It will thus be useful as a text in introductory food microbiology courses taught in a variety of programs and disciplines. Deb Rogers for excellent typing and editing in the preparation of the manuscript and to the students in the food microbiology class for their helpful suggestions, including new material in the second edition.
The Author
S ECTION I Introduction to Microbes in Foods
DISCOVERY OF MICROORGANISMS
In the 19th century, as a result of the Industrial Revolution, improved microscopes became more readily available, prompting many curious minds to observe and describe the creatures they discovered under a microscope. Although, like bacteria, the existence of submicroscopic viruses was recognized in the mid-19th century, they were observed only after the invention of the electron microscope in the 1940s.
WHERE ARE THEY COMING FROM?
The believers of abiogenesis rejected Spallanzani's observation, which suggested that there was not enough life force (oxygen) present in the sealed jar for animal cells to appear by spontaneous generation. Later Schulze (1830, by passing air through acid), Theodore Schwann (1838, by passing air through red-hot tubes) and Schröeder (1854, by passing air through cotton) showed that bacteria did not appear in boiled meat broth, even in the presence of air.
WHAT ARE THEIR FUNCTIONS?
Spallanzani (1765) showed that boiling the meat stock in broth in a jar and sealing the jar immediately prevented the appearance of these microscopic organisms, thus disproving Needham's theory. John Tyndall, in 1870, showed that boiled stock could be stored in dust-free air in a box without microbial growth.
DEVELOPMENT OF EARLY FOOD MICROBIOLOGY (BEFORE 1900 A.D.)(BEFORE 1900 A.D.)
1885 Theodor Escherich isolated Bacterium coli (later called Escherichia coli) from the stool and suggested that some strains were associated with infant diarrhea. Gartner isolated Bacterium (later Salmonella) enteritidis from the organs of a sick man as well as from the meat the man ate.
FOOD MICROBIOLOGY: CURRENT STATUS
Food Fermentation/Probiotics
Foodborne Diseases
Miscellaneous
- CONCLUSION
- INTRODUCTION
- CLASSIFICATION OF MICROORGANISMS
- NOMENCLATURE
- MORPHOLOGY AND STRUCTURE OF MICROORGANISMS IN FOODS
A food microbiologist should have a good understanding of current developments in food microbiology and of the characteristics of microorganisms that are important in food. Bacteria, yeasts, molds and viruses are important in food because of their ability to cause foodborne illness and food spoilage and to produce food and food ingredients.
Yeasts and Molds
In some European journals more than one letter is used, but there is no fixed system (e.g. Lact. lactis, Lc. lactis, Leu. lactis, Lb. lactis, List. monocytogenes). In rare cases, a minor modification is used (for example, Lactococcus lactis and Lactobacillus lactis are written as Lac. lactis and Lab. lactis for the two genera, respectively).
Bacterial Cells
Viruses
- IMPORTANT MICROORGANISMS IN FOOD A. Important Mold Genera
Molds are important in food because they can grow even in conditions in which many bacteria cannot grow, such as low pH, low water activity (Aw) and high osmotic pressure. Some of the most common genera of molds found in food are listed here (see also Figure 8.1).
Important Yeast Genera
Important Viruses
Important Bacterial Genera
- IMPORTANT BACTERIAL GROUPS IN FOODS
It is found in the intestinal contents of humans, animals and birds and in the environment. Slightly curved bars; some cells stain unevenly; facultative anaerobes; motionless; mesophiles; found in the environment, plants and animals.
Lactic Acid Bacteria
For convenience, we have arbitrarily divided the bacteria important in foods into several groups based on similarities in certain properties.
