GENERAL AND FOOD MICROBIOLOGY PART II - L. V. Krasnikova - 2016

TOPIC 4. CHARACTERISTICS OF SPOILAGE AGENTS IN MEAT, MEAT, AND DAIRY PRODUCTS

The spoilage of meat and dairy products is caused by the proliferation of various groups of microorganisms within them. Spoilage in products of animal origin and poultry is induced by putrefactive and lactic acid Bacteria, micrococci, enterococci, Yeasts, Molds, actinomycetes, as well as agents of specific Fermentation types (butyric, acetic acid, and other bacteria). There are several forms of microbial meat spoilage, of which putrefaction (decay) is the most widespread. Putrefaction can be internal or superficial. Internal putrefaction is caused by obligate and facultative anaerobes, initiating in areas where Connective Tissue accumulates around large Blood Vessels. Superficial putrefaction occurs more frequently than internal and is driven by the proliferation of aerobic microorganisms.

4.1. Putrefactive Bacteria

The principal agents responsible for the spoilage of animal-derived products are putrefactive (proteolytic) bacteria. These bacteria cause the decay of meat, sausage products, milk and dairy products, fish and fish products, as well as eggs and egg products.

Among the Structural components of meat, Muscle tissue and offal are the most susceptible to putrefaction. Connective, adipose, and Bone Tissues contain minimal protein and are consequently less vulnerable to this process.

Putrefactive bacteria (owing to the presence of active proteases) break down Proteins into Polypeptides, Peptides, peptones, and Amino Acids. The degradation of amino acids yields organic acids, aldehydes, ketones, amines, ammonia, hydrogen sulfide, carbon dioxide, indole, skatole, mercaptans, and other substances. Some of the compounds produced during putrefaction impart unpleasant tastes and odors to foods, while many are highly toxic.

Interactions among various species of putrefactive bacteria occur via metabiosis (the succession of one species by another). In the Initial Stages of putrefaction, spherical bacterial forms multiply on the meat surface. These are subsequently replaced by aerobic rod-shaped bacteria, followed later by anaerobic bacteria.

Based on morphological and biochemical characteristics, putrefactive bacteria are divided into four groups: spore-forming aerobes; spore-forming anaerobes; non-spore-forming facultative anaerobes; non-spore-forming aerobes.

The CHARACTERISTICS OF THE most common putrefactive bacteria are presented in Table 4.1, and their microscopic appearance is shown in Figs. 4.1-4.7.

Enterococci, micrococci, molds, yeasts, and actinomycetes also possess The ability to degrade proteins. The characteristics of these microorganisms were examined in the general microbiology course.

Class="center">Table 4.1. Characteristics of Putrefactive Bacteria

Non-spore-forming aerobes

Fig. 4.1. Microscopic preparation of Bacillus subtilis

Fig. 4.2. Microscopic preparation of Bacillus licheniformis

Fig. 4.23. Microscopic preparation of Bacillus cereus

Fig. 4.4. Microscopic preparation of Pseudomonas fluorescens

Fig. 4.5. Clostridium sporogenes

4.6. Microscopic slide of Proteus vulgaris

Fig. 4.7. Clostridium perfringens

Putrefactive bacteria are widespread in nature. They are found in soil, Water, air, the intestines of humans and animals, and in food products.

4.2. Lactic acid bacteria

Lactic acid bacteria (LAB) proliferate on fresh beef, other red meat, and poultry when vacuum-packed or packaged in a modified atmosphere (MAP). The consumption of sugars is crucial for the proliferation of LAB in meat. The most meat-specific strains include both obligately heterofermentative LAB of the genera Leuconostoc, Weissella, and Carnobacterium, and facultatively homofermentative species, notably Lactobacillus sakei (Fig. 4.8) and L. curvatus. The population of obligately homofermentative LAB is significantly smaller, and they rarely dominate spoilage microbial communities. Obligately heterofermentative LAB produce lactate, ethanol or acetate, and carbon dioxide from glucose. Facultatively homofermentative LAB produce two molecules of lactate from glucose, and lactate, acetate, and carbon dioxide from pentoses (such as ribose).

