MICROBIOLOGY Study Guide - 2012

CHAPTER 15. FOOD MICROBIOLOGY

15.4. MICROBIOLOGY OF FISH AND FISHERY PRODUCTS

Fresh fish. Microorganisms are typically absent from the Muscle tissue and muscle juice of newly caught fish. However, significant numbers of them are found on the outer mucous membranes, gills, and gastrointestinal tract. Bacterial counts in these areas can range from 10² to 10⁶ per 1 cm². The degree of microbial contamination depends on Water Temperature and microflora, the depth at which the fish lives, the season, and the fishing method.

The Skin and outer gills of fish harvested from northern seas harbor Bacteria of the genera Vibrio, Pseudomonas, Achromobacter, Alcaligenes, Acinetobacter, Flavobacterium, Corynebacterium, Micrococcus, Bacillus. The outer mucus layer may also contain luminous bacteria, such as Photobacterium phosphoreum. Yeasts can be present on the oral mucosa, skin, and outer gills of freshly caught fish, though they are suppressed by bacteria during subsequent storage. The isolated yeasts have been classified into the genera Debaryomyces, Torulopsis, Candida, Rhodotorula, Pichia, Cryptococcus. The intestinal microflora is relatively stable and generally independent of the environment. The number of Microorganisms in the intestine ranges from 1 x 10⁵ to 1 x 10⁸ Cells per 1 g. The predominant species include С. sporogenes, С. putrificum, С. perfringens, С. bifermentans, alongside food poisoning agents such as В. cereus, S. aureus, С. botulinum.

The dominant microflora of freshly caught fish consists of bacteria from the family Achromobacteriaceae (accounting for up to 60% of the total microflora). Bacteria of the genus Pseudomonas make up 22–30% of the total volume. Flavobacteria, corynebacteria, micrococci, bacilli, and vibrios account for less than 10% of the surface microflora. Anaerobic bacteria are generally absent On the surface of fish.

In 1951, cases of fish contamination with Vibrio parahaemoliticus—a CAUSATIVE AGENT OF human gastroenteritis—were described for the first time in Japan. Marine vibrios, particularly V. parahaemoliticus and V. vulnificus, may be present in Mollusks and the fish that feed on them. Because these vibrios are fragile and rapidly destroyed by refrigeration or heat Treatment, infection through them is likely only when consuming raw seafood.

Since inland water bodies and coastal marine waters are frequently polluted by sewage, fish can become contaminated with salmonellae, shigellae, yersiniae, enterococci, coliforms, as well as C. botulinum and Listeria monocytogenes. Listeriae readily infect fish and are widespread in fish products, especially cold-smoked fish and marinated fish preserves.

Freshly caught fish out of water dies quickly, after which its body undergoes several sequential changes: mucus secretion, rigor mortis, autolysis, and microbial spoilage. Immediately after death, mucus is secreted onto the body surface; its main component is the glycoprotein mucin, which serves as a nutrient medium for Microbial growth. Rigor mortis involves the contraction and tension of muscle fibers due to The formation of the Actin-Myosin complex, causing the fish body to become rigid. Autolytic and microbiological processes begin after rigor mortis. Autolysis causes profound structural Changes in the fish Tissues: they soften, delaminate, separate from the bones, develop spaces between fibers, and the belly may rupture. This period is highly favorable for the proliferation of microorganisms, which penetrate from the surface deep into the tissues. Under the action of Proteolytic Enzymes from putrefactive bacteria, the tissue Proteins break down to form ammonia, hydrogen sulfide, amines, organic acids, and Other Compounds. Bacterial protein breakdown leads to the accumulation of toxic degradation products, which can cause non-specific poisoning. This intoxication is driven by biogenic amines. Specifically, The breakdown of fish protein produces free Histidine, which undergoes decarboxylation to form histamine. Most bacteria that produce biogenic amines belong to the family Enterobacteriaceae (Escherichia coli, Еnterobacter aerogenes, Morganella morganii, Proteus vulgaris), as well as members of the genera Clostridium and Bacillus. In cases of food intoxication, histamine concentrations can reach up to 600 mg/kg.