Acetic Acid Bacteria
Propionic Acid Bacteria
Butyric Acid Bacteria
Proteolytic Bacteria
Lipolytic Bacteria
Saccharolytic Bacteria
Thermophilic Bacteria
- Psychrotrophic Bacteria
Thermoduric Bacteria
Halotolerant Bacteria
Aciduric Bacteria
Osmophilic Bacteria
Gas-Producing Bacteria
Slime Producers
Spore Formers
Aerobes
Anaerobes
Facultative Anaerobes
Coliforms
Fecal Coliforms
- Enteric Pathogens
- CONCLUSION
- PREDOMINANT MICROORGANISMS IN DIFFERENT SOURCES A. Plants (Fruits and Vegetables)
State the general differences in the morphology of yeasts, molds, bacteria, and bacteriophages important in food. Name two genera from each of the following groups: (a) Gram-negative aerobic rods, (b) Gram-negative facultative anaerobic rods, (c) Gram-positive cocci, (d) Gram-positive endospore-forming rods, (f. ) Gram -positive non-sporulating rods.
Animals, Birds, Fish, and Shellfish
In addition to natural microorganisms, food can be contaminated by various types of microorganisms that come from external sources such as air, soil, sewage, water, feed, people, food ingredients, equipment, packaging and insects. Pathogens, especially enteric types, may be present when soil is contaminated with untreated sewage.
Soil
Spores of Bacillus spp., Clostridium spp., and molds and cells of some Gram-positive bacteria (eg Micrococcus spp. and Sarcina spp.), as well as yeasts, may be predominantly present in air. If the environment contains a source of pathogens (eg animal and poultry farms or a sewage treatment plant), various types of bacteria, including pathogens and viruses (including bacteriophages), can be transmitted through the air.
Sewage
Airborne microbial contamination of food can be reduced by removing the potential sources, controlling airborne dust particles (using filtered air), using positive air pressure, lowering humidity and installing UV light.
Water
However, chlorinated drinking water (drinking water) must be used in processing, washing, sewage and as ingredients. To overcome the problems, many food processors use water, especially as an ingredient, that has a higher microbial quality than drinking water.
Humans
Although drinking water does not contain coliforms and pathogens (mainly enteric species), it may contain other bacteria that can cause food spoilage, such as Pseudomonas, Alcaligenes and Flavobacterium.
Food Ingredients
- Equipment
These dead spots can serve as sources of both pathogenic and spoilage microorganisms in food. Small equipment, such as cutting boards, knives, spoons and similar items, can be sources of cross-contamination due to improper cleaning.
Miscellaneous
- CONCLUSION
- RAW AND READY-TO-EAT MEAT PRODUCTS
- RAW AND PASTEURIZED MILK
- SHELL EGG AND LIQUID EGG
- FISH AND SHELLFISH
- VEGETABLES, FRUITS, AND NUTS
- CEREAL, STARCHES, AND GUMS
- CANNED FOODS
- SUGARS AND CONFECTIONERIES
- SOFT DRINKS, FRUIT AND VEGETABLE DRINKS, JUICES, AND BOTTLED WATER
- MAYONNAISE AND SALAD DRESSINGS
- SPICES AND CONDIMENTS
- CONCLUSION
Briefly discuss how an understanding of the microbial sources in food can be useful to a food microbiologist. Many of the microorganisms can cause different types of spoilage (different types of rot) of raw products.
S ECTION II Microbial Growth Response in the Food
MICROBIAL REPRODUCTION OR GROWTH A. Binary Fission
Division can take place in one or more planes, depending on the type and arrangement of cells. The yeast cell produces a bud that is initially much smaller and remains attached to the surface of the original cell.
Generation Time (or Doubling Time)
Specific Growth Rate
Optimum Growth
The growth rate slows on either side of the optimum growth temperature until growth stops. The area below the two points on either side of the optimum growth conditions where minimum growth occurs is the growth temperature range.
Growth Curve
- NATURE OF MICROBIAL GROWTH IN FOOD A. Mixed Population
The growth range and optimal growth of a microorganism under a specific parameter provide valuable information for its inhibition, reduction or stimulation of growth in a food. The growth characteristics of a mixed population differ in several respects from a pure culture (a single strain of a species).
Sequence of Growth
Many foods are more often spoiled by bacteria than by yeast and mould, because bacteria generally have a shorter generation time. In a mixed population, the internal and external environments dictate which one, two, or few of the original mixed population will become dominant and produce specific changes in a food.
Growth in Succession or Diauxic Growth
These aspects are very important in the control of microbial spoilage of foods and in the production of bioprocessed (or fermented) foods.3,4.
