FUNDAMENTALS OF MICROBIOLOGY - E. Y. Tyumentseva - 2015

TOPIC 7. FOOD MICROBIOLOGY

Fruits, vegetables, meat, fish, milk, and dairy products play a significant role in Human Nutrition while simultaneously being highly susceptible to microbial spoilage due to their favorable chemical composition.

To rapidly detect and identify the pathways by which spoilage microorganisms penetrate production facilities, pinpoint their sources and proliferation rates at various Stages of the technological process, and prevent The Development of extraneous microflora through various preventive measures, microbiological monitoring is carried out using standard Procedures.

7.1. Microbiological Control

Fruits and vegetables are living organisms capable of resisting microbial action. Their Immunity is determined by specific intrinsic properties: high acidity of the pulp juice, presence of glucosides, Essential Oils, Tannins, phytoncides, and others. Furthermore, the immunity of fruits and vegetables is supported by Phenolic Compounds formed at the sites of wounds and pathogen invasion. These compounds inhibit many pathogens affecting fruits and vegetables.

When studying the MICROBIOLOGY OF MEAT, it is essential to understand that its physicochemical properties make it a favorable medium for Microbial growth, rendering it highly susceptible to bacterial spoilage.

In studying fish microbiology, it should be noted that fish are stored whole and therefore harbor A wide variety of microbes located not only on their surface—within the slime layer—but also internally, in the intestines and gills. Even while alive, diseased fish contain more microorganisms than healthy ones, and they spoil faster after catch.

When studying the microbiology of milk and dairy products, one must understand how microorganisms enter milk, what determines the bactericidal properties of fresh milk and under what conditions these properties are preserved over time, what normal and abnormal milk microflora are, how the microflora dynamics change during storage, and what bacteriological requirements pasteurized milk intended for sale must meet.

When studying the microflora of canned foods, it is necessary to understand what residual microflora consists of, which types of microorganisms are most frequently found in canned goods after sterilization, what microorganisms and under what conditions cause biological Swelling (bombage) of cans, and what Other types of bacterial spoilage canned foods may undergo.

The objective of microbiological control is to rapidly detect and identify the pathways by which spoilage microorganisms penetrate production facilities, locate their sources and proliferation rates at various stages of the technological process, and prevent the development of extraneous microflora through the application of various preventive measures.

Microbiological control is carried out systematically by factory laboratories. In the absence of an in-house microbiological laboratory, such monitoring may be performed under a commercial contract with State Sanitary and epidemiological Surveillance authorities or laboratories accredited for microbiological testing. It is conducted at all stages of the technological process, from raw Materials to the finished product, based on approved state standards (GOST), technical specifications (TU), instructions, biomedical requirements, and sanitary quality standards for food raw materials and food products, as well as other regulatory documentation. Specific production facilities have their own microbiological control schemes, which define the objects of control, sampling points, monitoring frequency, required microbiological indicators, and established standards for these microbiological parameters.

Many food products serve as a favorable environment for the GROWTH AND DEVELOPMENT of extraneous microorganisms. Failure to comply with technological Processing regimes for raw materials, poor Sanitary and hygienic conditions in production, and violations of storage conditions and shelf life can lead to the intensive accumulation of toxin-producing microorganisms, which is a cause of food poisoning.

In addition, if food industry workers fail to observe sanitary rules and standards, pathogenic microorganisms—the causative agents of foodborne infections—can enter the products. Therefore, the most critical CHARACTERISTICS OF FOOD products are their safety and microbiological stability.

Safety is defined as the absence of harmful chemical and biological impurities, including pathogenic microorganisms and toxic byproducts of their metabolic activity. METABOLISM/2.html">THE CONCEPT OF "microbiological stability" refers to the potential ability of a product to resist spoilage over time.

The microflora of food products represents a complex, dynamic system interacting with the external environment, which significantly complicates the Methods used for its study and the interpretation of obtained results.

To assess the quality of food products as well as their production and storage conditions, quantitative and qualitative indicators are used. Quantitative indicators indicate the total count of microorganisms of specific groups per 1 g (cm³) of the product. Qualitative indicators indicate the absence (or presence) of specific microbial species in a given mass of the product.

