, food safety, and storage and supply chain of food. The main objectives of this series are to disseminate knowledge pertaining to recent technologies developed in the field of food science and food process engineering to students, researchers and industry people. ~is will enable them to make crucial decisions regarding adoption, implementation, economics and constraints of the different technologies.
As the demand of healthy food is increasing in the current global scenario, so manufacturers are searching for new possibilities for occupying a major share in a rapidly changing food market. Compiled reports and knowledge on bioprocessing and food products is a must for industry people. In the current scenario, academia, researchers and food industries are working in a scattered manner and different technologies developed at each level are not implemented for the benefits of different stake holders. However, the advancements in bioprocesses are required at all levels for betterment of food industries and consumers.
The volumes in this series will be comprehensive compilations of all the research that has been carried out so far, their practical applications and the future scope of research and development in the food bioprocessing industry. The novel technologies employed for processing different types of foods, encompassing the background, principles, classification, applications, equipment, effect on foods, legislative issue, technology implementation, constraints, and food and human safety concerns will be covered in this series in an orderly fashion. These volumes will comprehensively meet the knowledge requirements for the curriculum of undergraduate, postgraduate and research students for learning the concepts of bioprocessing in food engineering. Undergraduate, post graduate students and academicians, researchers in academics and in the industry, large- and small-scale manufacturers, national research laboratories, all working in the field of food science, agri-processing and food biotechnology will benefit.
Publishers at ScrivenerMartin Scrivener ([email protected])Phillip Carmical ([email protected])
Microbes in the Food Industry
Edited by
Navnidhi ChhikaraAnil PanghalandGaurav Chaudhary
This edition first published 2023 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA© 2023 Scrivener Publishing LLCFor more information about Scrivener publications please visit www.scrivenerpublishing.com.
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Library of Congress Cataloging-in-Publication Data
ISBN 9781119775584
Cover images: Petri dish: Luchschen | Dreamstime.com, Bacteria strain: Alexey Romanenko | Dreamstime.com.Bacteria: Irochka | Dreamstime.com, Microbes on vegetables: Chernetskaya | Dreamstime.comCover design by Kris Hackerott
Preface
The “Bioprocessing in Food Science” series of books is an attempt to address the recent developments in food sciences. As the global demand of healthy food is increasing, manufacturers are searching for new possibilities to occupy a major share in a rapidly changing food market. Microbes in the Food Industry is primarily focused on different aspects in food microbiology, food science and related subjects for individuals in the food industry, researchers, academics, and students up to the master’s level and assumes some knowledge of basic microbiology. The present book comprehends different topics incorporating technologies from different fields and at the same time, taking into account consumers’ concerns about food safety, quality, and sensory attributes. The microbes are considered as key component in food processing industry. Researchers around the globe would be able to use the information as a guide in establishing the direction of future research on food processing considering various aspects related to microbes. The main reason for writing this book now is to disseminate the wealth of knowledge on newer techniques in microbiology and its relation to the food industry. It is envisioned for scientists, technologists, and engineers working in the area of food processing, microbiology, environmental science, process equipment design, and product development. Also included are students of microbiology, food science & technology, nutrition, health science and engineering.
International peers having both academic and professional expertise in the field extended their knowledge in the form of chapters of this book. The book illustrates a very clear and concise discussion on microbes in food industry and microbiology. Food microbiology is a broad subject encompassing the study of both beneficial and harmful microorganisms in food, and their effects on the quality and safety of food. The chapters highlight different aspects of microbes in food industry and their impact on food safety, processing, and to the resulting finished products. Each of the chapters is supported with references owing to be an invaluable resource for the reader. Self-explanatory illustrations and tables of each chapter are an added advantage to understand the technology process and its outcome easily. We have also rationalized the index, which we decided was excessive and contained too many esoteric or trivial entries.
We are indebted to our numerous colleagues for their quality chapters and helping us to complete the book. We also thank the authorities of Chaudhary Charan Singh Haryana Agricultural University, Hisar and Guru Jambheshwar University of Science and Technology for their support.
Finally, we also express indebtedness and thankfulness to Scrivener Publishing and Wiley for their unfailing guidance and assistance provided in the finalization and publication of the book.
Navnidhi ChhikaraAnil PanghalGaurav Chaudhary
1Food Microbiology: Fundamentals and Techniques
Raina Jain, Prashant Bagade*, Kalpana Patil-Doke and Ganesh Ramamurthi
National Commodities Management Services Limited, Hyderabad, India
Abstract
Food microbiology is a broad subject encompassing study of both beneficial and harmful microorganisms in food, and their effects on the quality and safety of food. Beneficial microbes present in food offer an array of health benefits to humans and are important sources for fermentation, probiotics and bio- preservatives. Contrarily, harmful bacteria lead to food spoilage and a countless number of foodborne diseases which may even prove to be lethal, if uncontrolled. Food microbiology uses a number of testing methods to detect, enumerate and identify the microorganisms present in food. Conventionally, it involved culturing of microbes on suitable media and analyzing the results on the basis of physical or biochemical tests. However, such techniques are time-consuming and laborious. As a result, rapid and high-throughput techniques with use of advanced equipment and strategies have been developed to ensure quality and safety of food in real time. The chapter presents the long history of the development of Food Microbiology as a subject, along with classical and advanced techniques used to identify and quantitate foodborne microorganisms.
