Milk and dairy products are valuable products nutrition of animal origin. However, it should be remembered that milk obtained from sick animals can be a source of human infection with zooanthroponotic (common to humans and animals) diseases, in addition, if sanitary regulations and technologies for obtaining, processing and storing, milk can cause food toxicosis and toxic infections.
The source of primary contamination of dairy products with microorganisms is milk - raw materials. Microbes enter the milk from the external environment through the excretory ducts, the milk cistern and the nipple canal. The nonspecific microflora of milk is made up of bacteria, yeasts, and mold fungi. The contamination of milk with microorganisms occurs already in the process of milking and its intensity depends on the level of hygiene on the farm, the quality of washing and disinfection of milking equipment. A large number of microbes are found on the surface of the skin of the animal. Microbes on the surface of the skin come from food, bedding, manure, air.
Poor milk storage conditions also contribute to the growth of microflora in it. Freshly milked, fresh milk has bactericidal properties, i.e. the ability to delay the reproduction of bacteria entering the milk and even kill them. To preserve the bactericidal properties of fresh milk, it is cooled. At a temperature of +30°C, bactericidal activity lasts for 3 hours, at +15°C - about 8 hours, at +10°C - about 24 hours. Milk is cooled immediately after milking and stored at +2 to +6°C until dispatch. During storage, the antimicrobial properties of milk disappear, and if the storage rules are not followed, conditions are created in it for the development of undesirable microflora, as a result of which the product deteriorates.
Pathogenic microorganisms may be introduced into milk during its production and transport from the environment, or may be contained in the milk of sick animals. Especially many different microbes are found in the milk of animals with mastitis (staphylococci, streptococci, etc.). Microorganisms can enter milk through the air and through contact with sick animals with tuberculosis, salmonellosis, etc. And therefore, along with protein, fat and acidity, bacteria content (or QMAFAnM) is one of the most important indicators of milk quality and safety.
Good milk has a correspondingly low bacteria content. However, it must be remembered that raw milk cannot have zero bacteria content. Milk is a living product that is obtained from animals, and bacteria are integral companions of any living organism, and, as a result, its metabolic products. milk containing a large number of bacteria, even non-pathogenic and not changing organoleptic characteristics, cannot be considered complete. An increased bacterial contamination of the product indicates the multiplication of microorganisms, among which there may be pathogens that cause spoilage of the product. High content microorganisms can also cause food poisoning with signs of diarrhea and gastroenteritis.
The requirements for raw milk in terms of bacterial contamination are established by the regulatory documents of the Russian Federation and the Technical Regulations of the Customs Union. Bacillus contamination of milk - the quantitative content of bacteria in 1 cm³ raw milk. Microbiological indicators of milk according to TMC (total microbial number) or QMAFAnM (number of mesophilic aerobic and optional anaerobic microorganisms) must comply with the requirements of the Technical Regulations of the Customs Union "On the Safety of Milk and Dairy Products" (TR CU 033/2013) dated 09.10.2013 and be no more than 5.0 × 10 5 (500,000) CFU / cm³.
The bacterial contamination of the harvested milk is determined using a reductase test. The method is based on the fact that the reductase enzyme secreted by the microflora of milk decolorizes the methylene blue dye. A relationship has been established between the amount of microflora and the rate of discoloration of milk, to which methylene blue is added. The higher the bleaching rate, the large quantity microorganisms is found in milk and, consequently, its quality is worse.
In testing laboratories according to GOST 32901-2014 “Milk and dairy products. Methods of microbiological analysis”, to determine the bacterial contamination of raw milk as an arbitration method, the standard cup method of seeding certain dilutions of the original milk on a solid nutrient medium is used, followed by cultivation for 72 hours at 30 ± 1 ° C and counting the colony forming units (CFU) of mesophilic aerobic and facultative anaerobic microorganisms (QMAFAnM).
Thus, the determination of QMAFAnM in milk indicates the sanitary and hygienic state of the product, the degree of its contamination with microflora, makes it possible to judge the state of health of the animal, the state of the udder, the effectiveness of washing and disinfection of equipment, the observance of sanitary and hygienic conditions of production and the rules of personal hygiene of workers, about the conditions of storage, transportation finished products. Therefore, this indicator is normalized for all dairy products, with the exception of products produced using technically useful microflora (microflora of starter cultures).
Somatic cells are permanent components of milk and are represented by: epithelial cells of the mucous membrane of the mammary glands, alveoli and small milk passages, which are large rounded cells (from 12 to 100 microns in size and more), usually in the form of groups or layers, less often in the form of single cells; degenerated epithelial cells of an indefinite form of a destroyed structure; blood cells: leukocytes (mainly lymphocytes, neutrophils, eosinophils, etc.) and erythrocytes. It is known that somatic cells do not multiply in milked milk (unlike bacteria).
The morphological and cytological composition and the quantitative content of somatic cells in the milk of each animal varies greatly depending on various factors: the age of the animal (there are fewer somatic cells in the milk of first-calf heifers than in cows with a large number of lactations), lactation period (in the milk of a healthy cow minimal amount somatic cells observed for 2 - 6 months. lactation, and increased - during the colostrum period, at the end of lactation and during the start-up period), breeds and individual features animal, as well as the state of animal health (especially the state of the udder), the level and modes of feeding, etc.