When conducting microbiological testing of food products, one can be guided by the biomedical requirements and sanitary standards for the quality of food raw materials and food products (SanPiN 2.3.2.560-96). Microbiological standards for baby food products are presented in the methodological guidelines (MUK 4.2.577-96) "Methods of MICROBIOLOGICAL CONTROL OF Baby and Dietary Foods and Their Components."

7.2. Groups of Microbiological Food Safety Criteria

1. Group of sanitary condition indicators.

Direct detection of pathogenic microorganisms (causative agents of foodborne infections) in food products is often impossible due to their low concentration compared to the saprophytic microflora. Therefore, sanitary evaluation of food products relies on indirect methods that determine the level of product contamination by human excretions. The higher this level, the greater the likelihood that pathogenic microorganisms—causative agents of intestinal infections—have contaminated the object.

The sanitary evaluation of food products is carried out using two microbiological parameters: total bacterial count (TBC / TVC) and the presence of coliform Bacteria (coliform group).

Total bacterial count (TVC) — the number of mesophilic aerobic and facultatively anaerobic microorganisms per 1 g or 1 cm³ of the product. Regulatory documentation specifies the permissible limit of these microorganisms in CFU (colony-forming units) units.

High bacterial contamination of food products indicates inadequate heat Treatment of raw materials, insufficient washing and disinfection of equipment, and unsatisfactory storage and transportation conditions.

Total bacterial count is determined in products that lack technically beneficial microflora (starter cultures). To determine this indicator, universal nutrient media are used: meat-peptone Agar (MPA) or media designated for enumerating mesophilic aerobic and facultatively anaerobic microorganisms.

The presence of coliform bacteria (coliforms) is determined in all liquid products, in all products of animal origin (with the exception of sterilized ones), and in many plant-based products. Coliforms combine Representatives of the normal human intestinal microflora and belong to the family Enterobacteriaceae, genera Escherichia, Citrobacter, Enterobacter, Klebsiella, and Serratia. Coliforms serve as an indicator of fecal contamination and are classified as sanitary-indicator microorganisms.

The Selection of coliforms as sanitary-indicator microorganisms for assessing the sanitary condition of food products is no coincidence. Sanitary-indicator microorganisms must meet the following requirements:

- these microorganisms must be representatives of the normal microflora of the Organism, developing and multiplying within it;

- they must be excreted from the organism in large quantities;

- they must retain their viability in the environment for a long time without multiplying;

- they must not be altered by environmental factors, nor suppressed or stimulated by other microorganisms;

- these microorganisms must be evenly distributed in the studied environmental objects;

- the determination of these microorganisms must be carried out using simple methods.

Regulatory documents usually specify the quantity of product in which coliforms are not allowed. When the level of product contamination with coliforms is high, the probability of finding pathogenic microorganisms—causative agents of intestinal infections (dysentery, typhoid fever, cholera, etc.) increases. To determine coliforms, Kessler's enrichment medium is used, and the identification of these bacteria is carried out using Endo's differential diagnostic medium.

2. Group of opportunistic microorganisms.

This group includes microorganisms that cause food poisoning, such as Proteus vulgaris, Clostridium perfringens, Bacillus cereus, Staphylococcus aureus, and Clostridium botulinum.

Opportunistic microorganisms are constantly present in the environment and in living macro-organisms. A favorable environment for the growth and development of these microorganisms is meat and meat products, which is why these particular foods are most often the cause of food poisoning. Thus, many of the aforementioned microorganisms are regulated in sausage products and other meat products.

In canned meat and many canned plant products, the content of sulfite-reducing clostridia, which develop under anaerobic conditions, is regulated.

In protein-rich dairy products (e.g., cottage cheese, cheese), the content of coagulase-positive Staphylococcus aureus, the CAUSATIVE AGENT OF food intoxication, is regulated.

When identifying opportunistic microorganisms, elective nutrient media are used. For example, the presence of Staphylococcus aureus is detected using milk-salt agar (MSA) or egg-yolk-salt agar (EYSA).

