2.

FOOD MICROBIOLOGY
MORPHOLOGY AND STRUCTURE OF MICROORGANISMS IN FOODS The microbial groups important in foods consist of several species and types of bacteria, yeasts, molds, and viruses. 2.1. THE BACTERIAL CELL Bacteria constitute a large domain of prokaryotic microorganisms. Bacteria are unicellular, most ca. 0.51.0 2.010 mm in size, and have three morphological forms: spherical (cocci), rod shaped (bacilli), and curved (comma) They can form associations such as clusters, chains (two or more cells), or tetrads. They can be motile or nonmotile. Cytoplasmic materials are enclosed in a rigid wall on the surface and a membrane beneath the wall. It also forms intrusions in the cytoplasm (mesosomes). The cytoplasmic material is immobile and does not contain organelles enclosed in a separate membrane. The ribosomes are 70S type and are dispersed in the cytoplasm. The genetic materials (structural and plasmid DNA) are circular, not enclosed in nuclear membrane, and do not contain basic proteins such as histones. Both gene transfer and genetic recombination occur, but do not involve gamete or zygote formation. Cell division is by binary fission. Procaryotic cells can also have flagella, capsules, surface layer proteins, and pili for specific functions. Some also form endospores On the basis of Gram-stain behavior, bacterial cells are grouped as Gramnegative or Gram-positive. Gram-negative cells have a complex cell wall containing an outer membrane (OM) and a middle membrane (MM) The OM is composed of lipopolysaccharides (LPS), lipoprotein (LP), and phospholipids. Phospholipid molecules are arranged in a bilayer, with the hydrophobic part (fatty acids) inside and hydrophilic part (glycerol and phosphate) outside. LPS and LP molecules are embedded in the phospholipid layer. The OM has limited transport and barrier functions. The resistance of Gramnegative bacteria to many enzymes (lysozyme, which hydrolyzes mucopeptide), hydrophobic molecules (SDS and bile salts), and antibiotics (penicillin) is due to the barrier property of the OM. LPS molecules also have antigenic properties. Beneath the OM is the MM, composed of a thin layer of peptidoglycan or mucopeptide embedded in the periplasmic materials that contain several types of proteins. Beneath the periplasmic materials is the plasma or inner membrane (IM), composed of a phospholipid bilayer in which many types of proteins are embedded. Gram-positive cells have a thick cell wall composed of several layers of mucopeptide (responsible for thick rigid structure) and two types of teichoic acids. Some species also have a layer over the cell surface, called surface layer protein (SLP). 83

The wall teichoic acid molecules are linked to mucopeptide layers, and the lipoteichoic acid molecules are linked to both mucopeptide and cytoplasmic membrane. Teichoic acids are negatively charged (because of phosphate groups) and may bind to or regulate the movement of cationic molecules in and out of the cell. Teichoic acids have antigenic properties and can be used to identify Gram-positive bacteria serologically. Because of the complexity in the chemical composition of the cell wall, Gram-positive bacteria are considered to have evolved before Gram-negative bacteria.

Summary of characteristics of typical bacterial cell structures Predominant chemical Structure Function(s) composition Flagella Swimming movement Protein Pili Stabilizes mating bacteria Protein Sex pilus during DNA transfer by conjugation Attachment to surfaces; Common pili or protection against Protein fimbriae phagotrophic engulfment Attachment to surfaces; Capsules protection against phagocytic (includes Usually polysaccharide; engulfment, occasionally "slime layers" occasionally killing or digestion; reserve of and polypeptide nutrients or protection glycocalyx) against desiccation Cell wall Prevents osmotic lysis of cell Peptidoglycan (murein) Gram-positive protoplast and confers rigidity complexed with teichoic bacteria and shape on cells acids Peptidoglycan prevents osmotic lysis and confers Peptidoglycan (murein) rigidity and shape; outer surrounded by Gram-negative membrane is permeability phospholipid proteinbacteria barrier; associated LPS and lipopolysaccharide proteins have various "outer membrane" functions Permeability barrier; transport Plasma of solutes; energy generation; Phospholipid and membrane location of numerous enzyme protein systems Sites of translation (protein Ribosomes RNA and protein synthesis) Often reserves of nutrients; Highly variable; Inclusions additional specialized carbohydrate, lipid, functions protein or inorganic Chromosome Genetic material of cell DNA Plasmid Extrachromosomal genetic DNA 84

material

SHAPE AND SIZE OF BACTERIA Most bacteria are 0.2 um in diameter and 2-8 um in length. The three basic bacterial shapes are coccus (spherical), bacillus (rod-shaped), and spiral (twisted), however pleomorphic bacteria can assume several shapes.

Arrangement of cocci Cocci may be oval, elongated, or flattened on one side. Cocci may remain attached after cell division. These group characteristics are often used to help identify certain cocci.

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Cocci that remain in pairs after dividing are called diplococci.

Cocci that remain in chains after dividing are called streptococci.

Cocci that divide in two planes and remain in groups of four are called tetrads.

Cocci that divide in three planes and remain in groups cube like groups of eight are called sarcinae.

Cocci that divide in multiple planes and form grape like clusters or sheets are called staphylococci.

Bacilli Since bacilli only divide across their short axis there are fewer groupings. Bacillus is a shape (rod shaped) but there is also a genus of bacteria with the name Bacillus. You wouldn't confuse the two, since you know the rules for writing the genus and species names of organisms, right????

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Most bacilli appear as single rods. Diplobacilli appear in pairs after division.

Streptobacilli appear chains after division.

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Some bacilli are so short and fat that they look like cocci and are referred to as coccobacilli.

Spiral bacteria Spiral bacteria have one or more twists.

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Vibrios look like curved rods.

Spirilla have a helical shape and fairly rigid bodies.

Spirochetes have a helical shape and flexible bodies. Spirochetes move by means of axial filaments, which look like flagella contained beneath a flexible external sheath. Other shapes

Stella are star-shaped.

Haloarcula, a genus of halophilic archaea, are rectangular.