Fig. 4.8. Microscopic slide of Lactobacillus sakei

During the storage of fresh meat in vacuum or MAP packaging, LAB lower its pH. The initial sign of meat spoilage caused by LAB activity is an atypical, off-odor—sour, tangy, or cheesy. Further spoilage leads to gas formation and slime accumulation within the package. Surface greening of meat products is associated with The production of hydrogen peroxide by lactic acid bacteria, which reacts with Myoglobin to form a greenish oxidized porphyrin. The greening of vacuum-packed meat rolls and frankfurters is most commonly caused by the LAB species Weissella viridescens. Mild greening can be induced by certain strains of Lactobacillus sakei that produce hydrogen sulfide, which reacts with muscle pigments to form sulfmyoglobin.

Among the microbiota of chilled meat, psychrotrophic LAB specific to livestock and poultry meat can be found. These include L. sakei, Leuc. carnosum, and Leuconostoc gelidum (Fig. 4.9).

Figure 4.9. Microscopic slide of Leuconostoc gelidum

The population of these acid-tolerant bacteria increases during the anaerobic refrigerated storage of meat, eventually becoming dominant. Lactic acid bacteria (LAB) of the species Carnobacterium piscicola and Carn. divergensis are highly specific to fresh meat and poultry in particular.

This leads to fermentative meat spoilage, manifested by a sour odor caused by the accumulation of lactic, acetic, and other organic acids. This type of spoilage may be accompanied by gas production, slime formation inside the package, and meat discoloration upon opening.

4.3. Butyric Acid Bacteria

Butyric acid bacteria belong to the genus Clostridium. In recent years, numerous new species have been described, bringing the total number in this genus up to 100.

Clostridium butyricum consists of large rods with rounded, sometimes pointed ends measuring 0.3–2.0 x 1.5–20.0 µm, occurring in pairs or short chains (Fig. 4.10). A distinctive feature of these bacteria is their ability to accumulate a starch-like substance, granulose, in their Cells prior to spore formation, which stains blue with iodine. They are Gram-positive and motile by means of peritrichous flagella. They form oval or spherical endospores whose diameter typically exceeds that of The Cell.

Fig. 4.10. Bacteria of the species Clostridium butyricum

Butyric acid bacteria are obligate anaerobes. Most species are chemoorganotrophic, while some can be chemoautotrophic or chemolithotrophic. Regarding Temperature requirements, they are mesophiles. The spores of butyric acid bacteria are heat-resistant and can withstand boiling for several minutes.

Butyric acid bacteria are the causative agents of Butyric acid fermentation, which yields butyric, acetic, lactic, propionic, and formic acids, ethyl, butyl, and propyl alcohols, as well as gaseous products such as hydrogen and carbon dioxide.

On meat and meat products, Clostridium pasteurianum and C. butyricum are found more frequently than others. Key characteristics of their proliferation include vigorous gas production and the unpleasant odor of butyric acid. Butyric acid bacteria cause the spoilage of various meat and dairy products during prolonged storage. When these bacteria multiply in canned foods, they cause a defect known as "flipping" or "bombage". In the dairy industry, they are responsible for the cheese defect known as "late blowing".

4.4. Yeasts

Yeasts typically constitute a minor fraction of the microbiota in dairy, meat, and poultry products, and rarely lead to their spoilage. This is because yeasts have a low growth rate, meaning their metabolic activity is rapidly suppressed by psychrotrophic bacteria. Yeasts can cause spoilage only when bacterial proliferation is inhibited by factors such as low water activity, low pH, high salt content, or the presence of chemical preservatives or Antibiotics. Many Yeast species are more resistant to these factors, and some are capable of multiplying at low temperatures. Psychrotrophic species and strains have been identified among asporogenous yeasts of the genera Candida, Cryptococcus, Rhodotorula, Torulaspora, and Trichosporon, as well as ascogenous yeasts of the genera Debaryomyces, Yarrowia, and Pichia. At the same time, yeasts require oxygen for optimal growth, making them unable to multiply during meat storage in vacuum or modified atmosphere packaging (MAP); however, their numbers increase during refrigerated storage with access to air.