3. Group of pathogenic microorganisms (Appendix 2).

Among pathogenic microorganisms, salmonellae are determined in food products. Studies for the presence of salmonellae are conducted by sanitary and epidemiological surveillance authorities. Usually, salmonellae are not allowed in 25 g (cm³) of the product. In certain baby and dietary food products, the presence of salmonellae is not permitted in 50 or even 100 g (cm³).

To determine salmonellae, enrichment nutrient media (selenite, Kauffmann, Müller) and differential diagnostic media (Ploskirev, Levin) are used.

4. Group of indicators of product microbiological stability.

This group includes microscopic Fungi and Yeasts, which, as is known, are agents of product spoilage. This indicator is regulated in many products derived from plant raw materials, as well as in products of animal origin with plant additives. The growth dynamics of fungi and yeasts must be studied when establishing shelf lives and storage regimes for new types of products. Molds and yeasts are determined using wort agar or Sabouraud medium, and the number of fungal and Yeast colonies grown on solid media is counted separately.

In addition to the aforementioned regulated microbiological indicators, for predicting the quality of manufactured food products, it is advisable to determine separate groups of microorganisms that are representatives of technically beneficial and technically harmful microflora.

Thus, in cheese production, putrefactive bacteria are periodically determined as the main agents of cheese spoilage, and the development of beneficial microorganisms (lactic acid and propionic acid bacteria) is monitored during the cheese-making process.

7.3. Concept of the Hazard Analysis Critical Control Point (HACCP) system

To guarantee the quality and safety of manufactured food products abroad, the Hazard Analysis Critical Control Point (HACCP) system is actively implemented as the basis for food expertise. HACCP stands for Hazard Analysis Critical Control Point.

A characteristic feature of this system is the systematic monitoring and control of food products with the preliminary identification of all possible factors associated with the complete food handling cycle. This monitoring begins with the control of animal-rearing conditions and plant-growing conditions, as well as the habitat of commercial animals and hydrobionts. Next, the conditions for obtaining raw materials and The production of a specific product from these raw materials are controlled. The monitoring concludes with the examination of the finished product after its preparation, storage, transportation, and distribution.

This system differs significantly from the previously used sanitary and hygienic control and inspection methods, which focused primarily on inspecting final products only.

Although the critical control point system was originally developed for microbiological food safety control, in recent years it has also been successfully applied to monitor and prevent chemical residues—including agricultural chemicals (such as fertilizers, herbicides, and pesticides), veterinary drugs, Hormones, and foreign chemical contaminants in food products.

The International Commission on Microbiological Specifications for Foods (ICMSF) has recommended that the World Health Organization (WHO) adopt HACCP as an international standard. Currently, EU countries require that imported meat and seafood be processed and produced under the HACCP system.

7.4. Scheme for Food Product Dilution and Microbiological Analysis

To prepare product dilutions, test tubes containing 9 cm³ of sterile Water are used. Alternatively, dilutions can be prepared using sterile solutions of diluted phosphate buffer, isotonic sodium chloride solution, peptone water, or sodium citrate. Using a sterile pipette, 1 cm³ of the product is added to the first test tube. Using a new sterile pipette, the Contents of the tube are thoroughly mixed (yielding a 1:10 dilution). Then, using the same pipette, 1 cm³ of liquid is withdrawn from the 1:10 dilution tube and transferred to a second tube containing water (yielding a 1:100 dilution). The number of dilutions is calculated so that between 30 and 300 colonies grow on the Petri dishes.

For instance, when analyzing pasteurized milk, it is recommended to prepare the 1st, 2nd, and 3rd dilutions of the product, as the standard permissible level of mesophilic aerobic and facultatively anaerobic microorganisms in drinking milk does not exceed 50–200 thousand CFU/cm³.

Figure 5 illustrates the Procedure for preparing product dilutions and inoculating them onto Petri dishes.

Class="center">Fig. 5. Scheme for preparing product dilutions and inoculating them onto Petri dishes

Recommendations for preparing the 1st dilution (1:10):

1) for cream-filled confectionery products and margarine.

