MICROBIOLOGY Study Guide - 2012

CHAPTER 15. FOOD MICROBIOLOGY

15.2. MICROBIOLOGY OF MEAT AND MEAT PRODUCTS

Microflora of fresh meat. Fresh meat serves as an excellent nutritional substrate for the growth and survival of saprophytic, opportunistic, and pathogenic microorganisms. Meat contains all the essential nutrients required by microorganisms: sources of carbon and nitrogen, Vitamins, and mineral salts. The available Water content (a_w) and pH of meat are also highly favorable for microbial proliferation, making it prone to rapid microbiological spoilage.

Microorganisms enter meat from various sources. A distinction is made between endogenous (ante-mortem) and exogenous (post-mortem) contamination of animal Organs and Tissues by microorganisms.

Endogenous contamination of carcasses is observed in animals suffering from infectious diseases. Pathogens spread through organs and tissues depending on the type of infection, its course, and the physiological state of the sick animal. For instance, in animals with anthrax, swine erysipelas, and other diseases, the pathogen first multiplies in specific organs and tissues, and subsequently invades the bloodstream, spreading to all organs and muscular tissue. In other conditions, the pathogen localizes in one or several organs (in tuberculosis—in the Lungs or udder; in leptospirosis—in the Kidneys and Liver, etc.). In healthy animals, ante-mortem endogenous contamination of organs and tissues occurs due to a decline in the body's defense mechanisms under METABOLISM/18.html">The Influence of adverse factors (overexertion, hypothermia, overheating, starvation, stress, transport-related injuries, etc.). Meat from diseased, weakened, or transport-fatigued animals becomes contaminated via endogenous migration of microorganisms from the gastrointestinal tract and open wounds into muscular tissue through the circulatory and lymphatic systems.

Exogenous contamination. Meat obtained from the slaughter of healthy and rested animals is practically devoid of microorganisms. Normally, microorganisms are absent in the Blood, Muscles, and Internal Organs of animals. During subsequent carcass dressing operations, the meat and organs become contaminated with various microorganisms. The sources of exogenous microbial contamination of meat are highly diverse. They include the animal's Skin, the Contents of the gastrointestinal tract, tools, equipment, utensils, personnel clothing and footwear, transport vehicles, water, air, etc.

The microflora of warm carcass meat from healthy animals comprises a limited number of microbial species. If Sanitary and hygienic conditions are inadequately maintained, coliforms—including Escherichia coli, Bacteria of the genus Proteus, saprophytic aerobic rods of the genus Bacillus, and micrococci—can be found on the carcass surface. The degree of microbial contamination in fresh meat varies depending on the degree of maturation, Temperature and humidity conditions during chilling, as well as the sanitary and hygienic conditions during Processing. The surface of the meat may harbor from 10³ to 10⁶ microbial Cells per 1 cm². The predominant flora consists of aerobic and facultatively anaerobic, non-spore-forming, Gram-negative rods belonging to the genera Pseudomonas, Flavobacterium, Alcaligenes, Aeromonas, alongside coliform bacteria, proteus bacteria, corynebacteria, and lactic acid bacteria. Aerobic and anaerobic spore-forming bacteria, micrococci, Yeasts, and mold spores are detected in smaller quantities. Most of these microorganisms produce active proteases and Lipids-hydrolyzing lipases that catalyze The breakdown of meat Proteins and lipids. According to many researchers, overt signs of meat spoilage appear when bacterial numbers reach 10⁷—10⁸ cells per 1 g or per 1 cm² of its surface. The time required to reach this "threshold" microbial concentration depends on the storage temperature and the initial population of microorganisms capable of multiplying at that specific temperature.

Meat may be contaminated with pathogenic (Listeria monocytogenes, Salmonella dublin, S. cholerasuis, S. typhimurium) and toxigenic microorganisms (Clostridium botulinum, Clostridium perfringens, Bacillus cereus, Staphylococcus aureus). Substantial proliferation of these bacteria can render the meat a source of foodborne infections or intoxications.