Chilled fish. Fish are chilled or frozen to preserve their quality. If the voyage is short, fish can be chilled with ice. Salt is frequently added to the ice to enhance the chilling effect. The temperature of fish packed in ice is generally above 0 °C, and during transport, it can rise to 6 °C. Despite being iced, psychrophilic microorganisms continue to multiply within the fish. Consequently, the microbial load can increase from 10⁴ to 10⁶ per 1 g during onboard storage. Bacteria of the genus Pseudomonas thrive under these conditions, alongside other bacteria belonging to the genera Achromobacter and Flavobacterium. The proliferation of these bacterial groups leads to protein and lipid degradation and a decline in fish quality. Storing fish in ice has several notable drawbacks: 1 — the fish cools relatively slowly and, when frozen within a mass of ice, is subjected to compression and occasional physical damage; 2 — psychrophilic microorganisms continue to multiply during ice chilling; 3 — the ice quickly becomes contaminated with mucus, scales, and consequently microorganisms; 4 — the fish may undergo additional microbial contamination from the ice and salt.

Chilled fish stored in cold rooms at a temperature of (0 ± 2) °C shows rapid proliferation of psychrotrophic bacteria of the genera Pseudomonas, Flavobacterium, Acinetobacter, Moraxella, Vibrio, Cytophaga. After 10 days of storage at this temperature, the microbial count increases from 1 x 10²–1 x 10⁴ to 1 x 10⁶–10⁷ per 1 g.

Frozen fish. There are various Methods for freezing fish. The most modern approach is tunnel freezing, where fish are conveyed through a chamber maintained at —20...—40 °C. Fish quality depends heavily on the freezing rate. Slow freezing leads to the formation of ice crystals within the Cell Cytoplasm, resulting in cell death. Typically, slow freezing destroys 60–90% of the fresh fish microflora. High freezing rates preserve microbial viability to a greater extent than slow rates. Furthermore, the lower the freezing and storage temperatures, the greater the microbial survival rate. Micrococci are particularly resistant to freezing and can persist in frozen fish for several months. The total microbial count of frozen fish ranges from 1 x 10⁴ to 1 x 10⁵ cells per 1 g, consisting primarily of yeasts, micrococci, Achromobacter, Pseudomonas, Flavobacterium, and bacilli (В. cereus, В. mycoides, В. subtilis).

During storage of fish at —18...—20 °C, the Number of viable microbial cells gradually decreases. Within 3 months of storage, bacteria of the genus Pseudomonas and the majority of Achromobacter strains die off. At the same time, the proportion of flavobacteria within the remaining microflora increases. Pathogenic bacteria also maintain their viability during frozen storage. Enteropathogenic E. coli and staphylococci have been detected in frozen fillets, while salmonellae have shown relative resistance to freezing. The shelf life of frozen fish depends on the storage temperature and the fat content of the fish. Lean fish can be stored at —20...—25 °C for 6 to 8 months, whereas fatty fish under the same conditions keep for 4 to 6 months. The quality of chilled and frozen fish is regulated by the following microbiological criteria: total viable count (TVC) — not exceeding 5 x 10⁴ CFU/g; coliforms must be absent in 0.01 g; S. aureus — in 0.1 g; pathogenic microorganisms — in 25 g; for marine fish, the content of Vibrio parahaemolyticus must not exceed 100 CFU/g (SanPiN 2.3.2.1078—01).

Salted fish. Salting is one of the oldest and most widespread methods of fish preservation. The preserving action of sodium chloride is due to the reduction of water activity (a_w) in the medium and depends on salt concentration, temperature, medium pH, and the presence of preservatives. Based on their sensitivity to sodium chloride, microorganisms are conventionally divided into three groups: halophobic, whose activity is suppressed at an NaCl concentration of 5–6%; halotolerant, capable of growing at salt concentrations from 6 to 15%; and halophilic, capable of growing in concentrated saline solutions or directly in dry salt.

Table 13 shows the sodium chloride concentrations that inhibit the growth of specific microorganisms.

Class="center">Table 13. Sensitivity of certain microorganisms to sodium chloride

Salting is applied primarily to specific types of fish (such as herring and salmon families) which, upon ripening, develop distinct flavor profiles and textures resulting from the enzymatic breakdown of fish proteins and Lipids.