1 g of cream or margarine is weighed under aseptic conditions and added to a test tube containing 9 cm3 of water. The tube is then placed in a water bath at 50–55 °C and kept there until the cream is completely melted. The contents of the tube are thoroughly mixed, and 1 cm³ of the liquid beneath the fat layer is collected for subsequent dilutions;

2) for products with a solid and heterogeneous consistency (e.g., sausage products, canned vegetables).

1 g of the average sample of the test product is weighed under aseptic conditions and placed into a sterile mortar. 9 cm³ of sterile water is also added to the mortar, and the material is ground with sand for 10–15 minutes near a burner flame until a homogeneous mass is obtained. The suspension is then allowed to settle, and 1 cm³ of the supernatant is collected to prepare the 1:100 dilution.

7.5. Plate METHODS FOR QUANTITATIVE Enumeration of Microorganisms

The Essence of plate counting methods lies in inoculating product dilutions onto sterile solid nutrient media in Petri dishes, followed by incubation and counting the resulting colonies. Each colony is assumed to originate from the multiplication of a single Cell.

Recording results when using plate methods. The grown colonies are counted in each dish by placing it upside down against a dark Background, using a magnifying Glass with 4x to 10x magnification. When colonies are numerous and evenly distributed, the bottom of the dish is divided into sectors, the number of colonies in 2–3 sectors is counted, the arithmetic mean is calculated, and this value is multiplied by the dilution factor (10 for the first product dilution, 100 for the second, etc.).

If the incubated plates from the first dilution (1:10) show no colonies, the result is expressed as: less than 1x10 CFU/cm³ (CFU — colony-forming units).

If the Petri dishes from the 1st dilution (1:10) contain fewer than 15 colonies, the result is expressed as: microorganism count less than Mx10 CFU/g, where M is the number of colonies grown.

If the number of colonies exceeds 15, the colonies in the plates are counted, multiplied by the dilution factor, and the resulting value is rounded in accordance with GOST 26670-91 "Food products. Methods for CULTIVATION OF MICROORGANISMS":

- to the nearest multiple of 5, if the number of colonies in the plate is less than 100;

- to the nearest multiple of 10, if the number of colonies in the plate is greater than 100.

Example: The 1st dilution of the product (1:10) was inoculated. 194 colonies grew on the Petri dish. The resulting value is rounded to 200.

Microorganism count in the product: 200x10 = 2.0x103 CFU/g. Plate methods are used to determine the following microbiological parameters: total aerobic and facultatively anaerobic microorganisms (TAMRA/TVC), mold and yeast spore counts, putrefactive bacteria content, and coagulase-positive staphylococci.

7.6. Practical Identification of Microorganisms

Determination of mesophilic aerobic and facultatively anaerobic microorganisms (QMAFAnM)

Petri dishes are labeled prior to inoculation.

1 cm³ of each product dilution is added to Petri dishes. Hold the pipette with the inoculum at a 45 °C angle, touching the bottom of the dish with the tip. Then, pour 12-15 cm³ of melted meat-peptone agar—or specific media for determining mesophilic aerobic and facultatively anaerobic microorganisms, previously melted and cooled to 45 °C—into each dish. Immediately after pouring the agar, mix the contents thoroughly with a gentle rotary motion to evenly distribute the inoculum. If swarming growth of microorganisms is expected, overlay the solidified agar with a Second layer of nutrient medium or 3-5 cm³ of an aqueous agar solution. Once the medium solidifies, invert the Petri dishes (lids down) and incubate in a thermostat at (30±1) °C for 72 hours (a preliminary count is permitted after 48 hours, followed by the final count at 24 hours).

Determination of mold and yeast counts.

This is performed similarly to the QMAFAnM determination, except that wort agar or Sabouraud agar is used as the nutrient medium. Incubate the cultures at 24 °C for 5 days, with a preliminary reading after 3 days.

Determination of proteolytic (putrefactive) bacteria

An appropriate dilution of the product is inoculated onto milk agar, and the cultures are incubated at 30 °C for 72 hours. During growth on milk agar, proteolytic bacteria form clear zones where the agar is hydrolyzed (zones of proteolysis). Peptonizing bacteria form narrow zones of peptonization.