Yeast spoilage manifests as slime formation on the meat surface, off-odors, and the appearance of pigmented spots due to colony growth (pink, red, or black).

The yeast species most commonly found in meat and poultry are Candida famata (Fig. 4.11) and Kluyveromyces marxianus (Fig. 4.12). At low temperatures (-3 to -5 °C), the so-called "black yeasts" such as Monilia nigra (Fig. 4.13) can also proliferate.

Fig. 4.11. Candida famata yeast

4.12. Kluyveromyces marxianus yeast

Fig. 4.13. Monilia nigra yeast

In the dairy industry, yeasts play a dual role: some species participate in the production of fermented dairy products as biological agents that drive Alcoholic Fermentation, while other species contribute to dairy spoilage.

Yeasts present in dairy products are conventionally divided into three groups:

✵ Lactose-fermenting yeasts. These include sporogenous (spore-forming) yeasts of the species Saccharomyces lactis, Zygosaccharomyces lactis, Kluyveromyces fragilis, and Debaryomyces, as well as asporogenous (non-spore-forming) yeasts such as Torulopsis kefir, Torulopsis sphaerica, and Candida pseudotropicalis var. lactosa, among others.

✵ Non-lactose-fermenting yeasts that are nevertheless capable of fermenting Monosaccharides. Such yeasts can multiply in milk and dairy products alongside lactic acid bacteria, which break down lactose into glucose and galactose.

✵ Yeasts that neither ferment lactose nor other sugars, but instead cause their oxidation. They do not form spores and are incapable of alcoholic fermentation. This group primarily comprises yeasts of the genus Candida.

Lactose-fermenting yeasts are used in the production of mixed-fermentation beverages such as kumis, tan, ayran, matsoni, and kurunga, among others. In these products, they impart a specific flavor profile, synthesize Vitamins, and stimulate the growth of lactic acid bacteria.

Certain strains of yeasts multiplying in dairy products exhibit antagonistic activity against the tuberculosis pathogen as well as several opportunistic microorganisms.

Specific yeast species participate in cheese ripening as part of the cheese surface smear microflora.

Yeasts entering dairy products from external sources (so-called "wild yeasts") can cause the following types of spoilage:

1. Pigmentation: the appearance of colored colonies On the surface of meat, butter, cheese, and cottage cheese. Yeasts of the genus Rhodotorula are capable of forming pink, yellow, and black colonies (Fig. 4.14).

Fig. 4.14. Yeasts of the genus Rhodotorula

2. Swelling (bloating) of cottage cheese, sour cream, yogurt, and cheeses at an early stage of ripening. This type of spoilage is caused by The formation of carbon dioxide during alcoholic fermentation driven by yeasts.

Among the yeasts causing yogurt spoilage, the following species have been isolated: Torulopsis candida, Kluyveromyces fragilis, Debaryomyces hansenii, Candida krusei, and Saccharomyces cerevisiae (Fig. 4.15).

Fig. 4.15. Yeasts causing yogurt spoilage: a – Torulopsis candida; b – Kluyveromyces fragilis; c – Candida krusei; d – Saccharomyces cerevisiae

3. Swelling (sbombyazh) of sweetened condensed milk caused by sucrose-fermenting yeasts.

4. Rancidity, lipid oxidation, and off-flavors in fat-containing foods during cold storage are caused by the yeast Yarrowia lipolytica (Fig. 4.16), which exhibits lipolytic activity.

Fig. 4.16. Yeast Yarrowia lipolytica

4.5. Molds (Mycelial Fungi)

The growth of molds on food products depends on four critical factors: the availability of nutrients, temperature, active acidity (pH), and water activity (a_w), while also taking into account the interaction among these Physical and Chemical parameters.