Symbiotic Growth
Initially, Streptococcus thermophilus hydrolyzes milk proteins through its extracellular proteinase and generates amino acids, which are necessary for good growth of Lactobacillus delbrueckii subsp.
Synergistic Growth
Antagonistic Growth
- CONCLUSION
- INTRINSIC FACTORS OR FOOD ENVIRONMENT
34;In foods, microorganisms are present as a mixed population. What disadvantage does this situation entail in applying the results of pure culture studies in food systems? However, as previously mentioned, the factors in a food system are present together and exert a combination, either positively or negatively, on microbial growth.
Nutrients and Growth
Proteins are present in larger amounts in foods of animal origin than in foods of plant origin. Cholesterol is present in foods of animal origin or in foods containing ingredients from animal sources.
Growth Factors and Inhibitors in Food
Lysis of dead microbial cells in foods results in the release of intracellular lipases and oxidases, which can then carry out these reactions. In general, most foods contain a variety of carbohydrates, proteins, lipids, minerals, and vitamins in sufficient amounts to supply the necessary nutrients for the growth of molds, yeasts, and bacteria, especially Gram-negative bacteria, which are commonly present in foods.
Water Activity and Growth 1. Principle
When the Aw is reduced below the minimum level necessary for the growth of a microorganism, the cells remain viable for a while. This information is used to control spoilage and pathogenic microorganisms in food and to promote the growth of desirable types in food bioprocessing (such as adding salt in cured ham processing; see Chapter 34) and in laboratory detection of microorganisms (addition of salt to media). about Sta. aureus).
- EXTRINSIC FACTORS
Oxygen can be present in a food in gaseous form (on the surface, trapped inside) or in dissolved form. However, this varies greatly with concentrations of reducing components in a food and the presence of oxygen.
Temperature and Growth 1. Principle
- RESPIRATION AND FERMENTATION DURING GROWTH During growth in a food, microorganisms synthesize energy and cellular materials
- METABOLISM OF FOOD CARBOHYDRATES
Microbial growth and viability are important considerations in reducing food spoilage and improving safety against pathogens, as well as in food bioprocessing. Various energy generation and energy degradation processes that are important in food microbiology are briefly discussed here (also in Chapter 11).
Degradation of Polysaccharides
In foods rich in both carbohydrates and proteins, microorganisms usually use the carbohydrates first, producing acids and lowering the pH. Polysaccharides are broken down into mono- and disaccharides by extracellular microbial enzymes (e.g. α-amylase) which are secreted into the environment before they can be transported and metabolised.
Degradation of Disaccharides
Thus, microorganisms growing in foods rich in metabolizable carbohydrates use carbohydrates, but in foods low in metabolizable carbohydrates and rich in metabolizable proteins, they metabolize proteins (after metabolizing the carbohydrates). Subsequent microbial degradation of proteins can be prevented at low pH, which does not degrade proteins or create a protein-sparing effect.
Degradation of Monosaccharides
The EMP pathway is used by homofermentative lactic acid bacteria, Enterococcus faecalis, Bacillus spp., and yeasts. This pathway is used by Escherichia coli, Enterobacter aerogenes, Bacillus spp., and some lactic acid bacteria.
Anaerobic Respiration
Aerobic Respiration
Synthesis of Polymers
- METABOLISM OF FOOD PROTEINS
Proteins and large peptides in a food are hydrolyzed to amino acids and small peptides by microbial extracellular proteinases and peptidases. Small peptides are transported in the cell and converted into amino acids before being further metabolized.1,4,5.
Aerobic Respiration (Decay)
Protein compounds present in foods include various types of simple proteins (eg, albumin, globulin, zein, keratin, and collagen), conjugated proteins (eg, myoglobin, hemoglobin, and casein), and peptides containing two or more amino acids. In general, microorganisms can transport amino acids and small peptides (about 8 to 10 amino acids long) into cells.