Keywords: Food microbiology, food spoilage, food microbes, food regulations, microbiological techniques, probiotics, AI and ML in food, biosensors
1.1 Introduction
Food microbiology is the study of microorganisms that colonize, modify, process or spoil food. It deals with foods and beverages of diverse composition, combining a broad spectrum of environmental factors, which may influence microbial survival and growth. A variety of microorganisms having beneficial or harmful effects on food quality and safety are studied in food microbiology. This includes spoilage, pathogenic, fermentative, and probiotic bacteria; molds and yeasts; viruses, prions, and parasites.
Microorganisms, viz., bacteria, molds and viruses can contaminate foods across the food value chain. Consumption of such foods can cause food-borne diseases. Effective intervention technologies are being developed and implemented to ensure safety of consumers against foodborne diseases. Food spoilage occurs due to growth of microorganisms in food or due to the action of microbial enzymes. Incidences of food contamination and spoilage are very frequent, and are being reported on a regular basis. Such incidences are partly due to consumers’ changing palate in desiring undercooked, minimally processed, unpreserved foods, etc. Additionally, inadequate infrastructure along the supply chain, especially for temperature-sensitive foods, also plays a major role in food spoilage leading to economic losses.
Food bioprocessing is a technique of food and ingredient extraction, purification and production using processes that involve the application of enzymes and/or microorganisms. It is one the most primitive forms of food processing method, used by early Egyptians for production of wine, beer, and bread. Additionally, microbial enzymes were also being used to produce food and food additives. Since the past few years, by adoption of genetic recombination techniques and use of different microbial sources, enzymes of higher purity and activity are obtained. Nowadays, many types of additives from microbial sources are being developed and utilized in food processing. Some of these include single-cell proteins, essential amino acids, colour and flavour compounds, stabilizers, and organic acids [1]. Food bio-preservation through anti-microbial metabolites such as bacteriocins and organic acids like acetic, propionic and lactic acids are being developed and used, replacing preservatives of non-food origin. Probiotics – a rapidly emerging health food – contain live cells of bacteria that have apparent health benefits. The role of these bacteria for health and bacterial efficacy benefits are being researched upon.
1.2 Food Microbiology: A Historical Perspective
Foodborne disease and food spoilage have been part of the human experience since the dawn of our race. Several events spanning centuries led to the recognition of the role of microorganisms in foods. We are aware that early civilizations discovered and applied effective methods to preserve and protect their food. As far back as 7000 BC, Babylonians manufactured beer and wine. Egyptians, in 3000 BC, manufactured cheese and butter. Around the same time, use of salt to preserve meat and other foods became popular. In 1000 BC, the Romans discovered fermentation, salt, ice, drying and smoking to preserve shrimp and meat, though they did not know how these practices inhibited food spoilage or caused foodborne diseases. This was compounded by their belief that living things formed spontaneously from non-living matter.
In 1665, Francesco Redi demonstrated that maggots on putrefying meat did not arise spontaneously but were instead the larval stages of flies. This was the first effort opposing the doctrine of spontaneous generation. In 1765, Spallanzani disproved the theory of spontaneous generation of life by demonstrating that beef broth which was boiled and then sealed remained sterile. The French government offered 12,000 francs to anyone who could develop a practical way to preserve food in 1795. Nicholas Appert showed that meat could be preserved when it was placed in glass bottles and boiled. This was the beginning of food preservation by canning. Later, Schwann demonstrated that heated infusions remain sterile in the presence of air, again to disprove spontaneous generation. It is interesting to note that although Spallanzani and Schwann each used heat to preserve food, neither apparently realized the value of turning these observations into a commercial method for food preservation [2].
The first person to really appreciate and understand the cause- effect relationship between microorganisms in infusions and the chemical changes that took place in those infusions was Louis Pasteur. He convinced the scientific world through his experiments that all fermentative processes were caused by microorganisms. Later, he showed that souring of milk was caused by microbes and heat destroyed undesirable microbes in wine and beer. The later process is now used for a variety of foods and is called pasteurization. Because of the importance of his work, Louis Pasteur is known as the founder of food microbiology. Using his famous swan-necked flasks, he even demonstrated that air does not have to be heated to remain sterile, and this finally put an end to the theory of spontaneous generation. The knowledge that microbes were responsible for fermentation and putrefaction led Pasteur to argue that microbes were also causative agents in disease. These arguments eventually helped Joseph Lister to develop the first aseptic surgical procedures. Since that time, microbiological discoveries and developments began to proceed more rapidly, leading to implications of microbes in several diseases. This led governments to enact legislation to protect the quality of food.