MAJOR BACTERIAL GROUPS A. Lactic Acid Bacteria They are bacteria that produce relatively large quantities of lactic acid from carbohydrates. 88

Species mainly from genera Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, and Streptococcus thermophilus are included in this group. B. Acetic Acid Bacteria They are bacteria that produce acetic acid, such as Acetobacter aceti. C. Propionic Acid Bacteria They are bacteria that produce propionic acid and are used in dairy fermentation. Species such as Propionibacterium freudenreichii are included in this group. D. Butyric Acid Bacteria They are bacteria that produce butyric acid in relatively large amounts. Some Clostridium spp. such as Clostridium butyricum are included in this group. E. Proteolytic Bacteria They are bacteria that can hydrolyze proteins because they produce extracellular proteinases. Species in genera Micrococcus, Staphylococcus, Bacillus, Clostridium, Pseudomonas, Alteromonas, Flavobacterium, Alcaligenes, some in Enterobacteriaceae, and Brevibacterium are included in this group. F. Lipolytic Bacteria They are bacteria that are able to hydrolyze triglycerides because they produce extracellular lipases. Species in genera Micrococcus, Staphylococcus, Pseudomonas, Alteromonas, and Flavobacterium are included in this group. G. Saccharolytic Bacteria They are bacteria that are able to hydrolyze complex carbohydrates. Species in genera Bacillus, Clostridium, Aeromonas, Pseudomonas, and Enterobacter are included in this group. H. Thermophilic Bacteria They are bacteria that are able to grow at 50 C and above. Species from genera Bacillus, Clostridium, Pediococcus, Streptococcus, and Lactobacillus are included in this group. I. Psychrotrophic Bacteria They are bacteria that are able to grow at refrigerated temperature (5C). Some species from Pseudomonas, Alteromonas, Alcaligenes, Flavobacterium, Serratia, Bacillus, Clostridium, Lactobacillus, Leuconostoc, Carnobacterium, Brochothrix, Listeria, Yersinia, and Aeromonas are included in this group. J. Thermoduric Bacteria They are bacteria that are able to survive pasteurization temperature treatment. Some species from Micrococcus, Enterococcus, Lactobacillus , Pediococcus, Bacillus (spores), and Clostridium (spores) are included in this group. K. Halotolerant Bacteria 89

They are bacteria that are able to survive high salt concentrations ( 10%). Some species from Bacillus, Micrococcus, Staphylococcus, Pediococcus, Vibrio, and Corynebacterium are included in this group. L. Aciduric Bacteria They are bacteria that are able to survive at low pH (<4.0). Some species from Lactobacillus, Pediococcus, Lactococcus, Enterococcus, and Streptococcus are included in this group. M. Osmophilic Bacteria They are bacteria that can grow at a relatively higher osmotic environment than that needed for other bacteria. Some species from genera Staphylococcus, Leuconostoc, and Lactobacillus are included in this group. They are much less osmophilic than yeasts and molds. N. Gas-Producing Bacteria They are bacteria that produce gas (CO2, H2, H2S) during metabolism of nutrients. Species from genera Leuconostoc, Lactobacillus, Propionibacterium, Escherichia, Enterobacter, Clostridium, and Desulfotomaculum are included in this group. O. Slime Producers They are bacteria that produce slime because they synthesise polysaccharides. Some species or strains from Xanthomonas, Leuconostoc, Alcaligenes, Enterobacter, Lactococcus, and Lactobacillus are included in this group. P. Spore Formers They are bacteria having the ability to produce spores. Species from Bacillus, Clostridium, and Desulfotomaculum are included in this group. They are further divided into aerobic sporeformers, anaerobic sporeformers, flat sour sporeformers, thermophilic sporeformers, and sulfide-producing sporeformers. Q. Aerobes They are bacteria that require oxygen for growth and multiplication. Species from Pseudomonas, Bacillus, and Flavobacterium are included in this group. R. Anaerobes They are bacteria that cannot grow in the presence of oxygen. Species from Clostridium are included in this group. S. Facultative Anaerobes They are bacteria that are able to grow in both the presence and absence of oxygen. Lactobacillus, Pediococcus, Leuconostoc, enteric pathogens, and some species of Bacillus, Serratia, and coliforms are included in this group. T. Coliforms 90

Species from Escherichia, Enterobacter, Citrobacter, and Klebsiella are included in this group. They are used as an index of sanitation. U. Fecal Coliforms Mainly Escherichia coli is included in this group. They are also used as an index of sanitation. V. Enteric Pathogens Pathogenic Salmonella, Shigella, Campylobacter, Yersinia, Escherichia, Vibrio, Listeria , hepatitis A, and others that can cause gastrointestinal infection are included in this group.

IDENTIFICATION OF BACTERIA 1. Bacterial species a. Similar individuals: i. ii. A bacterial species is characteristics." "a population of cells with similar

Note that this definition is very different from how plant and animal species are often defined, which usually involves something to do with sex.

b. Resemble fossil species: i. ii. Bacterial species resemble the way fossil species distinguished (i.e., phylogenetic species concept). are

Normally plant and especially animal species are defined as populations of interbreeding or potentially interbreeding individuals that don't breed with individuals of other, likedefined populations. For fossils, on the other hand, it cannot be determined who interbred, or could have, with whom. Consequently, fossil species are defined only in terms of character resemblance, just as are bacteria.

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2. Bergey's manual a. Bergey's manual is a guide to distinguishing bacterial species based on phenotypic differences between isolates. 3. Strain a. A strain is a subset of a bacterial species differing from other bacteria of the same species by some minor but identifiable difference. b. A strain is "a population of organisms that descends from a single organism or pure culture isolate. Strains within a species may differ slightly from one another in many ways." (p. 392, Prescott et al., 1996) 91

c. Strains are often created in the laboratory by mutagenizing existing strains or wild-type examples of bacterial species. d. The term strain is also applicable to eucaryotic microorganisms , as well as to viruses . 4. Type strain a. "One strain of a species is designated as the type strain. It is usually one of the first strains studied and is often more fully characterized than other strains; however, it does not have to be the most representative member. Only those strains very similar to the type strain are included in a species." 5. Serovar [serotype] a. A serovar is a strain differentiated by serological means. b. Individual strains of Salmonella spp. are often distinguished and distinguishable by serological means. 6. Biovar [biotype] a. Biovars are strains that are differentiated by biochemical or other nonserological means. 7. Morphovar [morphotype] a. A morphovar is a strain which is differentiated on the basis of morphological distinctions. 8. Isolate a. An isolate is a pure culture derived from a heterogeneous, wild population of microorganisms . b. The term isolate is also applicable to eucaryotic microorganisms as well as to viruses . 9. Classification a. Placement of an organism within a scheme relating different types of organisms, such as that presented in Woese's universal tree , is know as classification. b. Organisms are classified for scientific purposes. 10. Identification a. Identification is the determination of whether an organism (or isolate in the case of microorganisms ) should be placed within a group of organisms known to fit within some classification scheme. b. Organisms are identified for practical purposes, such as diagnosis of disease . 92

