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

TOPIC 8. MICROBIOLOGICAL CONTROL IN FISH PROCESSING

8.1. Fish Microflora

The main objective of MICROBIOLOGICAL CONTROL OF fish and fish products is to study the species COMPOSITION AND PROPERTIES of Bacteria found on fish and fish products, their impact on fish Processing technologies, as well as to develop effective Sanitary and hygienic controls for facilities, Water analysis, equipment, inventory, packaging, and employee personal hygiene. Sanitary and microbiological control of fish production is subdivided into primary (preventive) and secondary.

Primary microbiological control involves the examination of incoming raw Materials, auxiliary materials, finished products, and the sanitary condition of the production environment. It is carried out systematically, within timeframes specified by regulatory and technical production documents and regulatory authorities in accordance with the law.

Secondary microbiological control of production is carried out in cases of persistently elevated microbial contamination of the finished product in order to detect and eliminate the source of contamination, as well as when the presence of foodborne pathogens and toxicoinfections in the product is suspected. Microbiological control is performed by the plant's internal laboratory or, in its absence, by certified third-party organizations.

Fish flesh has a loose consistency due to lower Connective Tissue content compared to the meat of warm-blooded animals. This facilitates the spread of microorganisms throughout the fish's body. The quantitative and species COMPOSITION OF THE microflora in freshly caught fish depends on the species, Nature of the water body, season of the year, region, fishing techniques, and other factors.

Muscle juice and Muscle tissue of a freshly caught, healthy fish are considered sterile. Significant numbers of bacteria are found in the outer mucous membrane, on the outer gills, and in the gastrointestinal tract. The bacterial count in these areas can range from 10³ to 10⁶ per 1 cm².

The slime covering The surface of the fish not only harbors microorganisms but also serves as a favorable medium for their proliferation.

In warm waters, mesophilic microbes—various species of bacilli, corynebacteria, and micrococci—are present On the surface of the fish. The outer mucous membrane may harbor luminous bacteria, such as Photobacterium phosphoreum.

Psychrophilic and psychrotrophic microorganisms dominate in temperate and cold waters. In river and marine waters, the number of psychrophiles per 1 cm² of fish Skin ranges from 10² to 10⁴. They are represented mainly by bacteria of the genera Pseudomonas, Alcaligenes, Acinetobacter, and Flavobacterium. Micrococci, corynebacteria, less commonly spore-forming rods of the genus Bacillus, as well as Yeasts and actinomycetes, are also encountered. Sturgeon species are sometimes contaminated with the botulism bacterium, C. botulinum. In coastal marine waters and inland water basins, the fish surface may be contaminated by bacteria of the Enterobacteriaceae family belonging to the genera Escherichia, Enterobacter, Citrobacter, Klebsiella, and Proteus.

Due to the deteriorating ecological situation worldwide and the discharge of municipal and industrial wastewater into natural water bodies, the risk of fish contamination with pathogenic bacteria, such as Shigella and Salmonella, has increased dramatically. This requires special attention to the sanitization of fish and changes in its microflora during storage. Freshwater fish can retain Salmonella in their bodies for extended periods. Mass endogenous contamination of fish with Salmonella species such as S. enteritidis or S. typhimurium causes pseudomembranous enteritis in them.

Enteropathogenic strains of staphylococci are usually isolated during fish processing, as these microorganisms account for about 40% of the microflora on the hands and nasopharynx of processing personnel. To prevent fish contamination with staphylococci, the fish storage Temperature should not exceed 10 °C.

Halophilic vibrios, specifically Vibrio parahaemolyticus—the CAUSATIVE AGENT OF gastroenteritis and toxicoinfection-type poisoning—are sometimes found on marine fish.

A large number of diverse microorganisms are always present in the gills and intestines of fish. The intestinal contents of a freshly dead fish contain up to 10⁵ - 10⁸ microbial Cells per 1 g. These include various putrefactive bacteria, including spore-forming species: C. sporogenes, C. perfringens, and C. bifermentans.

Yeasts may also be present on the outer gills and oral mucosa of freshly caught fish, but during subsequent storage, they are suppressed by bacteria. The isolated yeasts were identified as belonging to the genera Debaryomyces, Torulopsis, Candida, Rhodotorula, Pichia, and Cryptococcus.

MICROBIOLOGICAL EXAMINATION OF fresh fish begins with the preparation of impression smears (bacterioscopic method), followed by bacteriological analysis.