Most of the food industries hesitated in adopting microbiological food safety norms in their routine procedures until they were economically affected by outbreaks of foodborne diseases in their products. One similar case occurred in 1920s with the outbreak of Botulism, which affected food canning industries. This resulted in adoption of the 12D process for heat treatment of C.botulinum. At about the same time, the dairy industry was driven to implement microbiological control over safety in milk production, because of several nasty outbreaks of milk-borne diphtheria, tuberculosis, typhoid fever, and brucellosis. Regulatory bodies made it compulsory to address the risks with focus on animal health, sanitation, and pasteurization – which had an immediate and very effective impact on the problems.
In one of the cases of early food microbiology, the US government had institutionalized a woman who came to be known as “Typhoid Mary”. She was an asymptomatic typhoid carrier who worked as a cook for several families. Over 10 years, seven outbreaks of typhoid were directly traced to her and estimates suggest that she may have been responsible for 120 cases [3] of typhoid fever. New York authorities arrested her but eventually released her when she agreed never to work as a cook again. When another outbreak was traced to her a few years later, she was arrested as a threat to public safety and institutionalized until her death in 1938.
1.3 Beneficial Microbes in Food
The role of beneficial microbes is not given due recognition since it is a common perception to think of microbes only as harmful. Their presence in human gut play a significant role in maintaining human health by ensuring proper digestion apart from a range of benefits. The number of microorganisms that are present in the human GI tract is estimated to be over 1014 [4]. A recent study [5] has identified about 2,000 bacterial species in the human gut by using computational methods. However, these species are yet to be cultured in the lab. Beneficial microbes are used in the food industry for a variety of applications with simplest being in fermentation, which has been used since ancient times for production of wine, bread, cheese, etc., apart from a host of traditional dishes. Fermented foods are considered healthy due to the presence of various health promoting microorganisms. Based on the concept, a new trend that has taken over and is gaining popularity is probiotic foods. These are foods to which health- promoting microorganisms are added and are generally considered as “super foods”. A few such foods include Yogurt, Kefir (made by adding kefir grains to cow’s or goat’s milk), Sauerkraut (finely shredded cabbage fermented by lactic acid bacteria), Miso (made by fermenting soybeans with salt and koji, a fungus), etc. Furthermore, some microbes have been found to retard spoilage of food products when added in the appropriate proportion; they are known as microbial bio-preservatives. Though certain examples exist of such organisms, it is a field that is still in its infancy and needs more research and acceptance by consumers.
1.3.1 Factors Influencing Microbial Growth in Food
Ecology helps us learn the factors and their interactions determining growth of an organism in a given environment. Microbial growth in food is a complex process, and there are multiple genetic, biochemical and environmental factors affecting this [6]. They can be classified into four major classes:
Intrinsic factors – Characteristics of food itself, including naturally occurring compounds, added preservatives, nutrient content, water activity, pH, and the oxidation-reduction potential, are called intrinsic factors.
Extrinsic factors – They are related to the environment in which food is stored – relative humidity and temperature of atmosphere of food storage, composition of gases. Environmental temperature plays a critical role in influencing microbial growth, e.g., in a refrigerator, microbial cells grow at a much lower rate compared to room temperature.
Implicit factors – These include interactions between microbes contaminating the food and between these organisms and food itself. For example, a microorganism’s inner ability to utilize different nutrient sources and tolerate stress will define its growth in a particular food.
Processing factors – Food treatments, such as heating, cooling, and drying, which affect composition of food, also affect the microorganisms available in treated food.
Moreover, the combined effect of the four factors described above influence the microbial growth in food, in a more or less synergistic manner [7].
1.3.2 Food Fermentation
The science of fermentation is known as zymology or zymurgy. In the simplest terms, fermentation is the process of breaking down of carbohydrates such as starch and sugars to alcohols by either inherently present or externally added microbes. Despite being one of the most ancient techniques of food preservation, it is still in use for production of a variety of products apart from preservation. Countries with rich ancient civilizations have a host of traditional fermented food as part of their diet. Such foods and beverages serve as a rich source of nutrition apart from being capable of maintaining human health and preventing diseases. Though such foods have seen a decline in their consumption in the past, recently their consumption has increased owing to their health benefits. They are generally produced naturally or by addition of a starter culture resulting in food products that are considered superior nutritionally and/or organoleptically. However, such foods have limited appeal since culture plays a significant role in their consumption. Hence, most such foods are restricted geographically and culturally.