Fermentation (Putrefaction)
- METABOLISM OF FOOD LIPIDS
- CONCLUSION
- MOLD SPORES
- YEAST SPORES
- BACTERIAL SPORES
Some of the microorganisms that are important in food and can release lipases (hydrolytic enzymes) are found in the following genera: Alcaligenes, Enterobacter, Flavobacterium, Micrococcus, Pseudomonas, Serratia, Staphylococcus, Aspergillus,. Microorganisms important in food usually divide by binary fission (or elongation, as in non-septate forms).
Sporulation
Dormancy
Activation
Germination
Outgrowth
- IMPORTANCE OF SPORES IN FOOD
- STRESS ADAPTATION A. Definition and Observations
More such studies with many strains of the important species will provide more meaningful data to determine the potential of such a treatment to control bacterial spores in food. Explain how low hydrostatic pressure can be combined with other antibacterial treatments to destroy bacterial spores in food.
Mechanisms of Stress Adaptation 10,11
To overcome this problem, it is necessary to understand the basic mechanisms that confer resistance to stress-adapted cells and to develop methods to control them.10.
Importance of Stress-Adapted Microorganisms in Food 5,11
- SUBLETHAL STRESS AND INJURY A. Definition and Observations
However, if a pathogenic strain in a food is stress-adapted, even consumption of a much lower number will enable it to survive in the stomach and cause infection in the GI tract. However, first exposing the cultures to a mild stress to release stress proteins may enable the cells to survive subsequent freezing, freeze-drying or exposure to low pH in the stomach or in food products.
Manifestation of Bacterial Sublethal Injury
The survivors form colonies in the non-selective TS agar (they repair and multiply), but 80.1% of the survivors fail to form colonies in the selective TSD agar (due to their damage and developed sensitivity to deoxycholate). Among the survivors, however, 19.9% are normal or undamaged cells, as they grow equally well in both the TS and TSD agar media and are no longer sensitive to deoxycholate.
Sites and Nature of Injury
It is suggested that protein molecules in this structure probably undergo conformational changes in the damaged cells. Irradiation damage (UV and g) is mainly limited to DNA in the form of single-strand breaks and by some chemicals (H2O2, antibiotics or chlorine) to the lytic enzymes of the germinal system.
Repair of Reversible Injury
In some strains, autolytic enzymes can be activated by stress, causing cell death.1–3,14. Milder to stronger acid treatments can cause the spores to go dormant by removing Ca2+ from the spores and making them sensitive to heat.2,3. to destroy H2O2 produced by the cells) also improves repair and increases the number of repaired cells.
Injury in Yeasts and Molds
Importance of Sublethally Injured Microorganisms in Food
The cells included under this term cannot multiply in one selective environment, but multiply in another environment. Also, after resuscitation, they can proliferate in the selective environment (some similarities with sublethal injured cells).
Proponent Views
Due to the current controversy among researchers studying the VBNC phenomenon of bacterial cells, the views and observations of the groups are summarized in this chapter. Cells can be induced to proliferate by removing the VBNC-inducing factor or by supplying a revival-inducing factor to the culture medium.
Opponent Views
Once resuscitated in a suitable environment, the cells multiply rapidly like the cells in a normal population.
Current Views
Importance of VBNC Microorganisms in Food
- CONCLUSION
Kell, D.B., Kaprelyants, A.S., Weichart, D.H., Harwood, C.R., and Barer, M.R., Viability and activity of readily culturable bacteria: a review and discussion of practical issues, Ant. Mizunoe, Y., Wai, S.N., Ishikawa, T., Takade, A., and Yoshida, S., Revival of viable but nonculturable Vibrio parahaemolyticus cells induced at low temperature during starvation.
S ECTION III Beneficial Uses of Microorganisms in Food
MICROBIOLOGY OF FERMENTED FOODS
Food fermentation involves a process in which raw materials are converted into fermented food through the growth and metabolic activities of the desired microorganisms. The unused components of the raw materials and the microbial by-products (and sometimes microbial cells) together form fermented food.
LACTIC STARTER CULTURES
These microbial species, when used in controlled fermentation, are also called starter cultures. Three subspecies of Lactobacillus delbrueckii are used in the fermentation of dairy products such as some cheeses and yogurt.