The penetration of bacteria into the deeper layers of meat indicates a decline in its quality. This principle forms The basis of bacterioscopic analysis, which allows for the rapid determination of meat freshness.

For bacterioscopic examination, small pieces of meat are aseptically excised from varying depths and their cut surfaces are pressed against a Glass slide to prepare an impression smear. The smear is Gram-stained and examined microscopically to determine the bacterial count and the extent of muscular tissue degradation (Table 11).

Class="center">Table 11. Determination of meat freshness by bacterioscopy

Bacterioscopic examination of meat is essential for selecting the appropriate analytical method based on the sample's microbial load and the Morphology of the present microorganisms (rods, cocci, encapsulated or spore-forming variants, Gram-positive or Gram-negative).

Microflora of chilled meat. The species COMPOSITION OF THE microflora in meat entering cold storage is highly diverse. Among the bacteria isolated from fresh beef and poultry, the most frequent are: Pseudomonas, Aeromonas, Acinetobacter / Moraxella, Psychrobacter, Shewanella putrefaciens, Brochothrix thermosphacta, Micrococcus, Clostridium, lactic acid bacteria of the genera Lactobacillus, Leuconostoc, Weissella, as well as members of the family Enterobacteriaceae. Additionally, yeasts of the genera Cryptococcus, Candida, Torulopsis, Rhodotorula may be present on the meat surface.

The key factors influencing microbial proliferation during meat storage are temperature and the gas composition of the packaging environment. Storing meat at low positive temperatures significantly extends the lag phase of most microorganisms, although psychrotrophic species continue to multiply slowly. While a fraction of mesophilic microorganisms dies off, the vast majority merely slow down their metabolic processes and enter a state of anabiosis within the meat. This group includes numerous bacterial species from the family Enterobacteriaceae, as well as bacteria of the genera Micrococcus, Bacillus, and Clostridium.

Under aerobic conditions, the microbiota of raw chilled meat is primarily represented by putrefactive Gram-negative bacteria, dominated by pseudomonads and closely related species. Studies have demonstrated that after 14 days of chilled storage, bacteria of the genus Pseudomonas accounted for 84% of the total microbial population, compared to an initial level of approximately 4%. Among all bacteria recovered from spoiled chilled meat stored at —2 to 0 °C, 90% belonged to pseudomonads. Other psychrophilic microorganisms are represented by the genera Aeromonas, Achromobacter, Flavobacterium.

The species most commonly found on chilled meat are Pseudomonas fluorescens and Pseudomonas aeruginosa. They possess The ability to produce bacteriocins that inhibit the growth of other bacteria. Staphylococcus aureus, Proteus vulgaris, Achromobacter viscosus, Lactobacillus rhamnosus, and Lactobacillus acidophilus exhibit the highest sensitivity to pseudomonad bacteriocins. Consequently, both aforementioned pseudomonad species rapidly assume a dominant position among all psychrophilic bacteria. The antagonistic properties of pseudomonads manifest not only against bacteria but also toward certain filamentous Fungi. Conversely, the growth of pseudomonads is frequently suppressed by lactobacilli.

Psychrophilic species and strains have been identified among asporogenous yeasts of the genera Candida, Cryptococcus, Rhodotorula, Torulopsis, and Trichosporon. When multiplying on meat, yeasts of the genus Rhodotorula form pigmented colonies of yellow, pink, or red color. Representatives of the genera Candida, Cryptococcus, Torulopsis can synthesize extracellular Polysaccharides, manifesting as a slimy layer on the meat surface.

Ascosporogenous yeasts of the genera Debaryomyces and Pichia are also capable of growth at low temperatures. These yeasts multiply on meat products at —3 to —5 °C. Yeast proliferation on high-fat products results in a sharp, unpleasant, rancid odor.