A variety of bacteria, fungi and yeast are involved in food preservation by using food as the substrate and by producing citric acid, lactic acid and/ or acetic acid. Apart, they are also responsible for production of aroma in foods by metabolising foods into amino acids, fatty acids, and nucleotides [8]. Cheese, a globally consumed product, derives its flavor depending upon the variety, microflora and ripening conditions resulting in formation of free fatty acids [9]. Fermentation is also known to reduce the amount of anti-nutritional factors such as phytates, thereby improving the availability of nutrients present in food. Phytates are known to bind with certain important micronutrients such as iron and zinc, limiting their bio-availability. In a study [10] using Pearl millet, fermented dough was found to contain lower levels of phytate compared to flour. Apart, the process has resulted in increased content of polyphenols which are known to have higher antioxidant activity and are beneficial to human health.
1.3.3 Probiotics
Probiotics are live microorganisms that are intended to have health benefits when consumed or applied to the body. There are a wide range of natural probiotics foods which are being manufactured to cater to the needs of changing lifestyles. Such foods are manufactured by adding live microorganisms that improve gut health resulting in a wide range of benefits to the consumers. Though they may contain a variety of microorganisms, most of the bacteria belong to two groups – Lactobacillus and Bifidobacterium. The most common yeast used in probiotics is Saccharomyces boulardii. Research results of the Human Microbiome Project enabled the study of microbial communities that live in and on our bodies and the roles they play in human health and diseases. It has helped in mapping the normal gut microbiome forming a basis for establishing linkages between diseases and changing microbiome. Such information can be exploited to develop tailored probiotic foods which can address specific health disorders.
1.3.4 Microbial Bio-Preservatives
All foods have a shelf life beyond which they will be considered unfit for consumption due to various reasons such as development of unpalatable flavour, taste, texture, microbial growth, etc. To extend product shelf life, a wide variety of preservatives are added – chemical or otherwise. Considering the undesirable nature of chemical preservatives, a new trend in the form of bio-preservatives has taken root and is gaining attention. Bio-preservation is the use of antimicrobial active substances extracted from food or obtained by food-grade microbial fermentation to enhance safety and food quality [11]. The most commonly used bio-preservatives are Lactic acid bacteria (LAB) belonging to the order, Lactobacillales, which includes more than 200 species under 36 genera [12]. The most common genera to which LAB species belong are Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, Enterococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella. LAB species exert their effect by producing certain metabolites such as lactic acid, acetic acid, bacteriocins, etc. Their antagonistic effect is based on the principle that the majority of bacteria will stop multiplying when the pH reaches below 4. Apart from LAB, dairy propionibacteria (PAB) and Bacillus species have also been found to possess antifungal properties. Mycocins, also known as “killer proteins”, produced by yeasts have also been documented to produce antimicrobial compounds [13].
In a study [14] on seafood products, certain LAB species were found to be suitable as bioprotective agents in a hurdle technology strategy applied to cod and salmon-based products. In another study [15] on beef and lamb meat, microbial bio-preservatives were found to retard the growth of the major meat spoilage bacteria – Brochothrixthermosphacta, Pseudomonas species, and Enterobacteriaceae. Apart from being able to reduce the bacterial spoilage, they did not have any noticeable negative impact on the sensory properties of meat. Bacteriocins, ribosomally synthesized antimicrobial peptides, are produced by bacteria. These have been reported [16] to not only preserve food but also retain organoleptic and nutritional properties.
Though few such bio-preservatives are commercially available, their numbers have been significantly lower than the vast majority of strains described in literature. A few of the possible reason include mismatch in efficiency of such microbes under research conditions as compared to field applications. Consumer acceptance is another major constraint since microbes are generally considered as “harmful” and their intentional addition to food is not easily accepted by consumers. Also, regulatory aspects must be kept in mind since country-to-country requirements would vary.
1.4 Harmful Microbes in Food
Food is the most susceptible commodity targeted by microorganisms to grow and spoil. Raw food is a good home to all sort of microorganisms, may it be in the form of raw meat, milk or fresh fruits. The number of microbes existing on raw food decreases upon washing or processing or cooking or adding preservatives. However, adequate process efficiency and its packaging and storage are equally crucial in maintaining safety of the food. The cooked food, containing all nutrients required by microorganisms, easily attracts bacteria, yeasts & molds within a few hours after preparation, if left open [17].
On the other hand, unlike spoiled food, food contaminated with pathogens cannot be identified superficially by a general sense of appearance and smell. Consumption of such foods causes serious health hazards, known as foodborne diseases or foodborne illness. Overall, all the microorganisms which spoil the food to make it unacceptable for human consumption and/ or lead to development of diseases are classified as harmful microbes.
1.4.1 Factors Influencing Food Spoilage
As we have described in the previous section as well, the growth of microbes in food essentially depends on the extrinsic, intrinsic and implicit factors, which together decide the fate of food. Harmful bacteria challenge the shelf life of particular food. However, not all foods are susceptible to microorganisms; it mainly depends on the nature of food, its processing or preservative activities. Perishable foods, like dairy products, meat, poultry, fish etc., can be spoiled quickly by activity of microbes within a few days. Meat putrefies and sours because of bacterial growth on it and milk products are spoiled either by acid or mold growing in it. Similarly, fresh fruits and vegetables are spoiled because of degradation of pectin in them by pectin-degrading bacteria. Unlike perishables, semi-perishable foods have a relatively long shelf life, a few weeks or months, and include bread, butter, cake, canned fruits, pickle, jam juices, etc. Further, non-perishables having a very long shelf life of months or years include dried fruits and vegetables, peanut butter, etc.