Determination of coagulase-positive staphylococci.

This is performed in the same manner as the QMAFAnM determination. Salt-milk agar or egg-yolk-salt agar is used as the nutrient medium. Cultivation is carried out at 37 °C for 24-48 hours. When grown on egg-yolk-salt agar, pearlescent zones of agar opacification form around the colonies, whereas salt-milk agar yields small zones of peptonization.

Determination of aerobic spore-forming bacteria of the genus Bacillus

Before inoculation, the test material or product dilution is pasteurized at 75-85 °C for 20 minutes. The subsequent determination is conducted similarly to the QMAFAnM procedure. Vegetative Cells are destroyed by pasteurization, whereas spores germinate after inoculation onto meat-peptone agar and incubation at 37 °C, forming colonies within 24-48 hours.

Methods based on microbial enrichment followed by identification

These methods are used to detect microorganisms present in very small numbers relative to the total microbial population. The essence of these methods lies in inoculating the product or its dilutions into liquid enrichment media. If microbial growth is detected after cultivation (such as sediment formation, medium turbidity, or gas accumulation in Fermentation tubes), subsequent subculturing is performed from the tubes showing growth onto differential

diagnostic media to identify the microorganisms that grew in the enrichment medium.

Such methods include testing for the presence of coliforms and Salmonella species.

Determination of coliform bacteria (coliforms)

Inoculate the volume of product in which coliforms are expected to be absent (1 cm³ of milk or 1 cm³ of the primary milk dilution). Inoculation is performed into Kessler broth tubes equipped with Durham tubes (inverted vials). Place the cultures in an incubator at 37 °C for 24 hours.

If there are no signs of growth (gas production in the Durham tubes, turbidity of the medium), it is concluded that coliforms are absent and the tested product meets the standard requirements for coliforms.

Structure/127.html">Interpretation of Results. If Endo or Levine agar shows no typical coliform colonies (red with a metallic sheen on Endo agar; black with a metallic sheen, dark with a black center, or lilac with a dark center on Levine agar), the product is considered to comply with the standard. If typical colonies are present on Endo or Levine agar, they are Gram-stained and examined microscopically. The detection of Gram-negative, non-spore-forming rods indicates the presence of coliforms in the analyzed sample and non-compliance with the microbiological standard.

If the fermentation test is positive, to definitively confirm the presence of coliforms in the product, streak from the suspicious tubes onto Endo or Levine agar plates. Inoculate from each tube using an inoculation loop to obtain isolated colonies. Place the plates in an incubator.

Determination of Salmonella species

Aseptically weigh out analytical portions of dry components or sterilely measure out volumes of liquid components (typically 25 g or 25 cm³) and inoculate them into flasks containing magnesium broth or Müller broth (enrichment media for Salmonella), maintaining a product-to-medium ratio of at least 1:9.

For liquid products, media with a double concentration of ingredients may be used at a product-to-medium ratio of 1:1.

Place the inoculated flasks in an incubator at 37 °C for 18-24 hours.

Following incubation, subculture from the enrichment flasks onto The surface of differential diagnostic media (Ploskirev agar or bismuth sulfite agar). To obtain single colonies, take a minimal amount of inoculum with a loop and streak it onto the agar plates. Place the inoculated plates in an incubator at 37 °C. Check the cultures twice: 24 and 48 hours post-incubation.

Recording results. On Ploskirev agar, Salmonella colonies are colorless, transparent, and flat; on bismuth sulfite agar, they appear black with a distinctive metallic sheen, or greenish with a black rim, accompanied by black staining of the agar beneath the colony.

If typical Salmonella colonies are absent on all media, the final analysis result is recorded as "negative," meaning Salmonella is absent in the tested mass of the product.

If typical or suspected Salmonella colonies appear on any of the nutrient agar plates, they are subjected to further Identification based on biochemical and other characteristics.

Other Methods for determining qualitative microbiological indicators

To assess the quality of food products, other qualitative microbiological methods are also employed, such as the detection of anaerobic sulfite-reducing clostridia and bacteria of the genus Proteus.