OTHER STARTER CULTURES A. Bifidobacterium
They are present in large numbers in babies' stools within 2 to 3 days after birth and are usually present in high numbers in breastfed babies. The genus includes species in the classical or milky propionibacterium group and the skin or acne propionibacterium group.
YEASTS AND MOLDS
Yeasts
Molds
- MECHANISMS OF TRANSPORT OF NUTRIENTS
- TRANSPORT AND METABOLISM OF CARBOHYDRATES In lactic acid bacteria and other bacteria used in food fermentation, disaccharide
The beneficial microorganisms metabolize some of the components present in the starting materials (ie food, such as milk or meat) to produce energy and cellular materials and to reproduce. It will be advantageous to briefly recognize the cellular components involved in the transport of these substrates.
PEP-PTS System for Lactose Transport in Lactococcus lactis
In the PEP-PTS system for PTS sugars, the energy is extracted from PEP; in the permease system (for permease sugars, amino acids and probably small peptides) energy is derived from proton motive force. Similarly, in one species, some carbohydrates are transported by the PEP-PTS system while others are transported by the permease system.
Carbohydrates Available inside the Cells for Metabolism
Homolactic Fermentation of Carbohydrates
The overall reaction involves the production of two molecules each of lactic acid and ATP from one molecule of hexose. In addition to being an important component in the production of fermented foods (e.g. yogurt, cheeses and fermented sausages and vegetables), lactic acid is used as an ingredient in many foods (e.g. processed meat products).7 For this purpose, L (+)- lactic acid is preferred and approved by regulatory authorities as a food additive (since it is also produced by the muscle).
Heterolactic Fermentation of Carbohydrates
Genetic studies are currently being conducted to develop strains by inactivating the D-lactate dehydrogenase system in species containing both L. Some species that produce L(+)-lactic acid (>90% or more) are Lactococcus lactis sp.
Metabolism of Pentoses
Hexose Fermentation by Bifidobacterium
Diacetyl Production from Citrate
- Propionic Acid Production by Propionibacterium
- TRANSPORT AND METABOLISM OF PROTEINACEOUS COMPOUNDS AND AMINO ACIDS
- PLASMIDS AND PLASMID-LINKED TRAITS IN STARTER-CULTURE BACTERIA
The nature of genes and the mechanisms by which the genetic codes are translated into enzymes in lactic acid bacteria are discussed in Chapter 12. Initial research in the early 1970s revealed that many industrially important phenotypes in different lactic acid bacteria were plasmid linked.
Important Characteristics of Bacterial Plasmids
Starter culture bacteria, like other bacteria, carry genetic information (genetic code) in the circular chromosomal DNA, circular plasmids and transposons. Properties of plasmids and some plasmid-linked properties in starter culture bacteria are discussed here.1,2.
Some Characteristics of Small (ca. 10 kb) and Large (over 10 to ca
Presence of Plasmids in Some Starter-Culture Bacteria
Phenotype Assignment to a Plasmid
After curing, the transconjugants were converted to Lac-, and an analysis showed that these variants no longer had the 53-kb plasmid. From this series of experiments, it was determined that the 53-kb plasmid in the specific Lac.
Plasmid-Linked Traits in Starter-Culture Bacteria
Analysis of the plasmid profile, i.e. types of plasmids present as determined from their molecular weight (kb), showed that a 53-kb plasmid present in the Lac+ wild strain is missing in Lac-hardy variant. To further determine that the 53-kb plasmid actually encoded the Lac+ phenotype, the wild Lac+ strain was conjugally mated with a plasmidless Lac– strain, and several Lac+ transconjugants were obtained.
Cryptic Plasmids
Bac+, production of various bacteriocins (also their respective immune, processing and translocation characteristics; bacteriocin, like nisin, is encoded in a transposon). Resistance to various antibiotics (such as Kmr, resistance to kanamycin) Metabolism of various carbohydrates (such as Gal+, galactose utilization) Muc+, mucin production.