It is well known that the optimum growth temperature for fungi of the genus Aspergillus ranges from 25–30 °C, whereas for Penicillium species it is around 20 °C. Low temperatures generally suppress the growth of mycelial fungi, yet a considerable number of them are capable of proliferating under cold storage conditions at temperatures ranging from +5 to -10 °C. Studies have shown that the minimum growth temperature for the mold Thamnidium elegans is (-8...-10) °C (Fig. 4.17), while the mold species Cladosporium herbarum (Fig. 4.18) can grow within the temperature range of (-5...-8) °C.

Fig. 4.17. Mycelial fungus Thamnidium elegans: a — general view; b — sporangioles

Fig. 4.18. Mold Cladosporium herbarum

When meat is stored at minus 5 °C, colonies of Cladosporium herbarum appear within 10–19 days. This fungus can grow in the inner layers of butter if voids are present within the bulk, and it forms dark spot colonies on The surface of cheese. Possessing high proteolytic activity, Cladosporium herbarum not only spoils the appearance of the product but also triggers protein degradation within it.

The mold species Mucor racemosus (Fig. 4.19) belongs to the lower fungi of the class Zygomycetes. Mucoralean fungi are widespread in the topsoil and readily multiply on food products. Mucor racemosus can grow on the surface of foods at a minimum temperature of minus 4 °C.

Fig. 4.19. Mycelial fungus Mucor racemosus

The fungus Sporotrichum caris (Fig. 4.20) belongs to the class of higher imperfect fungi, Deuteromycetes. The minimum growth temperature for this fungus is minus 8 °C.

Fig. 4.20. Mycelial fungus Sporotrichum caris

Dairy mold Geotrichum candidum (Fig. 4.21) also belongs to the class of imperfect fungi, Deuteromycetes. It frequently proliferates on the surface of dairy products during cold storage at temperatures no lower than 0 °C.

Fig. 4.21. Microscopic preparation of Geotrichum candidum

The majority of mycelial fungi can grow across a wide pH range, meaning this factor alone has a relatively minor effect on their proliferation in food products. At neutral and alkaline pH levels, water activity is a significantly more crucial factor. The limiting a_w value for the growth of any microorganism species is approximately 0.6. Certain xerophilic molds (Xeromyces bisporus) are capable of growing even at an a_w of 0.60 (Table 4.2).

Table 4.2. Minimum water activity for the growth of selected mold species (C. Blackburn, 2008)

Foods infested with molds acquire an unappealing appearance, a musty taste, and an off-odor; consequently, moldy products are rejected, leading to substantial economic losses.

Topic Assignment:

1. To study the morphological and cultural characteristics of putrefactive bacteria.

2. To prepare fixed stained slides of certain species of lactic acid bacteria responsible for the spoilage of meat and meat products. Sketch the microscopic view.

3. To examine and describe the Characteristics of Lactic acid bacteria (LAB) colonies on solid media. Record the main morphological and PHYSICOCHEMICAL PROPERTIES OF lactic acid bacteria in your notebook.

4. To prepare a slide of butyric acid bacteria and review their characteristics.

5. To prepare fixed stained slides of yeasts causing the spoilage of meat products.

6. To prepare a stained fixed slide of the mold Geotrichum candidum.

7. To examine mold colonies grown on food products at an 8x magnification, describe their appearance, and make drawings.

8. To prepare "hanging drop" (or press-mount) slides of certain mold species.

Control Questions

1. What is meat putrefaction?

2. What products are formed during protein degradation?

3. What groups are putrefactive bacteria divided into?

4. Provide a brief description of putrefactive microorganisms and name their representative species.

5. Under what conditions can lactic acid bacteria multiply on meat products?

6. What species of lactic acid bacteria are found on meat products?

7. Name the morphological features of butyric acid bacteria.

8. What metabolic products do butyric acid bacteria accumulate in meat?

9. What species of yeast are found on meat products?

10. Under what conditions can yeasts multiply on meat?

11. What are the signs of meat spoilage resulting from yeast growth?

12. What factors determine the proliferation of molds on food products?