There are dry, mixed, and brine salting methods. Regardless of the method used, the fish loses water and absorbs salt.

Depending on the method of salt application, salting can be dry, wet, mixed, or via injection (for large, valuable fish); by temperature, it is classified as warm (10–15 °C), chilled (0–5 °C), or cold (—1...—4 °C); by salt content, it is categorized as heavy, medium, or light.

The salt used in the curing process can act as a source of microbial contamination. A single gram of salt may contain up to 1 x 10⁶ microbial cells, including halophilic bacteria.

In medium-strength brine (10–12% NaCl), the total microbial count averages 10²–10⁴ microbial cells per 1 cm³. In lightly salted fish products, elevated storage temperatures cause the total microbial count to rise, reaching anywhere from 10³ to 10⁸ cells per 1 g. The qualitative COMPOSITION OF THE microflora in such fish is highly diverse, including halophilic and halotolerant bacteria of the genera Micrococcus, Halobacterium, Streptococcus, Corynebacterium, as well as yeasts and molds. Food poisoning agents such as Staphylococcus aureus and enterococci may also be present. Spoilage of salted fish can be caused by pigment-producing microorganisms, such as Micrococcus roseus, which imparts a pinkish-red hue to salted herring; Halobacterium salinarium, which forms a foul-smelling red slime layer (a defect known as "fuxin" or pink slime); and molds of the genus Sporendonema, which cause brown spots to form on the fish surface. Lightly salted herring may develop a dingy white, smeary coating resulting from fat Hydrolysis driven by the lipases of aerobic halotolerant bacteria belonging to the genera Pseudomonas, Achromobacter, and others.

Preserves (Semipreserves). The primary preserving factor in fish preserves is common salt, used at concentrations ranging from 3 to 10%. To prevent spoilage, an antiseptic (sodium benzoate) is added at a concentration of 0.1% by weight of the fish. Sodium benzoate primarily inhibits putrefactive bacteria while having little effect on molds. Lactic acid bacteria (LAB) and yeasts are largely insensitive to this antiseptic. In some preserves, sodium benzoate is replaced by sorbic acid and the antibiotic nisin, which suppresses the growth of Gram-positive bacteria, including spore-formers.

During the initial days, the microflora of fish preserves is represented by spore-forming aerobic and anaerobic bacteria, lactic acid bacteria, and micrococci. Heterofermentative LAB participate in the ripening process, accumulating acetic and lactic acids as well as Aromatic Compounds through carbohydrate Fermentation. By the end of the ripening period, micrococci and LAB become the dominant groups in the preserves.

Microbial spoilage of preserves can be caused by lactic acid bacteria of the species Leuconostoc mesenteroides ssp. dextranicum and certain Gram-negative bacteria that produce slime in the brine As a result of synthesizing dextran or levan Polysaccharides from sucrose. Occasionally, Clostridium perfringens—introduced from the fish intestine or via spices—proliferates in the preserves. Through its metabolic activity, this Organism releases large amounts of gas, resulting in container Swelling (blown cans).

In some cases, lightly salted fish products (preserves) become contaminated with Staphylococcus aureus (S. aureus). This microbial contamination typically originates from food handlers. The multiplication of S. aureus and the subsequent production of enterotoxin can occur at salt concentrations of up to 10%. To inhibit toxin production by S. aureus, nisin should be used, and the finished product must be stored at temperatures not exceeding 10 °С.

Microbiological parameters for preserves: Total Plate Count (TPC) — not more than 5 x Ю⁴CFU/g; mass of the product (g) in which the following are not permitted: coliforms — in 0.01; S. aureus — in 1.0; sulfite-reducing clostridia — in 0.1; pathogens, including Salmonella — in 25.

Canned fish. Canned fish products (derived from the Latin conserve — to preserve) are categorized into: natural (produced without preliminary heat treatment, sauces, or brines); in oil (prepared from pre-treated fish with The addition of vegetable oil brines); in tomato sauce; fish-vegetable and fish-cereal mixes; pastes and pâtés.