Plasmid Mapping and Sequencing
- GENE TRANSFER METHODS IN STARTER-CULTURE BACTERIA Once the genetic basis of a phenotype in bacteria was understood, studies were
In theta-type replication, initiation begins with the formation of replication forks (as in the chromosome) due to base pair separation. Both strands are then replicated simultaneously, either in the same or opposite directions, and two copies of the plasmids are formed at the end of replication.
Transduction
Conjugation
These include plasmid size, plasmid incompatibility and instability in recipient strains, inability to express in hosts, inability to have proper donors and recipients, and, in some cases, inability to recognize the transconjugant. However, using a broad-host range plasmid (e.g. pAMb1, a plasmid from Enterococcus spp. encoding an antibiotic gene), it has been shown that plasmid transfer by conjugation is possible between lactic acid bacteria between the same species, between two different species in the same genus, or even between two different species from different genera.
Transformation
Protoplast Fusion
Electrotransformation
In addition, vectors carrying cloned genes from various sources have been successfully introduced into several species of lactic acid bacteria. This is currently the most preferred method for transferring DNA from a source to recipient cells of lactic acid bacteria.
Conjugative Transposons
- GENE CLONING
This method has been widely used in many lactic acid bacteria to introduce plasmids from different strains of the same species as well as from separate species and genera. This method is now used to transfer genes from different sources into lactic acid bacteria.
Cloning Vectors
A suitable plasmid (cloning vector) is selected which has one or more genetic markers (such as resistance to an antibiotic or metabolizing a carbohydrate) and a site that can be hydrolysed, preferably with the same restriction enzymes. This plasmid, carrying the genes from the donor, can then be introduced into a bacterial cell in several ways, most effectively by electroporation in lactic acid bacteria, as previously described.
Metabolic Engineering
To develop this strain, the nox gene (NADH oxidase) is first cloned from a suitable source under the control of the nisin-inducible niche A promoter (NICE) in the Lac. In the presence of a small amount of nisin, the nox gene is overexpressed in the stem.
Protein Targeting
In contrast, Leuconostoc mesenteroides secretes most of the mannitol into the environment because it has an efficient mannitol transport system (it also produces high amounts of mannitol). Many lactic acid bacteria are normally present in the gastrointestinal (GI) tracts of humans and food animals and poultry, and some of them have beneficial effects on the health and well-being of the hosts (see Chapter 15).
Protein Engineering
- GENOME MAPPING AND SEQUENCING
In order to understand the characteristics at the molecular level, efforts have been made in recent years to sequence the complete genome (chromosome) of several species of important lactic acid bacteria. These techniques have also helped to sequence the genomes of many phages and prophages of lactic acid bacteria.
Bacteriophages
The genome appears to have a fluid structure and can change through point mutation, DNA rearrangement, and horizontal gene transfer. This information can then be used to modulate gene expression and efficiently perform metabolic engineering to develop new strains for use in the production of new fermented products and important by-products.16–19.
The lac and las Genes
- HISTORY
- CONCENTRATED CULTURES
Renault, P., Genetic Engineering Strategies, in Lactic Acid Bacteria: Current Advances in Metabolism, Genetics and Applications, Bozoglu, T.F. With one example, explain the importance of (a) protein targeting and (b) protein engineering research in lactic acid bacteria.
Loss of a Desired Trait
In mixed-strain cultures, in which an initial culture contains two or more strains, the dominance of one over the others at a given condition can change the culture profile rapidly. Cultivators test for compatibility between desirable strains and develop mixed strain cultures with only compatible strains to avoid strain antagonism.4.
Cell Death and Injury
Inhibitors in Raw Materials
Bacteriophages of Lactic Acid Bacteria
- YEAST AND MOLD CULTURES
The phage DNA (called prophage) is carried by a bacterial cell DNA (lysogeny state), and as a bacterial cell multiplies, the phage also multiplies regardless of the presence of the phage in the bacterial strain (lysogenic strain) to show Recent genomic studies have shown that the DNA of a host strain can have five or more types of prophages.6 However, a prophage can be induced by a physical (such as UV) or a chemical (such as mitomycin C) agent, causing the phage DNA to separate from bacterial DNA and resume the lytic cycle (Figure 13.2).