c. Many identification techniques: i. Many different criteria may be employed for identification, though it is often desirable to employ the easiest techniques possible. What techniques and tests may be necessary, however, depend on what organism is being identified and how much detail into the organism's classification you are interested. Techniques include: 1. morphological identification 2. differential staining 3. use of differential media 4. serological methods 5. flow cytometry 6. phage typing 7. protein analysis 8. comparisons of nucleotide sequences 11. Morphological identification a. A number of morphological characteristics are useful bacterial identification. These include the presence or absence of: i. ii. iii. iv. endospores flagella glycocalyx etc. in

ii.

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b. Additional considerations include: i. ii. iii. iv. 12. colony morphology cell shape cell size etc.

Serological methods [agglutination test, ELISA, Western blot] a. Antibodies: i. Serological methods employ antibodies and include: 1. agglutination tests 93

2. ELISAs 3. Western blots ii. It is antibody binding that all serological tests ultimately detect.

b. The basic premise behind all of these tests is that antibodies are highly selective in terms of the proteins (or other cell structures) to which they bind, to the point that they are able to distinguish the proteins coming from one bacterial species among many species, or even one strain among many strain. 13. Flow cytometry a. Flow cytometry is a technique that can employ serological methods (but doesn't necessarily) that analyzes cells suspended in a liquid medium by light, electrical conductivity, or fluorescence as the cells individually pass through a small orifice. 14. Phage typing a. Bacteriophage (or phage ) are viruses that infect bacteria . b. Phage can be very specific in what bacteria they infect and the pattern of infection by many phage may be employed in phage typing to distinguish bacterial species and strains. 15. Protein analysis [gel electrophoresis, SDS-PAGE, establishment of clonality] a. The size and other differences between proteins among different organisms may be determined very easily employing methods of protein separation using methods collectively known as gel electrophoresis. b. SDS-PAGE: i. One popular technique goes by the name SDS-PAGE which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis Note that another name for SDS is sodium lauryl sulfate, a detergent you will find in many shampoos.

ii.

c. Such methods are very good at detecting small differences between isolates and are especially good at establishing clonality. 16. Comparison of nucleotide sequences [Southern blot, nucleic acid hybridization, RFLP, DNA fingerprinting] a. The actual sequence of bases (nucleotides) in the genome of organisms may be inferred or actually determined (nucleotide sequencing) by a variety of methods. b. Various methods of inference include: 94

i. ii. iii.

Southern blotting nucleic acid hybridization RFLP comparison (restriction fragment length polymorphism or DNA fingerprinting)

c. Another technique that is worth knowing about is PCR which stands for polymerase chain reaction, a method of amplifying specific regions of DNA found in an organisms genome by selectively catalyzing the replication of those regions. 17. Most Probable Number Method (MPN) The most probable number (MPN) is particularly useful for low concentrations of organisms (<100/g), especially in milk and water, and for those foods whose particulate matter may interfere with accurate colony counts. Only viable organisms are enumerated by the MPN determination. If, in the microbiologist's experience, the bacteria in the prepared sample in question can be found attached in chains that are not separated by the preparation and dilution, the MPN should be judged as an estimate of growth units (GUs) or colony-forming units (CFUs) instead of individual bacteria. The following assumptions are necessary to support the MPN method. The sample is prepared in such a way that the bacteria are distributed randomly within it. The bacteria are separate, not clustered together, and they do not repel each other. The growth medium and conditions of incubation have been chosen so that every inoculum that contains even one viable organism will produce detectable growth. The essence of the MPN method is the dilution of a sample to such a degree that inocula will sometimes but not always contain viable organisms. The "outcome", i.e., the numbers of inocula producing growth at each dilution, will imply an estimate of the original, undiluted concentration of bacteria in the sample. In order to obtain estimates over a broad range of possible concentrations, microbiologists use serial dilutions, incubating several tubes (or plates, etc.) at each dilution. MPN tables should omit those combinations of positive tubes (high for low concentrations and low for high concentrations) that are so improbable that they raise concerns about laboratory error or contamination. Selecting Three Dilutions for Table Reference An MPN can be computed for any numbers of tubes at any numbers of dilutions. MPN values based on 3 decimal dilutions, however, are very close approximations to those based on 4 or more dilutions. When more than three dilutions are used in a decimal series of dilutions, refer to the 3 dilution table according to the following two cases, illustrated by the table of examples below (with 5 tubes at each dilution). Case 1. One or more dilutions show all tubes positive. Select the highest dilution that gives positive results in all tubes (even if a lower dilution gives 95

negative results) and the next two higher dilutions (ex. a and b); if positive results occur in higher unselected dilutions, shift each selection to the next higher dilution (ex. c). If there are still positive results in higher unselected dilutions, add those higher-dilution positive results to the results for the highest selected dilution (ex. d). If there were not enough higher dilutions tested to select three dilutions, then select the next lower dilutions (ex. e). Case 2. No dilutions show all tubes positive. Select the 3 lowest dilutions (ex. f). If there are positive results in higher unselected dilutions, add those higher-dilution positive results to the results for the highest selected dilution (ex. g).