Before sterilization, the microflora of the product to be canned consists of microorganisms from the fish meat, brines, spices, and contaminants introduced during preparation. Seamed cans are sterilized in autoclaves. This sterilization must be carried out as soon as possible after sealing, as microbiological processes develop very rapidly and can render the product unfit for consumption. To ensure the safety of the finished product and its storage stability, factors such as the sterilization regimen, pH and a_w values of the product, and storage temperature play a crucial role. Canned fish are characterized by pH > 4.6 and a_w > 0.98. Such a pH level does not prevent the growth and toxin production of C. botulinum, which produces the most heat-resistant spores among all pathogenic microorganisms. Consequently, the minimum thermal process must ensure a reduction in C. botulinum spores to 10^-12 of their initial population. This treatment is known as the "12D concept." It involves sterilization at a temperature of at least 121 °С for a minimum of 3 min, which is required to reduce the probability of C. botulinum spore survival to 1 in a trillion (10^-12), where D represents the time required to reduce the microbial population by 90% (10-fold).

In addition to pathogenic bacteria, the maximum permissible level of surviving spoilage microorganism spores must be determined in canned foods.

After sterilization, isolated bacterial cells survive in canned fish, constituting the residual microflora. In most cases, this residual microflora (especially aerobic spore-forming bacilli) does not multiply immediately after sterilization because the cells are weakened by thermal Processing, a low oxidation-reduction potential, and other factors. Such canned goods are considered commercially sterile and are cleared for distribution. The detection of isolated B. subtilis spores in canned products, in the absence of swelling (bombardment) and with normal organoleptic properties, does not preclude their release from the manufacturing plant and subsequent consumption.

Sterile canned foods must be free from pathogenic and toxigenic microorganisms, as well as spoilage agents such as thermophilic bacilli and clostridia spores.

The effectiveness of sterilization depends on the initial microbial load of the product, the species composition of the microflora, The chemical composition of the product, its pH, and the size of the can.

Under certain favorable conditions, microorganisms surviving the sterilization process begin to multiply, causing significant biochemical and organoleptic changes in the final product. Most commonly, this occurs within the first 10—15 days after canning, though microbial reactivation can sometimes unfold over a prolonged period. The proliferation of residual microflora leads to microbiological spoilage, which includes flippers, springers, or swells, as well as flat-sour and sulfide spoilage.

Swelling (Bombardment) refers to the bulging of the can's bottom and lid that does not disappear upon pressing; a springer is a bulge on the bottom or lid that flattens when pressed, accompanied by a characteristic clicking sound. The cause of swells and springers is an increase in internal pressure inside the can due to gas accumulation by gas-producing spore-forming bacteria such as C. sporogenes, C. thermosaccharolyticum, C. perfringens, C. thermoaceticum, and others. Microbiological swells can also be triggered by yeasts or cocci that enter the can as a result of a compromised hermetic seal. The organoleptic Properties of the canned product alter drastically due to the accumulation of microbial metabolites, manifesting as tissue maceration, acidic or putrid odors, and foaming. Typically, such alterations are observed at high levels of proliferated microorganisms — 10⁷—10⁸ cells per 1 gram of product. Such canned goods are easily detected and rejected by visual inspection.

Flat-sour spoilage involves alterations in the organoleptic properties of canned goods without gas production. The products develop a sour odor and taste, accompanied by A change in color. The causative agents of this type of spoilage are spore-forming aerobic bacilli, including B. subtilis, B. cereus, B. stearothermophilus, B. megatherium, B. aerothermophilus, and others. These microorganisms break down CARBOHYDRATES to produce various organic acids without gas evolution.

Sulfide spoilage is characterized by the accumulation of hydrogen sulfide in canned goods. This type of spoilage is caused by thermophilic spore-forming bacteria of the species Desulfotomaculum nigrificans. They degrade Sulfur-Containing Amino Acids, releasing hydrogen sulfide. Sulfide spoilage in canned fish is relatively rare; it occurs more frequently in canned shrimp because they contain a high concentration of sulfur-containing amino acids. In sulfide spoilage, the product develops a foul odor and undergoes blackening.

In some instances, organoleptic changes are not clearly defined, yet the canned products may still contain exotoxins — metabolic byproducts of certain microbial species.

Alongside microbial reactivation, certain types of canned products experience a die-off of microorganisms during storage, rendering the canned food fully sterile.