Psychrophilic filamentous fungi capable of growing on foods during cold storage have been identified among Mucorales and imperfect fungi (Deuteromycetes). Species capable of growth at low temperatures include: Mucor mucedo at —2 °C, Mucor racemosus at —4 °C, and Thamnidium elegans at —5 °C. Among imperfect fungi, visible growth of Cladosporium herbarum and Botrytis cinerea was observed at —5 °C on days 10–19 of chilled meat storage, Alternaria tenuis grew exclusively at —2 °C, Trichoderma lignorum at 1–2 °C, and Geotrichum spp. at approximately 0 °C.

The proliferation of psychrophilic microorganisms on chilled meat gradually leads to a microbial population increase that vastly exceeds initial levels, altering the organoleptic Properties of the meat. Microbial growth on the meat surface can be detected by Touch through The formation of slime, with microbial counts ranging from 10⁶ to 10⁸ cells per 1 cm². When the surface concentration reaches 10⁹ microorganisms per 1 cm², the slime layer thickens, its odor and color change, and the meat becomes unfit for consumption.

The shelf life of chilled meat can be extended by storage in a gaseous atmosphere containing traces of ozone, nitrogen, or carbon dioxide. These measures help suppress the growth of aerobic microorganisms (bacteria, yeasts, Molds). Specifically, utilizing carbon dioxide (at CO₂ concentrations up to 10% in the cold room air) extends the lag phase of aerobic bacteria by a factor of 4 to 5, while exerting a particularly strong inhibitory effect on psychrophilic bacteria of the genera Pseudomonas and Achromobacter. The inhibitory action of CO₂ on psychrotrophic microflora is attributed to the intracellular formation of carbonic acid, which suppresses cellular Enzymes and disrupts Cell Membrane Functions.

The bacteriostatic and bactericidal action of ozone stems from its ability to dissociate into atomic oxygen, which acts as a powerful oxidizing agent. Bacilli, lactobacilli, and coliforms exhibit the highest sensitivity to ozone, whereas leuconostocs and pseudomonads show the lowest.

Vacuum packaging represents another effective measure for preserving the freshness of chilled meat; while it prevents the surface proliferation of aerobic bacteria of the genus Pseudomonas, it permits the growth of lactic acid bacteria belonging to the genera Lactobacillus and Carnobacterium. The METABOLIC ACTIVITY OF lactobacilli and carnobacteria degrades the quality of meat products, resulting in a sour odor and discoloration. The proliferation of Lactobacillus viridescens leads to The Development of a greenish hue on the meat surface.

Currently, to maintain the freshness of chilled meat, some countries apply UV irradiation and Antibiotics prior to packaging.

In most cases, qualitative changes in meat products during the Initial Stages of microbial proliferation can be detected organoleptically. These changes correspond to a specific microbial count: for bacteria, it ranges from 10⁶ to 10⁸ per 1 cm² of the surface or 1 g of the product; for yeasts, it is 10⁶ per 1 g. The time required to reach this concentration depends primarily on the storage temperature and the initial microbial load capable of multiplying on the product at a given temperature.

Microflora of frozen meat. As the temperature drops, the processes of enzyme production and microbial reproduction gradually slow down and eventually cease entirely. This occurs at —10 °C, although certain mold fungi continue to multiply down to —15 °C. Consequently, the storage of frozen meat requires maintaining a temperature of —18 °C or lower. Frozen meat is typically stored at temperatures ranging from —18 °C to —25 °C.

The composition of the microflora and the quantitative ratio between groups of microorganisms on frozen meat products are determined by the death rate of each group comprising the initial microbiota of the meat. The rate of microbial destruction depends on the freezing speed and the final temperature. A higher number of microorganisms die at a low freezing rate down to temperatures not lower than —10...—12 °C (see Section 7.1). The degree of microbial mortality alone does not yet indicate an improvement in the microbiological parameters of the product, since the released enzymes and toxins are not destroyed and can negatively affect product quality after thawing.

If frozen meat is subjected to a prolonged temperature increase from —18 to —5 °C during storage, mold fungi may proliferate on its surface: Cladosporium herbarum appearing as small black spots; Thamnidium elegans forming a fluffy grayish bloom; and Penicillium glaucum developing into grayish-green colonies. The presence of mold on frozen meat indicates that temperature regimes were violated during storage.