Freshly dead fish rapidly undergo microbial spoilage, which occurs faster the higher its storage temperature and the greater the microbial load on the fish. Putrefactive aerobic bacteria actively multiply on the fish surface, consuming oxygen and thereby creating conditions for anaerobes to develop, shifting the putrefactive process inward. The proliferation of putrefactive microflora leads to an alkaline shift in pH, which favors the multiplication of many other microorganisms. Therefore, fish should be rapidly cooled after catching. A sharp drop in the fish's body temperature halts the multiplication of most microorganisms. Thus, while at 18 °C the bacterial count increases to 10⁸ - 10⁹ per 1 g of fish within one day, at a temperature around 0 °C Microbial growth is delayed for 24–48 hours, after which the proliferation of psychrophilic microorganisms begins on the surface of the chilled fish and gills. Bacteria of the genus Pseudomonas are the primary agents of fish spoilage. After 10 days, the proportion of pseudomonads rises to 50% of the total count, and after 18 days, to 96%. By causing the putrefactive breakdown of protein, pseudomonads produce significant amounts of volatile compounds, including foul-smelling trimethylamine, as well as gases such as H₂S and NH₃. Pseudomonads are characterized not only by a high growth rate but also by elevated enzymatic activity toward Proteins and Lipids.

Ice combined with sodium chloride is frequently used for chilling fish, which in turn can serve as sources of contamination by extraneous microorganisms.

Freezing ensures longer-term preservation of fish. If freshly caught fish is subjected to rapid freezing, all microflora remains localized on the surface of its body, whereas the muscle tissue interior remains free of microorganisms. Storing such frozen fish at temperatures no higher than minus 12–15 °C allows its quality to be maintained for a long time (several months) and significantly reduces the microbial count.

During freezing and prolonged cold storage, some microorganisms die off, but many retain viability in a state of anabiosis. Moreover, the higher the freezing rate and the lower the storage temperature, the greater the number of microorganisms that will remain viable.

Frozen fish predominantly harbors various micrococci, spore-forming and non-spore-forming rod-shaped bacteria, and mold fungal spores. The permissible saprophytic microbial content on frozen fish is up to 10⁵ CFU/g.

8.2. Bacterioscopic Examination of Fresh Fish

Sampling. Samples for microbiological examination are taken in accordance with regulatory documentation from each batch (of the same species, grade, name, etc.). The number of packaging units to be opened is established by current production regulatory and technical documents (at least 5% or 5 units of the total batch quantity). Before sampling, the entire batch must be inspected, individual packaging units opened, an organoleptic evaluation of the product performed, and then the sample collected. Samples for microbiological analyses are taken using sterile instruments into sterile containers. Samples of small fish, non-fish marine harvest objects, roe sacs (yastyki), milt, etc., are taken in amounts of 3–10 specimens from different locations of the test batch to form a composite sample. The composite sample is prepared by mincing, mixing, and grinding the collected specimens.

A 1 g analytical portion is taken from the composite sample and gradually added to 9 cm³ of liquid (isotonic sodium chloride solution) to obtain an initial dilution of 10^-9. The suspension is thoroughly mixed or shaken and left at room temperature for 3–5 minutes. The supernatant liquid is then examined. If necessary, subsequent dilutions are prepared using a fresh pipette each time.

Large fish and large specimens of non-fish marine harvest are sampled in quantities of no more than 3 pieces. From each specimen, tissue pieces containing skin and muscle, measuring approximately 4 cm² in area and 4-5 mm in thickness, are excised from several locations without touching the intestine, and placed into a flask to prepare a composite sample. After gutting and washing, fish and marine harvest items are sampled by cutting out small pieces weighing no more than 300 g.

Each sample is wrapped individually in parchment paper, labeled with the date, sampling Location, and fish species, indicating the reason and purpose of the study, and sent to the laboratory.

For perishable foods such as fish and fishery products, the time interval between sampling and microbiological analysis must not exceed 6 hours at a temperature range of 0 to +4 °C. Such analyses are performed separately for the surface of the fish and its internal Tissues.

A flamed Glass slide is applied to the surface of the fish under study and pressed against it for 1 min. The slide is carefully removed, air-dried, fixed in a burner flame, and Gram-stained. A drop of immersion oil is applied to the smear preparation, which is then examined under a Microscope using an oil immersion objective (90x magnification) in at least thirty fields of view.

A smear preparation is similarly prepared from deep muscle tissue. To do this, the skin in the middle of the fish's back is scaled and cauterized with a red-hot scalpel. Using sterile scissors and forceps, a piece of fish muscle is excised at a depth of 1.0-1.5 cm with a total surface area of approximately 2 cm². Several smears are made using the excised piece. The piece is applied to a glass slide with different faces for 1 min, after which the preparation is processed as described above.