Moreover, the food environment itself sometimes favors or restricts the growth of harmful microbes. pH, water activity (Aw), oxidation-reduction potential, nutrients and inhibitory agents are the key factors for controlling spoilage. Furthermore, microorganisms used in food fermentation and pathogens can multiply in a food to reach the spoilage detection level and are capable of causing food spoilage. The best example is curd or yoghurt, which spoils because of overgrowth of its own organisms.
Apart from inherent properties of food responsible for spoilage, factors during processing are also a part of risk of spoilage. For microbial food spoilage to occur, microorganisms have to get into the food from one or more sources. It can be through a low-quality ingredient, unhygienic environment and handling at any stage, inadequate thermal processing or preservative dosages and many more. Packaging integrity, time of storage and any deviation in storage conditions are some more causes responsible for spoilage.
Different categories of bacteria thrive in varied food environments, such as raw meat, which is liked by Salmonella and Campylobacter, whereas fresh fruits and vegetables are liked by E. coli and dairy products are home for Listeria monocytogens. Clostridium perfringens is better known as “buffet germ” because it is found in foods which are made in larger batches and get inadequate heat treatments. Home canned foods are always threatened by anaerobic bacteria, Clostridium perfringens and Clostridium botulinum, which can cause gas formation and bulging of the can.
It is the nature of food-spoiling bacteria which affect different categories of food, as given below:
Psychrotrophic Bacteria – As the name indicates, these bacteria are capable of growing at 5°C and below, but multiply rapidly at 10 – 25°C. These bacteria grow on many foods, stored on ice and in a refrigerator and some are expected to have a long shelf life of 50 days or more. Examples are Pseudomonas fluorescens, Pseudomonas fragi, Acinetobacter, Moraxella, Flavobacterium and some molds and yeasts.
Anaerobic Bacteria – These bacteria grow in the absence of oxygen in foods stored anaerobically, also in the interior of prepared food. Examples are Lactobacillus viridescens, Lactobacillus sake, Lactobacillus curvatus, Leuconostoccarnosum, Leuconostocgelidum, some Enterococcus Spp., Alcaligenes Spp., Enterobacter Spp. and some Microaerophilic yeasts.
Thermoduric Psychrotrophs – include facultative anaerobes such as spores of Bacillus coagulans, Bacillus megaterium, Lactobacillus viridescens.
Mesophiles – grow in moderate temperature range of 20 to 45°C and include Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli.
Thermophilic Bacteria are – a group of bacteria which grow between 40 – 90°C, with optimum temperature at 55 – 65°C. Spores of these bacteria also germinate and start spoilage. Examples are spores of some thermophilic Bacillus and Clostridium Spp.
Thermoduric vegetative bacteria surviving low heat processing (such as pasteurization) or thermophiles can also multiply in these warm foods especially if the temperature is close to 50°C. These include some lactic acid bacteria such as Pediococcus Spp. and Streptococcus Spp., Bacillus and Clostridium Spp.
Aciduric Bacteria are the bacteria that can grow in food at pH 4.6 or below. They are associated with spoilage of acidic food products such as fruit juices, pickles, salsa, salad dressing and fermented sausages. Heterofermentative and homofermentative lactic acid bacteria have been associated with such spoilage. Yeasts and molds are aciduric and are also associated with spoilage of such foods.
1.4.2 Indicators of Food Spoilage
Once these huge numbers of microorganisms start releasing the extracellular and intracellular enzymes in the food environment, their effects are visible in the form of colour change, odour, texture changes with slime formation or gas accumulation, foam accumulation and many more. Molds form visible growth on surfaces; and affected food appears softened and rotten to the naked eye. Slime formation and Ropiness is formed after U-ring growth of Pediococcusdamnosus and Lactobacillus brevis. Some spoilages are associated with formation of hydrogen sulfite gas, giving a rotten egg smell to food. Other end products released from microbial metabolism of food nutrients include carbohydrates, CO2, H2S, H2O2, lactate, acetate, formate, succinate, butyrate, ethanol, propanol, butanol, diacetyl, dextran proteins and non-protein nitrogenous compounds, amines, ketoacids, putrescines, lipids, fatty acids, glycerol, hydroperoxides, aldehydes, ketones.