Meat spoilage caused by microorganisms. High-protein products undergo spoilage As a result of the metabolic activity of the aforementioned microorganisms. A special place is occupied by bacteria of the family Enterobacteriaceae, which includes both spoilage-causing and pathogenic species. Certain bacteria from this family are used as food safety indicators (coliform bacteria).

As a result of microbial proliferation, meat and meat products are subject to various types of spoilage (Table 12).

Table 12. Most common Types of microbial spoilage in meat

* Brochothrix thermosphacta are polymorphic bacteria which, depending on conditions, can range in shape from cocci and short rods to long threads and chains; they are Gram-positive, non-motile, and do not form spores. During glucose fermentation, they produce L-lactate. This species can be mistakenly identified as lactic acid bacteria of the genus Carnobacterium, but differs from them by being catalase-positive.

** Putrefactive meat spoilage is most frequently caused by bacteria of the genus Pseudomonas—P. fragi, P. lundensis, P. fluorescens, P. putida. Most pseudomonads multiplying on meat first break down glucose, then lactate, followed by the utilization of Amino Acids. A characteristic metabolite produced by pseudomonads during meat spoilage is dimethyl sulfide (DMS). Shewanella putrefaciens consists of aerobic, Gram-negative, motile rods with properties similar to those of the genera Pseudomonas and Aeromonas. This bacterial species does not multiply in meat with a pH value below 6.0. When utilizing Sulfur-Containing Amino Acids, this species produces hydrogen sulfide, which reacts with Myoglobin to form sulfmyoglobin, imparting a greenish hue to the meat.

Aerobic Gram-negative motile bacteria of the genera Aeromonas and Alcaligenes are also found in spoiled meat. Among the non-motile Gram-negative aerobic bacteria causing meat spoilage, non-pigment-producing genera such as Moraxella, Acinetobacter, Psychrobacter, and the yellow pigment-producing genus Flavobacterium are noteworthy.

Putrefactive meat spoilage can also be caused by facultatively anaerobic Gram-negative rods, particularly numerous bacteria of the family Enterobacteriaceae. Among enterobacteria, meat spoilage is most commonly caused by the species Serratia liquefaciens, Hafnia alvei, and Pantoea agglomerans (formerly Enterobacter agglomerans). Like pseudomonads, these species first degrade glucose and subsequently amino acids, accumulating amines, sulfides, and H₂S in the process. In addition to forming off-flavor compounds, enterobacteria and shewanellas carry out protein ammonification, releasing large amounts of ammonia that contribute to the odor of spoiled meat.

Microflora of sausage products. During the manufacture of sausage products, the microbial count in the raw Materials gradually increases in the initial Stages of the technological process. During deboning and trimming, microorganisms contaminate the meat from tools, workers' hands and clothing, equipment, and the air. To prevent rapid microbial proliferation, the cutting process should be performed quickly at a reduced ambient temperature while strictly observing sanitary and hygienic requirements. The microbial count increases significantly during mincing and the preparation of the meat batter upon The addition of backfat and spices. Spices themselves may carry a high microbial load and serve as a source of undesirable microflora contamination in the batter. According to some researchers, 1 g of non-sterile spices can contain up to 10⁶—10⁷ microorganisms, with spore-forming forms predominating. Raw sausage batter typically contains 10⁵—10⁷ bacteria per 1 g, dominated by Gram-negative non-spore-forming rods. After stuffing the batter into casings, no further microbial contamination of the batter occurs.

In The production of cooked sausages, the sausage links are subjected to settling, smoking, cooking, and cooling; semi-smoked sausages are smoked and dried after cooking. During the heat Treatment of sausage products, the total microbial population is reduced by 90—95%. As a rule, micrococci, enterococci, and spore-forming rods retain their viability.

For cooked sausage products, the total viable count (TVC) must not exceed 1 x 10³ CFU/g; coliforms (coli-form bacteria) and S. aureus are not permitted in 1 g; sulfite-reducing bacteria in 0.01 g; and pathogenic bacteria, including salmonellae (as well as Listeria monocytogenes in frankfurters and small sausages), in 25 g.