Detection of anaerobic sulfite-reducing clostridia

Using aseptic techniques, 1 cm³ of the appropriate dilutions of the test product is added to test tubes containing 9 cm³ of Wilson-Blair solid medium previously melted and cooled to 45 °C. The contents of the tube are thoroughly mixed, placed in an incubator, and cultured at 37 °C for 24 hours. The highest dilution of the product that yields blackening of the medium in the culture is taken as the positive titer.

Detection of bacteria of the genus Proteus

This is performed using Shukevich's method. To test, 0.5 cm³ of the analyzed suspension (dilution) is inoculated onto the Condensation water of a freshly sloped agar slant without touching the surface of the medium.

The vertically positioned tubes are incubated at 37 °C for 24 hours. On the agar slant, Proteus bacilli grow as a bluish, veil-like film. Microscopy of the preparation reveals Gram-negative, non-spore-forming rods.

Laboratory Procedure

Objective: to examine the microflora of selected food product samples (fruits and vegetables, flour, milk, meat, fish, etc.) using previously mastered methods of Qualitative and quantitative analysis.

Materials, Reagents, and equipment: food samples to be tested (cream, margarine, mayonnaise, drinking milk, sausage products, infant formula, canned vegetables); test tubes with 9 cm³ of sterile water; 1 cm³ sterile pipettes and Petri dishes; test tubes with sterile nutrient media: MPA or medium for total viable count (TVC) determination, Sabouraud medium or wort agar, Kessler medium with Durham tubes, salt agar, etc.; Gram staining kit; bacteriological loops and dissection needles; filter paper; glass slides and cover slips; Microscope; spirit lamp; staining rack; wash bottle; incubators.

Experiment No. 1. Cream confectionery products

Creams used for cakes and pastries are highly perishable products that can cause food poisoning. In addition to various spore-forming and non-spore-forming bacteria, yeasts, and mold spores, pathogenic microorganisms may be present in creams. Custard is particularly hazardous because, unlike other creams, it features a lower sugar concentration, higher moisture content, and the presence of flour. Besides spoiling rapidly due to the growth of acid-producing bacteria, custard can support the growth of toxigenic Staphylococcus aureus and certain opportunistic pathogens (e.g., enteropathogenic Escherichia coli). It should be noted that toxin accumulation in cream-based products occurs at temperatures between 15 and 22 °C.

Cream contamination may stem from raw materials (milk, cream, sugar, butter, eggs). Breaches of processing protocols and sanitary regulations during the production and storage of creams and cream-based products lead to the intensive proliferation of microorganisms introduced via raw materials as well as those entering the cream during manufacture and storage. Therefore, in accordance with regulations for the storage and shelf-life of perishable goods, cakes and pastries with various creams may be stored at temperatures not exceeding 6 °C for a limited time (for example, protein-whipped cream for no more than 72 hours).

Finished cream products are subject to microbiological control. Depending on the type of cream, the total viable count (TVC) must not exceed 1x10⁴ – 1x10⁵ CFU/g; coliforms must be absent in 0.01 g; and Staphylococcus aureus must be absent in 1 g of custard and 0.01 g of butter cream. Pathogenic microorganisms, including Salmonella, must be absent in 25 g of cream.

Experiment No. 2. Drinking milk

The microflora of drinking milk consists of residual microflora from pasteurized milk (represented by bacillar and clostridial spores, as well as heat-resistant lactic acid rods) and secondary contamination microflora (coliform bacteria, psychrophilic putrefactive bacteria, mesophilic lactic acid streptococci and rods, yeasts, etc.). Microorganisms entering drinking milk can cause defects in its consistency, taste, and color. The regulated quality indicators for drinking milk are presented in Appendix 5. For example, for bottled and pouched pasteurized milk of Group A, the total bacterial count (TVC) must not exceed 5x10 CFU/cm³, coliforms and Staphylococcus aureus must be absent in 1 cm³ of milk, and pathogens, including Salmonella, must be absent in 25 cm³.