To identify spoilage of food, microbiological analysis is the first tool where enumeration techniques reveal the status of deterioration. Aerobic plate count indicates the effectiveness of sanitary procedures used during processing and handling and before storage of a product. A similar conclusion is drawn by yeast & mold count. But the major disadvantage of microbiological enumeration methods is that it takes several days. To overcome this problem, other indirect methods have been used [18]. Examples of such methods are determination of lipopolysaccharides (LPS) in a food (for Gram-negative bacteria), measurement of ATP as its concentration is increased with high numbers of viable cells. Chemical indicators of food spoilage are also useful in determining food spoilage. Pasteurization is done to ensure effective treatment of milk. A quick indicator of pasteurization is use of Alkaline Phosphatase (ALP), an enzyme naturally present in all raw milks, which is inactivated upon complete pasteurization. Heat stability of ALP is greater than that of pathogens which may be present in milk; the enzyme serves as an indicator of product safety. MBRT and resazurin tests are similar tabletop tests in the dairy industry which indicate bacterial population [19].
1.4.3 Foodborne Infections and Intoxications
Food infection is caused by consumption of a food that contains viable pathogenic cells, present in large numbers. Food pathogens can be bacteria and viruses like hepatitis A & E. Depending upon the origin of the food and extent of process of preservation, pathogenic bacteria remain viable on food and get into the human body. Upon consumption, within 24 hours till 3 days, bacteria colonize and grow on the gastrointestinal tract of the host and then lead to localized infections, tissue damage and deeper infections to cause systemic infections [20]. Visible effects of such infections are mostly nausea, vomiting, diarrhea, abdominal cramps and fever. Further typical infections are bacteria-specific – Hepatitis, Salmonellosis, vibriosis, yersiniosis, campylobacteriosis, listeriosis, etc. As per the type of infection, food poisoning can be categorized as non-inflammatory, inflammatory and systemic/penetrative (Table 1.1).
Food intoxication is another type of foodborne disease; however, unlike food infection, intoxication is caused due to toxins released by bacteria in food. Actual bacterial cells need not be present at the time of consumption but the toxins produced by them are ingested and cause symptoms. Staphylococcal enterotoxin, botulinum toxin, and Bacillus cereus toxin are a few of the examples which cause botulism, staphylococcal poisoning, and Bacillus cereus poisoning, respectively. Botulinum is believed to be lethargic since it is a kind of neurotoxin and a very little dose is also enough to cause severe effects on health. Additionally, mycotoxins – aflatoxins, and ochratoxins – are also part of food toxins which are produced by fungi. Foodborne toxico-infection, the combination of food intoxication and infection, is caused when a pathogen is consumed and then it releases toxins in the GI tract, resulting in illnesses.
Table 1.1 Different types of food poisoning caused by food pathogen infections.
Mechanism
Pathogens
Illness
Noninflammatory
Vibrio cholerae
, Enterotoxigenic
E. coli
(ETEC), Enteroaggregative
E. coli
,
C. perfringens
,
Bacillus cereus
,
S. aureus
, Rotavirus, norovirus, Enteric adenoviruses,
Giardia lamblia
, Microsporidia
Watery diarrhea
Inflammatory
Shigella, Salmonella,
C. jejuni
, Shigatoxigenic verotoxigenic
E. coli
(EHEC),
Enterocolitica
,
Vibrio parahaemolyticus
,
C. difficile
,
E. histolytica
Dysentery/Inflammatory diarrhea
Penetrating
Salmonella typhi
,
Yersinia enterocolitica
,
Campylobacter fetus
Enteric fever
Neurological
C. botulinum
, Ciguatera toxin, Combroid neurotoxic shellfish poisons, mushroom toxins
Giddiness
Miscellaneous
Listeria monocytogenes
, Group A streptococci, Hepatitis A virus,
Brucella
spp. toxin,
Trichinella spirali
Stillbirth, fever
1.4.4 Food Preservation to Control Spoilage
Food Preservation is equally important as food preparation or manufacturing to assure long-term storage and complete food safety upon consumption. Food preservation has been practiced since ancient times; pickles, jams, jellies, and alcoholic drinks maturing for years are quick examples of the understanding of preservation techniques by our ancestors. With the development of urbanization and industrialization, more techniques are being invented and practiced. Still, perfect preservation is an everyday challenge for the entire food industry.
Generally, it is believed that temperature and chemical preservatives can prevent food spoilage. But in actuality, there are many more ways of control including selection of the appropriate nature and quality of ingredients, quality of water being used for washing and preparation, effi-ciency of thermal processing/drying, effectiveness of food preservatives, hygienic handling, health status of the food handlers, hygienic premises and hygienic processing facilities, hygienic packaging, integrity of packaging materials, storage conditions and well-established shelf life period. All these factors are considered in food safety management systems and HACCP while manufacturing the food on industrial scale, and hence food safety is assured through adopting the standards like FSSC 22000 or BRC or similar standards globally [21].