2.2. YEASTS AND MOLDS Both yeasts and molds are eucaryotic, but yeasts are unicellular whereas molds are multicellular. Eucaryotic cells are generally much larger (20 to 100 mm) than procaryotic cells (1 to 10 mm). The cell wall does not have mucopeptide, is rigid, and is composed of carbohydrates. The plasma membrane contains sterol. The cytoplasm is mobile (streaming) and contains organelles (mitochondria, vacuoles) that are membrane bound. Ribosomes are 80S type and attached to the endoplasmic reticulum. The DNA is linear (chromosomes), contains histones, and is enclosed in a nuclear membrane. Cell division is by mitosis (i.e., asexual reproduction); sexual reproduction, when it occurs, is by meiosis. Molds are nonmotile, filamentous, and branched The cell wall is composed of cellulose, chitin, or both. A mold (thallus) is composed of large numbers of filaments called hyphae. An aggregate of hyphae is called mycelium. A hypha can be nonseptate, septate-uninucleate, or septate-multinucleate. A hypha can be vegetative or reproductive. The reproductive hypha usually extends in the air and form exospores, either free (conidia) or in a sack (sporangium). Shape, size, and color of spores are used for taxonomic classification. 96

Yeasts are widely distributed in nature. The cells are oval, spherical, or elongated, about 530 210 mm in size They are nonmotile. The cell wall contains polysaccharides (glycans), proteins, and lipids. The wall can have scars, indicating the sites of budding. The membrane is beneath the wall. The cytoplasm has a finely granular appearance for ribosomes and organelles. The nucleus is well defined with a nuclear membrane. 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. Many types of molds are found in foods. They are important spoilage microorganisms. Many strains also produce mycotoxins

Aspergillus. It is widely distributed and contains many species important in food. Members have septate hyphae and produce black-colored asexual spores on conidia. Many are xerophilic (able to grow in low Aw) and can grow in grains, causing spoilage. They are also involved in spoilage of foods such as jams, cured ham, nuts, and fruits and vegetables (rot). Some species or strains produce mycotoxins (e.g., Aspergillus flavus produces aflatoxin). Many species or strains are also used in food and food additive processing. Asp. oryzae is used to hydrolyze starch by a-amylase in the production of sake. Asp. niger is used to process citric acid from sucrose and to produce enzymes such as b-galactosidase. MAJOR FUNGI FOUND IN FOOD WITH MORPHOLOGY Alternaria. Members are septate and form dark-colored spores on conidia. They cause rot in tomatoes and rancid flavor in dairy products. Some species or strains produce mycotoxins. Species: Alternaria tenuis. Fusarium. Many types are associated with rot in citrus fruits, potatoes, and grains. They form cottony growth and produce septate, sickle-shaped conidia. Species: Fusarium solani. Geotrichum. Members are septate and form rectangular arthrospores. They grow, forming a yeastlike cottony, creamy colony. They establish easily in equipment and often grow on dairy products (dairy mold). Species: Geotrichum candidum. Mucor. It is widely distributed. Members have nonseptate hyphae and produce sporangiophores. 97

They produce cottony colonies. Some species are used fermentation and as a source of enzymes. They cause spoilage of vegetables. Species: Mucor rouxii.

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Penicillium. It is widely distributed and contains many species. Members have septate hyphae and form conidiophores on a blue-green, brush like conidia head. Some species are used in food production, such as Penicillium roquefortii and Pen. camembertii in cheese. Many species cause fungal rot in fruits andvegetables. They also cause spoilage of grains, breads, and meat. Some strains produce mycotoxins (e.g., Ochratoxin A). Rhizopus. Hyphae are aseptate and form sporangiophores in sporangium. They cause spoilage of many fruits and vegetables. Rhizopus stolonifer is the common black bread mold. Yeast genera: Saccharomyces. Cells are round, oval, or elongated. It is the most important genus and contains heterogenous groups Saccharomyces cerevisiae variants are used in baking for leavening bread and in alcoholic fermentation. They also cause spoilage of food, producing alcohol and CO2. Pichia. Cells are oval to cylindrical and form pellicles in beer, wine, and brine to cause spoilage. Some are also used in oriental food fermentation. Species: Pichia membranaefaciens. Rhodotorula. They are pigmentforming yeasts and can cause discoloration of foods such as meat, fish, and sauerkraut. Species: Rhodotorula glutinis. Torulopsis. Cells are spherical to oval. They cause spoilage of milk because they can ferment lactose (e.g., Torulopsis versatilis). They also spoil fruit juice concentrates and acid foods. Candida. Many species spoil foods with high acid, salt, and sugar and form pellicles on the surface of liquids. Some can cause rancidity in butter and dairy products (e.g., Candida lipolyticum). Zygosaccharomyces. Cause spoilage of high-acid foods, such as sauces, ketchups, pickles, mustards, mayonnaise, salad dressings, especially those with less acid and o less salt and sugar (e.g., Zygosaccharomyces bailii).

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2.3. MICROBIAL GROWTH IN FOOD Foods, because they provide nutrients for us, also are excellent environments for the growth of microorganisms. Microbial growth is controlled by factors related to the food itself, or intrinsic factors, and also to the environment where the food is being stored, or what are described as extrinsic factors The intrinsic or food-related factors include pH, moisture content, water activity or availability, oxidation-reduction potential, physical structure of the food, available nutrients, and the possible presence of natural antimicrobial agents. Extrinsic or environmental factors include temperature, relative humidity, gases (CO2, O2) present, and the types and numbers of microorganisms present in the food. 2.4. INTRINSIC FACTORS and EXTRINSIC FACTORS INTRINSIC FACTORS Food composition is a critical intrinsic factor that influences microbial growth. If a food consists primarily of carbohydrates, spoilage does not result in major odors. Thus foods such as breads, jams, and some fruits first show spoilage by fungal growth. In contrast, when foods contain large amounts of proteins and/or fats (for example, meat and butter), spoilage can produce a variety of foul odors. This proteolysis and anaerobic breakdown of proteins that yields foulsmelling amine compounds is called putrefaction

One major source of odor is the organic amine cadaverine (imagine the origin of that name). Degradation of fats ruins food as well. For example, the production of short-chained fatty acids from fats renders butter rancid and unpleasant. The pH of a food also is critical because a low pH favors the growth of yeasts and molds In neutral or alkaline pH foods, such as meats, bacteria are more dominant in spoilage and putrefaction.