Experiment No. 3. Margarine and mayonnaise

a) margarine. Microorganisms play a dual role in margarine production. Lactic acid bacteria, which form part of the water-milk phase, constitute the beneficial microflora of margarine as they impart a specific taste and aroma. All other microorganisms introduced via raw materials or from the external environment act as production contaminants, degrading margarine quality and reducing its storage stability. The primary sources of extraneous microflora in margarine are the Components of the water-milk phase, since fats and vegetable oils provide an unfavorable environment for microbial growth. Microbial spoilage of margarine is caused by putrefactive bacteria introduced with milk (causing a bitter taste), molds, yeasts, and fluorescent bacteria (causing a rancid taste and unpleasant odor, with molds also causing pigment spots on the margarine), as well as heat-resistant lactic acid bacteria (causing an excessively sour taste). Margarine quality is assessed by the absence of coliforms in 0.01 g, and the levels of yeasts (not more than 5x10³ CFU/g) and molds (not more than 20 CFU/g). Salmonella must be absent in 25 g of margarine.

б) mayonnaise. Industrially beneficial microorganisms are not used in mayonnaise production. This product may harbor only technically harmful microflora originating from equipment surfaces and the residual microflora of the mayonnaise ingredients. Representatives of the technically harmful microflora in mayonnaise cause gas production (spoilage agents include Heterofermentative lactic acid bacteria and yeasts), container swelling (caused by butyric acid bacteria of the genus Clostridium), and a bitter taste (putrefactive spoilage bacteria). In margarine [Note: referring to mayonnaise context], standards regulate the presence of coliforms (absent in 0.1 cm³), yeast content (not more than 5x10² CFU/cm³), mold count (not more than 10 CFU/cm³), and the absence of Salmonella (absent in 25 cm³).

Experiment No. 4. Canned vegetables

Depending on pH and chemical composition, canned vegetables can be classified into four groups: A, B, C, and D.

Before conducting MICROBIOLOGICAL ANALYSIS OF canned vegetables, it is necessary to determine to which of the aforementioned groups they belong. Canned products with a pH above 4.2–4.4, which can support the growth of food poisoning agents, require particularly thorough inspection.

The permissible total bacterial count (TVC) in canned foods prior to sterilization is regulated. The total bacterial count per 1 g (1 cm³) of the product must not exceed 10,000–50,000 (depending on the product type), and in canned baby food, it must not exceed 200. Clostridia must be absent in 0.5 cm³ of the can contents. Mesophilic bacilli are permitted in amounts of no more than 100–300/g.

When examining finished canned products, the cans are checked for hermetic sealing, incubated at 37 °C for 5 days, after which samples are taken from the cans for microbiological analysis. Maintaining a normal container appearance after incubation is an indicator of the microbiological Stability of the canned goods. The contents of defective cans showing signs of microbial spoilage (such cans are permitted in amounts of no more than 0.2%) are analyzed to determine The Nature of the defect.

Experiment No. 5. Sausage products

Sausages belong to products consumed without prior heat treatment; therefore, they must meet high sanitary standards. The sources of microflora in sausage products are raw materials and secondary contamination introduced during processing. Consequently, the manufacturing processes of sausage products are aimed at imparting the appropriate properties to them while destroying microorganisms.

The quantitative and qualitative COMPOSITION OF THE sausage microflora depends on the type and grade of the sausage. In cooked sausages subjected to high temperatures (68–70 °C at the core of the sausage), non-spore-forming bacteria are destroyed, but spores, partial coccoid forms, and isolated rods survive as they are protected by a layer of fat. The shelf-life stability of sausage products depends on their moisture content, sodium chloride concentration, the degree of impregnation with smoke antiseptics, and, above all, the level of microbial contamination. Provided that sanitary and hygienic requirements are met during production and high-quality raw materials are used, the bacterial count in freshly produced finished products is: for cooked sausages, 10³ per 1 g; semi-smoked, 10²; Liver sausages, 10⁴–10⁵. Sausage spoilage is caused by lactic acid bacteria (sourness), non-spore-forming putrefactive rods and micrococci (slime formation on casings), molds (mold growth), and other microorganisms.