During storage, liver and blood sausages, meat loaves, aspics, and HEAD cheeses are most susceptible to microbial spoilage. For instance, the microbial count on the surface of packaged loaves can reach 10⁴—10⁵ CFU/cm², with Escherichia regularly detected. The shelf life and commercial distribution periods of these products are strictly limited at specified temperatures both in the retail network and at public catering establishments.

Dry-cured sausages are characterized by a fairly high stability during storage, which is associated with a low water activity value (a_w), elevated salt content, and the antiseptic action of wood smoke components.

After stuffing the batter into casings, the sausage links are held for 5—7 days at a temperature of 2—4 °C to allow the batter to mature, followed by drying for 1.5 months and smoking at 18—22 °C for 2—5 days.

A significant number of microorganisms are involved in the production process of dry-cured sausage. They partially help ensure the storage Stability of the sausage and impart specific organoleptic properties to it. This function is performed by the beneficial and desirable microflora of dry-cured sausage. During settling, ripening, and drying, the total microbial load of the batter increases substantially, accompanied by Changes in the qualitative composition of the microflora. In the initial batter, the bulk of the microorganisms consists of Gram-negative rods of the genera Pseudomonas, Proteus, Escherichia, Enterobacter, Aeromonas, as well as Gram-positive bacteria of the genera Bacillus, Clostridium, Enterococcus, Lactobacillus, Pediococcus. After 10—30 days, the dominant microflora of the batter becomes represented by micrococci and lactic acid bacteria of the species L. plantarum, L. brevis, Pediococcus cerevisiae. The population of acid-producing microorganisms during this period can range from 10⁸ to 10⁹ per 1 g. Developing during ripening and drying, lactic acid bacteria (LAB) lower the ambient pH to 5.5 and below, which inhibits the growth of undesirable putrefactive microflora and suppresses the development of pathogens. Furthermore, they synthesize antibiotic substances called bacteriocins, which suppress the proliferation of coliforms, proteus, bacilli, and clostridia. Lactobacilli have a favorable effect on the texture and cohesion of the sausage batter. This is explained by alterations in the surface tension of the batter resulting from the action of lactic acid—accumulated by the end of fermentation—on soluble meat proteins. Another positive role of lactobacilli is that they promote the formation and retention of color in certain sausage products.

In some dry-cured sausages (cervelat, salami), yeasts of the genera Debaryomyces and Candida participate in the ripening process alongside the aforementioned bacterial groups. Yeasts of the genus Debaryomyces exhibit antagonism toward mycelial fungi.

To achieve a directed ripening process in dry-cured sausages, dry bacterial preparations (starter cultures) have been developed, incorporating various microorganisms: lactic acid bacteria (L. plantarum), micrococci (Micrococcus caseolyticus), pediococci (P. cerevisiae), and staphylococci (S. xylosus).

During the smoking process, non-spore-forming bacteria are primarily destroyed, whereas bacilli, clostridia, and mycelial fungi are quite resistant to wood smoke constituents.

Spoilage of sausage products (putrefaction, sour fermentation, molding) is caused by The activity of the same microorganisms that induce meat spoilage. However, unlike meat putrefaction, the putrefactive spoilage of sausage products occurs simultaneously throughout the entire thickness of the sausage link. The texture of the sausage becomes loose, and the odor turns unpleasant and putrid.

Rancidity is a defect characteristic of dry-cured and semi-dry sausages. This type of spoilage is caused by pseudomonads (P. fluorescens, P. putida, P. fragi), Serratia marcescens, and dairy mold (Endomyces lactis), which possess active lipases that hydrolyze fat with the accumulation of glycerol, Fatty acids, aldehydes, and ketones. Fat breakdown products impart a rancid, pungent taste and odor to the sausage.

Dry-cured and semi-dry sausages are most susceptible to mold formation, especially when stored in conditions of high humidity.