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The presence and availability of water affect the ability of microorganisms to colonize foods. Simply by drying a food, one can control or eliminate spoilage processes. Water, even if present, can be made less available by adding solutes such as sugar and salt. Water availability is measured in terms of water activity (aw). This represents the ratio of relative humidity of the air over a test solution compared with that of distilled water. When large quantities of salt or sugar are added to food, most microorganisms are dehydrated by the hypertonic conditions and cannot grow Even under these adverse conditions, osmophilic and xerophilic microorganisms may spoil food. Osmophilic[Greek osmus, impulse, and philein, to love] microorganisms grow best in or on media with a high osmotic concentration, whereas xerophilic[Greek xerosis, dry, and philein, to love] microorganisms prefer a low aw environment and may not grow under high aw conditions The oxidation-reduction potential of a food also influences spoilage. When meat products, especially broths, are cooked, they often have lower oxidationreduction potentials. These products with their readily available amino acids, peptides, and growth factors are ideal media for the growth of anaerobes, including Clostridium The physical structure of a food also can affect the course and extent of spoilage. The grinding and mixing of foods such as sausage and hamburger not only increase the food surface area and alter cellular structure, but also distribute contaminating microorganisms throughout the food. This can result in rapid spoilage if such foods are stored improperly. Vegetables and fruits have outer skins (peels and rinds) that protect them from spoilage. 100

Often spoilage microorganisms have specialized enzymes that help them weaken and penetrate protective peels and rinds, especially after the fruits and vegetables have been bruised. Many foods contain natural antimicrobial substances, including complex chemical inhibitors and enzymes. Coumarins found in fruits and vegetables exhibit antimicrobial activity. Cows milk and eggs also contain antimicrobial substances. Eggs are rich in the enzyme lysozyme that can lyse the cell walls of contaminating grampositive bacteria Tabasco and other hot red pepper sauces apparently have particularly desirable antimicrobial characteristics. Herbs and spices often possess significant antimicrobial substances; generally fungi are more sensitive than most bacteria. Sage and rosemary are two of the most antimicrobial spices. Aldehydic and phenolic compounds are found in cinnamon, mustard, and oregano. These compounds inhibit microbial growth. Other important inhibitors are garlic, which contains allicin, and cloves, which have eugenol. Unfermented green and black teas also have well documented antimicrobial properties because of their polyphenol contents, which apparently are diminished when the teas are fermented. Such teas are active against bacteria, viruses, and fungi and may have anticancer properties. EXTRINSIC FACTORS Temperature and relative humidity are important extrinsic factors in determining whether a food will spoil. At higher relative humidities microbial growth is initiated more rapidly, even at lower temperatures When drier foods are placed in moist environments, moisture absorption can occur on the food surface, eventually allowing microbial growth. The atmosphere in which the food is stored also is important. This is especially true with shrink-packed foods because many plastic films allow oxygen diffusion, which results in increased growth of surface associated microorganisms. Excess CO2 can decrease the solution pH, inhibiting microbial growth. Storing meat in a high CO2 atmosphere inhibits gram-negative bacteria, resulting in a population dominated by the lactobacilli. The observation that food storage atmosphere is important has led to the development modified atmosphere packaging (MAP). By the use of modern shrink-wrap materials and vacuum technology, it is possible to package foods with controlled atmospheres. With a carbon dioxide content of 60% or greater in the atmosphere surrounding a food, spoilage fungi will not grow, even if low levels of oxygen are present. Some oxygen is kept because if all the oxygen is removed, the psychrophileClostridium gasigenescan grow. This organism can produce gases in 14 days at 2C, which leads to swollen food packages. 101

1.

2. 3.

4.

Such MAP procedures, while assisting in controlling spoilage, also cause a shift in the general structure of the microbial community, from gramnegative to gram-positive organisms. 2.5. MICROBIAL GROWTH LAG PHASE o Cells are adjusting to environment. o Cells are synthesizing needed macromolecules. LOG PHASE OR EXPONENTIAL PHASE o Cells are undergoing binary fission. STATIONARY PHASE o Growth rate slows down. o Some cells die. o Due to depletion of nutrients and/or accumulation waste. DEATH PHASE o Cells die.

2.6. SERIAL DILUTION Viable Plate Count Sample serially diluted and dilutions plated Count multiplied by dilution factor Technique assumes that 1 cell will give rise to 1 colony (colony forming unit (CFU) Counts ONLY living (viable) organisms

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2.7. MICROBIAL SPOILAGE Microbial growth in foods can lead to visible changes, including a variety of colors caused by spoilage organisms Meat and dairy products, with their high nutritional value and the presence of easily usable carbohydrates, fats, and proteins, provide ideal environments for microbial spoilage. Proteolysis and putrefaction are typical results of microbial spoilage of such high-protein materials. 2.8. SPOILAGE OF MILK Unpasteurized milk undergoes a predictable four-step succession during spoilage; 1) Acid production by Lactococcus lactissubsp. Lactisis followed by additional acid production associated with the growth of more acid tolerant organisms such as Lactobacillus. 2) At this point yeasts and molds become dominant and degrade the accumulated lactic acid, and the acidity gradually decreases. 3) Eventually protein-digesting bacteria become active, resulting in a putrid odor and bitter flavor. 4) The milk, originally opaque, can eventually become clear In comparison with meat and dairy products 103