Major preservation techniques include heat processing, low- temperature storage, control of water activity, chemical preservatives, modification of atmosphere and irradiation [22]. Heat processing is probably the best way to eliminate microorganisms from a food. It has the capacity to kill microorganisms, their spores as well as denature their toxins. Nevertheless, during heat treatment of food it is also important to retain its nutritive, organoleptic and texture properties. Generally, the process of heating validation involves a thermal death curve, where a destruction pattern of microorganisms is plotted in the form of a graph or in the form of a formula and studied how much reduction occurs at what temperature and time stage. It is also calculated as D-value and Z-value. D value is the indicator of resistance of a microorganism to the heat. It is the time in minutes at a given temperature required to destroy 1 log cycle (90% population or 100 bacteria reducing to 10). On the other hand, Z value reflects the temperature dependence of the reaction. It is defined as the temperature change required changing the D value by a factor of 10. Retort technology, also called as autoclave, is a kind of a pressure vessel where sealed food is kept in and heated for 110 to 135°C. This is as good as commercial sterilization. Ready meals or low acid foods are preferably treated with retort technology to extend the shelf life and ensuring food safety.
Foods where high heating procedures are not suitable are subjected to low-temperature methods. This includes ice cooling, chill storage, freezing, freeze drying, etc. Low temperatures prevent and reduce growth and catalytic activities of microorganisms. It is a preferred method for meat, fish and seafoods. General cooling is achieved by use of ice, essentially made by potable water. But it is a very primary means of preservation and also poses a risk of heterogeneous chilling, cross-contamination and ice melting risk. Chilling storage for fruits and vegetables is used at 10 – 20°C with controlled humidity. Highly perishable foods are stored at 1 – 4°C, whereas other perishable foods are stored at 4 – 5°C. Freezing is done either slowly at -20 – -30°C or rapidly at -70°C. Nonetheless, there are many drawbacks of low-temperature treatments – microbial death is not assured, rate of death is unpredictable, does not affect endospore viability and cannot prevent germination. Moreover, these techniques are useful to preserve a food which is already having less microbial load.
Individually Quick Frozen (IQF), is a promising technology widely being used as flash-freezing [23]. In this technique, literally every individual food item is placed on a kind of conveyor belt that speedily moves and enters into a blast chiller that freezes the item very quickly. The temperature of a blast chiller is 0.5 to -4°C and the time is short; this forms smaller ice crystals unlike a slow freezing process where bigger ice crystals are formed that can damage the fibres of foods, squeeze the components of food and water outside and rupture cell walls. IQF saves all these reductions in ice crystallization and saves time as well. Fruits like berries, vegetables like corn and peas, fish, and prawns are processed using IQF techniques.
Control of water activity is one more means of achieving freedom from spoilage microorganisms. Microorganisms need moisture to grow. Most of the bacteria require water activity of more than 0.91, whereas molds need more than 0.80, and a few can grow at 0.75 too. But 0.60 is the activity where almost no microorganism can survive. Hence reducing water activity of a food till lowest is a promising way to control microorganisms and their spoilage. It is achieved by lesser additions of salt and sugar, which binds the water and makes it less accessible to microbes. This is the secret to pickles and jams preservation! An alternate way to reduce water activity is physical removal of water by means of drying. Drying can be natural dehydration by solar heat. It can be mechanical drying too, where drying ovens or drying tunnels or fluid beds, hot rolls are used [24]. Freeze drying or lyophilization is also an advanced technique of drying, where food is subjected to a process that involves freezing a substance at very low temperatures and then extracting liquid through sublimation, converting water from a solid to a gaseous state. Water extracted in such a way passes directly from solid to gas without going through the liquid phase and hence product integrity remains unchanged. This kind of preservation provides very much longer shelf life, till years also.
There are many chemicals which can kill or restrict and delay the growth of microorganisms, essentially classified as chemical preservatives. But not all chemicals can be used in food because either they are toxic for humans or it may affect the narcoleptic properties of the food. Hence in commercial food preparation, one cannot use any chemical without legislative norms. National and international food authorities have laid down a list of approved additives and preservatives and their dosage. Most of the preservatives are used in concentration of less than 0.2%. Commonly used chemical preservatives include salt, sugar & saccharides, acids like vinegar (acetic acid), lemon juice (citric acid), etc. Spices like cinnamon & cloves also impart antimicrobial properties to food along with the flavors. No limit is set for these preservatives by any regulatory food authority.
Acidulant preservatives include benzoic acid, sorbic acid, lactic acid, malic acid, tartaric acid. Where weak acids are used as preservative, they inhibit the outgrowth of both bacterial and fungal cells. Acids like sorbic acid inhibit germination and outgrowth of bacterial spores. Sodium benzoate is one of the most commonly used preservatives to inhibit yeast growth in acid or acidified foods like fruit juices, pickles, jams, etc. Sorbate is another popular preservative, since it is tasteless and odorless and nontoxic; it is an approved preservative by WHO with highest daily intake dosage.
Smoking of a few food types is also a traditional way of food preservation, which involves heating, drying and adding antimicrobial preservatives.