During microscopic examination, the number of bacterial cells (cocci and rods) and Yeast cells is counted separately in each observed field of view; the result is expressed as the average Total Cell Count across thirty fields of view. The presence or absence of signs of muscle tissue degradation in the field of view is also noted.

The Microscopy results are evaluated According to the data presented in Table 8.1.

Class="center">Table 8.1. Evaluation of the results of bacterioscopic analysis of fish

8.3. Bacteriological analysis of fresh, chilled, and frozen fish

The bacteriological method includes testing fish for total microbial count, coliform bacteria content, presence of salmonellae, Proteus group bacteria, and enterococci.

During bacteriological analysis, each sample is freed from adipose and Connective Tissues, immersed in alcohol, and then pieces measuring 2.0 x 1.5 x 2.5 cm are excised from deep layers at various locations using sterile scissors. All excised pieces are minced with sterile scissors. Composite samples of 15 g are prepared for inoculation.

Determination of total viable count (TVC). When examining fresh fish, sample dilutions from 10^-1 to 10^-4 are prepared.

To inoculate 0.1 g of the product (10^-1 dilution), the first tenfold dilution of the suspension is prepared: 1 cm³ of the suspension is collected with a sterile pipette and transferred into a test tube containing 9 cm³ of sterile saline (1 cm³ of the resulting solution contains 0.1 g of the product).

To inoculate 0.01 g of the product (10^-2 dilution), the second tenfold dilution is prepared: the Contents of the test tube with the first dilution are mixed using a sterile pipette, 1 cm³ is collected and transferred into a test tube containing 9 cm³ of sterile saline (1 cm³ of the resulting solution contains 0.01 g of the product).

To inoculate 0.001 g of the product, the third tenfold dilution (10^-3) is prepared: the contents of the test tube with the second dilution are mixed using a sterile pipette, 1 cm³ is collected and transferred into a test tube containing 9 cm³ of sterile saline (1 cm³ of the resulting solution contains 0.001 g of the product).

To inoculate 0.0001 g of the product, the fourth tenfold dilution (10^-4) is prepared: the contents of the test tube with the third dilution are mixed using a sterile pipette, 1 cm³ is collected and transferred into a test tube containing 9 cm³ of sterile saline (1 cm³ of the resulting solution contains 0.0001 g of the product).

Table 8.2 shows the microbiological safety parameters for fish.

Table 8.2. Microbiological parameters of fresh, chilled, and frozen fish

1 cm³ of each dilution is inoculated into Petri dishes with pre-labeled lids and poured with 10–15 cm³ of meat-peptone Agar (MPA) melted and cooled to 40–45 °C. Immediately after pouring the agar, the contents of the Petri dishes are thoroughly mixed by gentle swirling to evenly distribute the inoculum. After the agar solidifies, the Petri dishes are inverted and placed in this position in an incubator at 30 °C for 72 hours.

Upon completion of cultivation, the number of colonies grown on the MPA plates is counted. A magnifying glass with 4-10x magnification or a dedicated colony counter is used for this purpose. If There is a large number of colonies evenly distributed in the agar, four or more identical sectors are drawn on the bottom of the Petri dish, the number of colonies in two to three sectors is counted (covering at least 1/3 of the dish surface), the arithmetic mean of the colonies is calculated, and it is multiplied by the total number of sectors across the entire dish.

8.4. Microbiology of salted fish

Salting is the most common method of fish preservation. Based on their sensitivity to sodium chloride, microorganisms are conventionally divided into three groups:

1. Halophobic. This group includes microorganisms whose metabolic activity is inhibited at a sodium chloride concentration of 5-6 %. These primarily include most putrefactive bacteria, lactic acid bacteria, coliforms, and certain pathogenic bacteria of the genera Salmonella and Vibrio. At a sodium chloride content of about 6 % in the medium, the multiplication of bacteria of the genera Pseudomonas and Achromobacter is suppressed, although they remain viable.

2. Halotolerant microorganisms (facultative halophiles). These microorganisms can grow at salt concentrations ranging from 6 to 15%. Staphylococci are characterized by a high resistance to salt; their reproduction is suppressed only at a salt concentration of 16–18% in the medium. Molds also exhibit significant resistance to sodium chloride. Specifically, the growth of Aspergillus niger is inhibited at 17% sodium chloride, and Penicillium glaucum at 19–20%.

3. Halophilic microorganisms (obligate halophiles). For the reproduction of this group of microorganisms, the optimal salt concentration lies within the range of 3–6%. They are capable of growing in concentrated salt solutions or directly in salt itself. It is hypothesized that elevated osmotic pressure or reduced water activity in the nutrient medium stimulates the growth of these microorganisms. This group includes Micrococcus roseus, which imparts a red-pink color to salted herring.