2.9. SPOILAGE OF FRUITS AND VEGETABLES: Most fruits and vegetables have a much lower protein and fat content and undergo a different kind of spoilage. Readily degradable carbohydrates favor vegetable spoilage by bacteria, especially bacteria that cause Soft rots, such as Erwiniacarotovora, which produces hydrolytic enzymes. The high oxidation-reduction potential and lack of reduced conditions permits aerobes and facultative anaerobes to contribute to the decomposition processes. Bacteria do not seem important in the initial spoilage of whole fruits; instead such spoilage often is initiated by molds. These organisms have enzymes that contribute to the weakening and penetration of the protective outer skin. Food spoilage problems occur with minimally processed, concentrated frozen citrus products. These are prepared with little or no heat treatment, and major spoilage can be caused by Lactobacillus and Leuconostocspp., which produce diacetyl-butter flavors. Saccharomyces and Candida can also spoil juices. Concentrated juice has a decreased water activity (aw _ 0.8 to 0.83), and when kept frozen at about _9C, the juices can be stored for long periods. However, when concentrated juices are diluted with water that contains spoilage Molds are a special problem for tomatoes. Even the slightest bruising of the tomato skin, exposing the interior, will result in rapid fungal growth. Frequently observed genera include Alternaria, Cladosporium, Fusarium,and Stemphylium. This growth affects the quality of tomato products, including tomato juices and ketchups. Molds can rapidly grow on grains and corn when these products are held under moist conditions Infection of grains by the ascomycete Claviceps purpura causes ergotism, a toxic condition. Hallucinogenic alkaloids produced by this fungus can lead to altered behavior, abortion, and death if infected grains are eaten. 2.10. FUNGAL TOXINS: Alfatoxin Fungus-derived carcinogens include the aflatoxins and fumonisins. Aflatoxinsare produced most commonly in moist grains and nut products. Aflatoxins were discovered in 1960, when 100,000 turkey poults died from eating fungus-infested peanut meal. Aspergillusflavuswas found in the infected peanut meal, together with alcohol-extractable toxins termed aflatoxins. These flat-ringed planar compounds intercalate with the cells nucleic acids and act as frameshift mutagens and carcinogens. This occurs primarily in the liver, where they are converted to unstable derivatives Aflatoxins B1 and B2, after ingestion by lactating animals, will be modified in the animal body to yield the aflatoxins M1 and M2, 104

a)

If cattle consume aflatoxin-contaminated feeds, these also can appear in milk and dairy products. The aflatoxins are potent hepatocarcinogens, which have been linked to effects on immunocompetence, growth, and disease resistance in livestock and laboratory animals

b) Fumonisins: More recently discovered fungal contaminants of corn are the fumonisins, These are produced by Fusariummonili forme and cause leukoencephalomalacia in horses, pulmonary edema in pigs, and esophageal cancer in humans. The fumonisins function by disrupting the synthesis and metabolism of sphingolipids, important biochemically active compounds, which influence a wide variety of cell functions. The fumonisins inhibit ceramide synthase, a key enzyme for the proper use of fatty subtances in the cell. Thus it is extremely important to store corn and corn products under dry conditions where these fungi cannot develop. OTHER FUNGAL TOXINS Algal toxins contaminate fish and thus affect the health of marine animals higher in the food chain; they also can contaminate shellfish and fin fish, which are later consumed by humans. Most toxins are produced by dinoflagellates, but some diatoms also are toxic. Major human diseases that result from algal toxins in marine products include amnesic, diarrhetic, and neurotoxic shellfish poisoning These toxins are known to cause peripheral neurological system effects, often in less than one hour after ingestion. 2.11. FOOD BORNE DISEASES Recent estimates indicate that Norwalk-like viruses, Campylobacter jejuni and Salmonella are the major causes of food-borne diseases. In addition, Escherichia coli O157:H7 and Listeria are important food-related pathogens. There are two primary types of food-related diseases: food-borne infections food intoxications. All these food-borne diseases are associated with poor hygienic practices. Whether by water or food transmission, the fecal-oral route is maintained, with the food providing the vital link between hosts. Fomites, such as sink faucets, drinking cups, and cutting boards, also play a role in the maintenance of the fecal-oral route of contamination. A food-borne infection involves the ingestion of the pathogen, followed by growth in the host, including tissue invasion and/or the release of toxins.

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SALMONELLOSIS Salmonellosis results from ingestion of a variety of Salmonella serovars, particularly typhimurium and enteritidis Gastroenteritis is the disease of most concern in relation to foods such as meats, poultry, and eggs, and the onset of symptoms occurs after an incubation time as short as 8 hours. Salmonella infection can arise from contamination by workers in foodprocessing plants and restaurants, as well in canning processes Campylobacter jejuniis considered a leading cause of acute bacterial gastroenteritis in humans and can affect persons of all ages. This important pathogen is often transmitted by uncooked or poorly cooked poultry products often occurs when kitchen utensils and containers are used for chicken preparation and then for salads. Contamination with as few as 10 viable Campylobacter jejuni cells can lead to the onset of diarrhea. Campylobacter jejuni also is transmitted by raw milk, and the organism has been found on various red meats. LISTEROSIS Listeriosis, is caused by Listeria monocytogenes The outbreak was traced to pinhole leaks in the heat exchangers of a pasteurizing unit. The leaks allowed incoming raw milk to contaminate the pasteurized milk before production of the cheese. Listeria is difficult to work with because an extended incubation of samples is required for growth and detection. ESCHERICHIA COLI Escherichia coli is now recognized as an important food-borne disease organism. Enteropathogenic, enteroinvasive, and enterotoxigenic types can cause diarrhea. E. coli O157:H7 with its specific somatic (O) and flagellar (H) antigens is thought to have acquired enterohemorrhagic genes from Shigella, including the genes for shiga like vero cytotoxins. 106

Enterohemorrhagic E. coli has been found in meat products such as hamburger and salami, in unpasteurized fruit drinks, on fruits and vegetables, and in untreated well water. Prevention of food contamination by E. coli O157:H7 is essential from the time of production until consumption. Hygiene must be monitored carefully in larger-volume slaughterhouses where contact of meat with fecal material can occur. Even fruits and vegetables should be handled with care because disease outbreaks have been caused by imported produce. It may be possible to reduce this threat by destroying the pathogen with gamma irradiation, a food preservation method under consideration for wider use.

PRIONS: An infectious agent of increasing worldwide concern with respect to food safety is a prion that causes the poorly understood new variant CreutzfeldtJakob Disease (vCJD). This is one of a group of progressively degenerative neuronal diseases termed transmissible spongiformencephalopathies (TSEs), and is associated with beef cattle. It is often called the mad cow disease.