Preservation using gaseous chemicals like Sulphur dioxide is another example of a food preservative. CO2, a colorless, odorless non-combustible gas, is also sometimes used as a food preservative. It inhibits the growth of many psychotropic bacteria and is introduced as a direct additive in storage of fruit and vegetables. Additionally, if CO2 is introduced in packaging environment of food along with other gases like nitrogen, it can prevent further spoilage of food. The technique is called as modification of atmosphere and is employed for a few food types, where it is used to replace oxygen in packaged conditions. Unavailability of oxygen discourages microbial growth; additionally CO2 acts as an inhibiting agent too. When CO2 is compressed, it results in dry ice, a very effective way to maintain chilling conditions in sealed containers. Hence it is used for transport of food samples.
Irradiation is a modern technique of food preservation, which can be called as cold sterilization. Foods are exposed to ionizing radiations, including gamma rays, X-rays or electron beams, which eliminate microorganisms by destroying them and provide spoilage protection. Also, it destroys insects and prevents food from sprouting and ripening. Radiations create energy waves in the product and collide with other particles on a targeted surface. Chemical bonds between these particles are broken and generate short-lived radicals leading to disruption of microorganisms’ structures, nuclear materials and hence growth is altered or stopped. Visually, the product appears the same as before; there is no change in appearance, taste, or nutritive quality of food after irradiation. Norms of dosage are to be followed as laid out by a regulatory body while using irradiation technique.
Different technologies are being used for food preservation based on different principles. Every method has its own advantages and disadvantages. A thought was put out in 1976 by Leistner for combining two or more technologies for better preservation of food and to achieve a total quality, known as hurdle technology. The principle of hurdle technology indicates that hurdles are created to disturb microbial well-being or homeostasis, and multiple challenges are in the way of microbial growth, where microbes cannot jump over all the hurdles [25]. By combining hurdles to address multiple targets, individual techniques, e.g., removing moisture and lowering water activities, maintaining pH, addition of preservatives, disadvantages of individual techniques are overlapped or minimized. This can fulfill the demand of next-generation consumers to get a minimally processed “as natural as original food” as can be possible.
1.5 Classical Food Microbiological Techniques
Classical techniques of microbial analysis originated with the invention of the microscope by Leeuwenhoek in 1674. These are mainly observational techniques for detection of microbes. Notably, less than 5% and perhaps as little as 1% of all bacteria can be cultured in the laboratory. If one includes all viruses infecting all species on earth, the number becomes lower by several orders of magnitude. Hence, one caveat of classical microbial methods is that the tools used to describe microbial development and provide systematic organization are based on a statistically minor portion of all bacteria [26, 27]. Still, these techniques have been routinely utilized for a long time in major food microbiology laboratories throughout the world, despite development of advanced techniques, and are biased for detection of medically important species infecting humans.
The design of the laboratory plays a pivotal role in any food microbiological analysis [28]. Laboratories must be constructed with materials that will restrict microbial growth and shall have a unidirectional flow, with a separate area for each activity. It is imperative that the work environment is maintained in a hygienic condition by rigorous housekeeping procedures and regular fumigation. During each step of analysis, appropriate temperature and humidity levels are maintained to discourage colonization of bacteria and mold. Such setup will result in minimizing false negatives and false positives resulting in accuracy of results.
Microbial media plays an important role and is crucial for obtaining good results. It acts as “food” for microorganisms. All growth media consists of peptones – extracts of protein that are obtained from animal tissues, milk casein and various plant sources like soybean, which serve as basic building material in the growth of microorganisms. Apart from peptone, they also contain yeast extract as nitrogen source and other growth supporters such as minerals, vitamins and growth factors. A media with such composition is known as primary enrichment media, supportive to almost all types of bacteria in food samples. Upon addition of certain inhibitory components, primary enrichment media becomes selective for growth of select microorganisms. Such media are known as secondary or selective enrichment media. A differential media consists of certain dye indicators which result in color changes upon encountering specific conditions such as acidic conditions. Bacterial colonies grown on such media can be visually differentiated based on their color [29]. Typical colonies of select microorganisms are shown in Figure 1.1. It is vital to select appropriate media depending upon the organism of interest. All prepared media are subjected to autoclave sterilization prior to their use in analysis. However, it is important to note that certain media contain heat labile compounds which can degrade during autoclave sterilization. Such media must be sterilized by boiling.
Microbial analysis is performed either qualitatively (detection) or quantitatively (enumeration) [30]. Qualitative techniques are used mainly for detection of pathogens, whereas quantitative techniques are utilized for analysis of bioburden or hygiene parameters, for example, total bacterial count, total yeast & mold count, Enterobacteria count, etc. Enumeration of microbes is normally performed using one of the following three inoculation techniques:
Pour plate technique: Measured sample volume is transferred to a diluent and serial dilutions (
Figure 1.2