Table 8.3 presents the sodium chloride concentrations at which the growth of certain microorganisms is suppressed.

Table 8.3. Sodium chloride concentrations inhibiting the growth of various microorganism species

The preserving action of salt depends on its concentration, the temperature of the medium, the pH of the medium, and the presence of preservatives.

The following salting Methods are distinguished:

✵ warm, chilled, and cold;

✵ saturated and unsaturated;

✵ dry, mixed, and wet (brine).

With warm salting, a salt concentration of 15–20% is achieved in fish muscle within a day; with chilled salting, it takes more than a day, and cold salting is used for large and fatty fish.

In saturated salting, the salt concentration in the brine must exceed 20%.

In dry salting, the fish is mixed with salt crystals; in wet and brine salting, it is immersed in a salt solution; in the mixed method, the fish is first mixed with salt, and then the salt solution is added immediately.

The latter method is finding increasingly wide application, because this type of salting creates a fish-salt-brine system in which The transfer of NaCl from the brine into the fish tissues proceeds efficiently, accompanied by the simultaneous osmotic transfer of water from the fish cells into the brine. This two-way transfer continues until an equilibrium salt concentration is established in both the brine and the muscle tissue.

During the salting process, fish loses water and absorbs salt. Over the first 10–15 days, the microbial count in the brine increases, and then decreases after 2–3 months.

In medium brine, the total microbial count is 102–104 microbial cells per 1 cm³. In lightly salted fish products, as the storage temperature rises, the total number of microorganisms increases and can range from 10 to 10 cells per 1 g of product. The qualitative composition of the microflora in such fish is quite diverse. It includes halophilic bacteria of the genera Micrococcus, Streptococcus, and Corynebacterium, as well as yeasts and molds. Food poisoning pathogens—such as Staphylococcus aureus, salmonellae, and enterococci—may also be present.

Under heavy dry salting conditions, 90% of the microflora is represented by bacteria of the genus Micrococcus. The remaining 10% consists of bacteria of the genera Flavobacterium, Achromobacter, Pseudomonas, and Sarcina.

8.5. Microflora of Fish Preserves (Prèserves)

The primary preserving factor in preserves is sodium chloride, with an allowable content ranging from 3 to 10%. To protect preserves from spoilage, an antiseptic (benzoic acid or sodium benzoate) is added at a concentration of 0.1% by mass of the fish. Sodium benzoate predominantly suppresses putrefactive bacteria and, to a lesser extent, molds. Lactic acid bacteria and yeasts have low sensitivity to the antiseptic. In some preserves, acetic acid is added instead of sodium benzoate, which enhances The Effect of sodium chloride.

Microbial spoilage of Preserves

1. Slime formation in the brine. This defect is caused by the polymerization of sucrose, resulting in The formation of a levorotatory compound (levan) or a dextrorotatory compound (dextran). In the brine, the slime consists mainly of levan. Gram-negative rods are responsible for levan production, whereas Leuconostoc mesenteroides ssp. dextranicum is responsible for dextran formation.

2. Gas production. In small-pack preserves, gas formation is observed, driven by the growth of Heterofermentative lactic acid bacteria or leuconostocs (Leuconostoc citrovorum).

Pathogenic Microorganisms in Preserves

Due to the growth of lactic acid bacteria, the product becomes acidified; consequently, C. botulinum multiplies very rarely. Contamination of preserves with salmonellae is of no significant concern, as the existing salt concentrations (up to 8%) prevent the proliferation of these microbes. S. aureus exhibits high Salt Tolerance. Contamination of preserves with Staphylococcus aureus occurs primarily from processing personnel. The multiplication of Staphylococcus aureus and its enterotoxin production can occur at salt concentrations of up to 10%. To inhibit toxin production by S. aureus in preserves, they must be stored at temperatures below 10 °C.

Topic Assignment:

1. Perform a bacterioscopic analysis of fresh fish. Draw a Conclusion regarding the freshness of the tested fish samples.

2. Conduct a bacteriological analysis of raw fish. Inoculate nutrient media to determine the total viable count (TVC) and the titer of coliform bacteria (coliform group).

Control questions

1. What microorganisms are present on the surface of fresh fish, in the gills, and in the intestines?

2. Which microorganisms dominate on chilled fish during storage?

3. Into what groups are microorganisms categorized based on their attitude to sodium chloride?

4. List the methods of fish salting.

5. Name the types of spoilage in semi-preserved fish products.