Sprouts: Care must be taken especially when the seeds are germinated, because a newly germinating seed releases organic matter and creates a spermosphere that stimulates microbial growth in a way similar to that which occurs in the rhizosphere. Contaminated alfalfa, beans, watercress, mungbean, mustard, and soybean sprouts can be major sources of typhoid and cholera. Shellfish and finfish also present major concerns. Raw sewage can contaminate shellfish-growing areas; in addition, waterborne pathogens such as Vibrio are more prevalent in the water column during the warm months Viruses also can be a problem. Oysters are filter feeders that process several liters of water per day, leading to the potential concentration of at least 100 types of enteric viruses. Reverse transcriptase PCR (RT-PCR) can be used to detect RNA viruses in oysters based on the presence of their nucleic acids. FOOD-BORNE INTOXICATIONS Intoxication produces symptoms shortly after the food is consumed because growth of the disease-causing microorganism is not required. 1) Toxins produced by Staphylococcus: Most Staphylococcus aureu s strains cause a staphylococcal enteritis related to the synthesis of extracellular toxins The main reservoir of S. aureusis the human nasal cavity. Frequently S. aureus is transmitted to a persons hands and then is introduced into food during preparation. 107

Growth and enterotoxin production usually occur when contaminated foods are held at room temperature for several hours. 2) Toxin Produced by Spore forming Bacteria:Three gram-positive rods are known to cause food intoxications: Clostridium botulinum, C. perfringens, and Bacillus cereus Clostridium perfringens food poisoning is one of the more widespread food intoxications. These microorganisms, which produce exotoxins, must grow to levels of approximately 106 bacteria per gram or higher in a food to cause disease. At least 108 bacteria must be ingested. They are common inhabitants of soil, water, food, spices, and the intestinal tract. Upon ingestion the cells sporulate in the intestine. The enterotoxin is a sporespecific protein and is produced during the sporulation process. Enterotoxin can be detected in the feces of affected individuals. Clostridium perfringens food poisoning is common and occurs after meat products are heated, which results in O2 depletion. If the foods are cooled slowly, growth of the microorganism can occur. At 45C, enterotoxin can be detected 3 hours after growth is initiated. Onset of the symptomswatery diarrhea, nausea, and abdominal cramps usually occurs in about 8 to 16 hours. Bacillus cereus also is of concern in starchy foods. It can cause two distinct types of illnesses depending on the type of toxin produced: an emetic illness characterized by nausea and vomiting with an incubation time of 1 to 6 hours, and a diarrheal type, with an incubation of 4 to 16 hours. The emetic type is often associated with boiled or fried rice, while the diarrheal type is associated with a wider range of foods.

2.12. FERMENTED FOOD PRODUCTS The major fermentations used in food microbiology are the lactic, propionic, and ethanolic fermentations Fermented Milks These fermentations are carried out by mesophilic, thermophilic, and therapeutic lactic acid bacteria, as well as by yeasts and molds Mesophilic- Buttermilk Production Mesophilic milk fermentations result from similar manufacturing techniques, in which acid produced through microbial activity causes protein denaturation. To carry out the process, one usually inoculates milk with the desired starter culture incubates it at optimum temperature (approximately 20 to 30C), and then stops microbial growth by cooling. Lactobacillus spp. And Lactococcuslactiscultures are used for aroma and acid production. The organism Lactococcus lactis subsp. Diacetilactis converts milk citrate to diacetyl, which gives a special buttery flavor to the finished product. The use of these microorganisms with skim milk produces cultured buttermilk 108

 Thermophilic- Yogurt Production In addition to mesophilic milk fermentations, thermophilic fermentations can be carried out at temperatures around 45C. An important example is yogurt production. Yogurt is made using a special starter culture in which two major bacteria are present in a 1:1 ratio: S. thermophilus and L. bulgaricus. With these organisms growing in concert, acid is produced by Streptococcus, and aroma components are formed by the Lactobacillus. Freshly prepared yogurt contains 109 bacteria per gram. Therapeutic- Acidophilus milk Fermented milks may have beneficial therapeutic effects. Acidophilus milk is produced by using Lactobacillus acidophilus. L. acidophilus may modify the microbial flora in the lower intestine, thus improving general health, and it often is used as a dietary adjunct. Many microorganisms in fermented dairy products stabilize the bowel microflora, and some appear to have antimicrobial properties. They may involve minimizing lactose intolerance, lowering serum cholesterol, and possibly exhibiting anticancer activity. Several lactobacilli have antitumor compounds in their cell walls. Another interesting group used in milk fermentations are the bifidobacteria. The genus Bifidobacteriumcontains irregular, nonsporing, gram-positive rods that may be club-shaped or forked at the end Bifidobacteria are nonmotile, anaerobic, and ferment lactose and other sugars to acetic and lactic acids. Bifidobacteria are thought to help maintain the normal intestinal balance, while improving lactose tolerance; to possess antitumorigenic activity; and to reduce serum cholesterol levels In addition, some believe that they promote calcium absorption and the synthesis of B-complex vitamins.

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Yeast-lactic fermentations include kefir, a product with an ethanol concentration of up to 2%. Kefir products tend to be foamy and frothy, due to active carbon dioxide production. This process is based on the use of kefir grains as an inoculum. These are coagulated lumps of casein that contain yeasts, lactic acid bacteria, and acetic acid bacteria In this fermentation, the grains are used to inoculate the fresh milk and then recovered at the end of the fermentation.

Cheese Production Cheese is one of the oldest human foods and is thought to have been developed approximately 8,000 years ago. About 2,000 distinct varieties of cheese are produced throughout the world, representing approximately 20 general types Often cheeses are classified based on texture or hardness as soft cheeses (cottage, cream, Brie), semisoft cheeses (Muenster, Limburger, blue), hard cheeses (cheddar, Colby, Swiss), or very hard cheeses (Parmesan).

All cheese results from a lactic acid fermentation of milk, which results in coagulation of milk proteins and formation of a curd. Rennin, an enzyme from calf stomachs, but now produced by genetically engineered microorganisms, can also be used to promote curd formation. 110

After the curd is formed, it is heated and pressed to remove the watery part of the milk or whey, salted, and then usually ripened The cheese curd can be packaged for ripening with or without additional microorganisms. Cheese curd inoculation is used in the manufacture of Roquefort and blue cheese. In this case Penicillium roqueforti spores are added to the curds just before the final cheese processing. Sometimes the surface of an already formed cheese is inoculated at the start of ripening; for example, Camembert cheese is inoculated with spores of Penicillium camemberti. The final hardness of the cheese is partially a function of the length of ripening. Soft cheeses are ripened for only about 1 to 5 months, whereas hard cheeses need 3 to 12 months, and very hard cheeses like Parmesan require 12 to 16 months ripening. The ripening process also is critical for Swiss cheese. Gas production by Propionibacterium contributes to final flavor development and hole or eye formation in this cheese. Some cheeses are soaked in brine to stimulate the development of specific fungi and bacteria; Limburger is one such cheese.