The regulated microbiological parameters for sausage products include TVC, the presence of coliforms (coliform bacteria), Staphylococcus aureus, sulfite-reducing clostridia, and pathogenic microorganisms, including salmonellae (Appendix 5).

Experiment No. 6. Dry infant formulas

As shown in the table in Appendix 5, infant and dietary foods are subject to much stricter requirements than mass-consumption products. Accordingly, industrial raw materials and components used for manufacturing baby food are also subject to elevated microbiological standards.

Thus, in dry dairy products intended for infants, the following are regulated: TVC (from 2x10³ to 2.5x10⁴ CFU/g), mold count (from 5x10 to 1x10² CFU/g), yeast content (from 10 to 5x10 CFU/g), Bacillus cereus spore count (no more than 1x10²–2x10² CFU/g); coliforms must be absent in 1 g, E. coli in 10 g, Staphylococcus aureus in 1–10 g, and pathogenic microorganisms, including salmonellae, in 50–100 g.

Presentation and analysis of research results

Perform microbiological analyses of Food Products and document them in your laboratory notebook According to the following outline:

1. Preparation of the sample for microbiological analysis.

2. Method for isolation of microorganisms.

3. Method for cultivation of microorganisms.

4. Method for enumeration of microorganisms.

5. Method for identification of microorganisms.

6. Fill in Table 3.

7. Draw a Conclusion regarding the microbiological status of the studied product.

Table 3. Cultural and Morphological Characteristics of colonies grown on agar plates

Upon completing this topic, students will be able to assess the quality and microbiological safety of various types of products.

Control questions

1. What is the primary objective of microbiological control of raw materials, semi-finished products, and finished goods at food industry enterprises?

2. By whom and on The basis of which documents is microbiological analysis of food products carried out?

3. Define The concepts of "safety" and "microbiological stability" of food products.

4. List the groups of microbiological safety criteria for food products.

5. Which microbiological indicators belong to the group of sanitary condition indicators for food products?

6. What is total bacterial count (TVC / QMAFAnM)? What is the purpose of determining this indicator?

7. In which products is TVC not determined?

8. What is the purpose of determining coliforms (CC) in food products?

9. What requirements apply to sanitary-indicator microorganisms?

10. Which pathogenic microorganisms are regulated in food products?

11. For what purpose and in which products are opportunistic pathogens determined?

12. How are food product dilutions prepared?

13. For what purpose is the content of molds and yeasts determined in food products? Are these indicators regulated in all food products?

14. Define the Hazard Analysis Critical Control Point (HACCP) system.

15. Which microbiological indicators are regulated in food products such as cream confectionery, sausages, drinking milk, margarine, baby food, canned goods, etc.?

16. What is the essence of plate count methods for determining microorganisms in food products?

17. Which microbiological indicators are determined using plate count methods?

18. What is the essence of methods based on the enrichment of microorganisms followed by identification? Which microbiological indicators are determined by these methods?

19. What methods are used to determine TVC, the presence of coliforms, and the titer of anaerobic sulfite-reducing clostridia?

20. How are the cultural characteristics of microbial colonies grown on plates described?

21. How is the determination of coagulase-positive staphylococci carried out?

22. What constitutes the epiphytic microflora of fruits and vegetables?

23. What Types of microbial spoilage of potatoes and vegetables are most common?

24. Explain why molds are primarily the causative agents of fruit and vegetable spoilage.

25. What is The Role of microbes in the fermentation (salting and pickling) of fruits and vegetables?

26. How does contamination of the meat surface by microorganisms occur?

27. What is The Significance of bacterioscopic examination of meat, and how is it performed?

28. Which microorganisms are most frequently found in meat?

29. What is the microflora of fish?

30. Why is fish less resilient during storage than the meat of warm-blooded animals?

31. What is normal and abnormal milk microflora?

32. Which diseases can be transmitted through milk?

33. What characterizes the residual microflora of canned foods?

34. Which bacteria cause the spoilage of sausage products?

35. What determines the shelf life and stability of sausage products?

36. What are the primary sources of extraneous microflora in margarine?