Production of Alcoholic Beverages A variety of plants that contain adequate carbohydrates can be used to produce alcoholic beverages. When carbohydrates are available in readily fermentable form, the fermentation can be started immediately. For example, grapes are crushed to release the juice or must, which can be allowed to ferment without further delay. The must also can be sterilized by pasteurization or the use of sulfur dioxide, and then the desired microbial culture added. In contrast, before cereals and other starchy materials can be used as substrates for the production of alcohol, their complex carbohydrates must be hydrolyzed. They are mixed with water and incubated in a process called mashing. The insoluble material is then removed to yield the wort, a clear liquid containing fermentable sugars and other simple molecules. Much of the art of beer and ale production involves the controlled hydrolysis of protein and carbohydrates to provide the desired body and flavor of the final product. Wines and Champagnes Wine production, or the science of enology [Greek oinos, wine, and ology, the science of], starts with the collection of grapes, continues with their crushing and the separation of the liquid (must) before fermentation, and concludes with a variety of storage and aging steps

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All grapes have white juices. To make a red wine from a red grape, the grape skins are allowed to remain in contact with the must before fermentation to release their skin-coloring components. Wines can be created by using the natural grape skin microorganisms. This natural mixture of bacteria and yeasts gives unpredictable fermentation results. To avoid such problems, one can treat the fresh must with a sulfur dioxide fumigant and add a desired strain of Saccharomyces cerevisiaeor S. ellipsoideus. After inoculation the juice is fermented for 3 to 5 days at temperatures varying between 20 and28C. Depending on the alcohol tolerance of the yeast strain, the final product may contain 10 to 18% alcohol. Clearing and development of flavor occur during the aging process. A critical part of wine making involves the choice of whether to produce a dry (no remaining free sugar) or a sweeter (varying amounts of free sugar) wine. This can be controlled by regulating the initial must sugar concentration. With higher levels of sugar, alcohol will accumulate and inhibit the fermentation before the sugar can be completely used, thus producing a sweeter wine. During final fermentation in the aging process, flavoring compounds accumulate and influence the bouquet of the wine. Microbial growth during the fermentation process produces sediments, which are removed during racking. Racking can be carried out at the time the fermented wine is transferred to bottles or casks for aging or even after the wine is placed in bottles. Many processing variations can be used during wine production. The wine can be distilled to make a burned wine or brandy. Acetobacter and Gluconobactercan be allowed to oxidize the ethanol to acetic acid and form a wine vinegar. 112

In the past an acetic acid generator was used to recirculate the wine over a bed of wood chips, where the desired microorganisms developed as a surface growth. Today the process is carried out in large aerobic submerged cultures under much more controlled conditions. Natural champagnes are produced by continuing the fermentation in bottles to produce a naturally sparkling wine. Sediments that remain are collected in the necks of inverted champagne bottles after the bottles have been carefully turned. The necks of the bottles are then frozen and the corks removed to disgorge the accumulated sediments. The bottles are refilled with clear champagne from another disgorged bottle, and the product is ready for final packaging and labeling.

Beers and Ales Beer and ale production uses cereal grains such as barley, wheat, and rice. The complex starches and proteins in these grains must be changed to a more readily usable mixture of simpler carbohydrates and amino acids. This process, involves germination of the barley grains and activation of their enzymes to produce a malt. The malt is then mixed with water and the desired grains, and the mixture is transferred to the mash tun or cask in order to hydrolyze the starch to usable carbohydrates. Once this process is completed, the mash is heated with hops (dried flowers of the female vine Humuluslupulis), which were originally added to the mash to inhibit spoilage microorganisms. The hops also provide flavor and assist in clarification of the wort. In this heating step the hydrolytic enzymes are inactivated and the wort can be pitchedinoculatedwith the desired yeast. Most beers are fermented with bottom yeasts, related to Saccharomyces carlsbergensis, which settle at the bottom of the fermentation vat. The beer flavor also is influenced by the production of small amounts of glycerol and acetic acid. Bottom yeasts produce beer with a pH of 4.1 to 4.2 and requiring 7 to 12 days of fermentation 113

With a top yeast, such as Saccharomyces cerevisiae, the pH is lowered to 3.8 to produce ales. Freshly fermented (green) beers are aged or lagered, and when they are bottled, CO2 is usually added. Beer can be pasteurized at 140F or higher or sterilized by passage through membrane filters to minimize flavor changes. In many places there is increased interest in specialty beers. Local Braumeisters develop unique products with special brewing techniques and ingredients.

Distilled Spirits Distilled spirits are produced by an extension of beer production processes. The fermented liquid is boiled, and the volatile components are condensed to yield a product with a higher alcohol content than beer. Rye and bourbon are examples of whiskeys. Rye whiskey must contain at least 51% rye grain, and bourbon must contain at least 51% corn. Scotch whiskey is made primarily of barley. Usually a sour mash is used; the mash is inoculated with ahomolactic (lactic acid is the major fermentation product) bacterium such as Lactobacillus delbrueckii, which can lower the mash pH to around 3.8 in 6 to 10 hours. This limits the development of undesirable organisms. Vodka and grain alcohols are also produced by distillation. 114

Gin is vodka to which resinous flavoring agentsoften juniper berrieshave been added to provide a unique aroma and flavor.

Sauerkraut: Sauerkraut or sour cabbage, is produced from wilted, shredded cabbage Usually the mixed microbial community of the cabbage is used. A concentration of 2.2 to 2.8% sodium chloride restricts the growth of gramnegative bacteria while favoring the development of the lactic acid bacteria. The primary microorganisms contributing to this product are Leuconostoc mesenteroides and Lactobacillus plantarum. A predictable microbial succession occurs in sauerkrauts development. The activities of the lactic acid-producing cocci usually cease when the acid content reaches 0.7 to 1.0%. At this point Lactobacillus plantarum and Lactobacillus brevis continue to function. The final acidity is generally 1.6 to 1.8, with lactic acid comprising 1.0 to 1.3% of the total acid in a satisfactory product.

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