The content of somatic cells is an important indicator of the safety of milk and shows its suitability for processing. The presence of a large number of somatic cells in milk leads to a serious decrease in its quality indicators: biological usefulness is lost, technological properties deteriorate during processing. In addition, the acidity of milk decreases, there are losses of fat, casein, lactose. Milk becomes less heat-resistant, coagulates worse rennet, slows down the development of beneficial lactic acid bacteria. It is impossible to make high-quality products from such milk (cheese, cottage cheese, yogurt, kefir, etc.). Somatic cells affect not only the quality of milk, but also the productivity of cows.
From July 1, 2017, the content of somatic cells in raw milk should be no more than 7.5 × 10 5 in 1 cm3, while for raw milk intended for production baby food, cheeses and sterilized milk - no more than 5 × 10 5 cells in 1 cm3.
It is very important that the content of somatic cells in milk can be easily and quickly determined. To identify mastitis milk impurities in raw materials, direct and indirect methods are used, based on determining the number of somatic cells. Indirect methods for determining the number of somatic cells in milk include methods for their detection when interacting with a number of reagents. Currently, the determination of the number of somatic cells in milk is regulated by GOST 23453-2014 “Raw milk. Methods for determining somatic cells” and is carried out using diagnostic preparations such as “Mastoprim” visually and using a viscometer. The standard was developed by the State Scientific Institution "VNIIMS of the Russian Agricultural Academy".
The method is based on the effect of sulfanol (a surfactant that is part of the Mastoprim preparation) on the cell membrane of somatic cells, leading to a violation of its integrity and the release of cell contents into the external environment. In this case, the viscosity (consistency) changes, which is fixed visually or with a viscometer. For analysis, PMK-1 plates are used with subsequent visual assessment or capillary-type viscometers calibrated by the device manufacturer to determine the number of somatic cells in raw milk.
Visual assessment is extremely simple, but it does not make it possible to obtain specific numerical indicators of the number of somatic cells in milk. With a visual assessment, we can only determine the margins of safety, according to the instructions of the reagent.
In our laboratory, the content of somatic cells in milk is determined using the Somatos-V.2K viscometer. The course of the determination is as follows: 5 ml of the solution of the drug "Mastoprim" and 10 ml of the analyzed raw milk are taken with pipettes and added to the viscometer flask. Before sampling, the analyzed raw milk must be thoroughly mixed and, if necessary, cleaned of mechanical impurities. A mixture of analyzed raw milk with a solution of the drug "Mastoprim" in the viscometer flask is stirred for (30 ± 10) s in manual or automatic mode. At the end of mixing, the number of somatic cells in the analyzed raw milk is determined by the time the mixture flows out of the capillary. The duration of the outflow is determined by the viscosity of the mixture of raw milk with the Mastoprim solution, which correlates with the initial content of somatic cells in it. The range for determining the number of somatic cells using capillary viscometers is from 90 to 1500 thousand per 1 cm3 of raw milk, and the duration of the mixture flowing out of the capillary ranges from 12 to 58 s.
Viscometer readings of less than 90 thousand in 1 cm3 indicate falsification of raw milk both by chemicals and by exposure to temperature:
The addition of hydrogen peroxide, urea, soda and other substances to milk, used to falsify certain indicators of raw milk, leads to a directly proportional decrease in the values of the viscometer, depending on their concentration;
Any heating of milk to thermization or pasteurization temperatures leads to a failure of the instrument readings, and the viscometer shows values of less than 90 thousand cells per 1 cm3 of milk, regardless of their true content.
These features must be taken into account when analyzing the results obtained.
The content of somatic cells is the most important indirect indicator of udder health, since during the inflammatory process in milk, the number of blood cells, in particular leukocytes and neutrophilic granulocytes, sharply increases. Inflammatory processes are the cause of the development of subclinical mastitis. With subclinical mastitis, there are no visible symptoms of inflammation in the udder, but the content of somatic cells in milk increases. Thus, changes in chemical composition milk are often proof of the presence of the same mastitis. The most common causative agents of subclinical mastitis are streptococci and staphylococci. Subclinical mastitis can last for a long time, causing permanent harm to both the health of the udder and the farm (reduced productivity, lower milk prices), and can also turn into clinical mastitis.
There are other factors that influence the content of somatic cells in milk, for example: milking errors, defects in milking equipment, insufficient hygiene, maintenance errors, feeding errors, etc.
In conclusion, I would like to present some figures: since the beginning of this year, more than 1,500 samples of raw cow's milk from farms, of which only 7 samples had to be rejected according to the indicators "QMAFAnM" and "Content of somatic cells". This speaks of good quality milk sold by agricultural producers of our region.
The dynamic development of the economy of the food industry is impossible without increasing the competitiveness of goods and services. The determining factor for consumers is the quality of products. Manufacturers must know and study the requirements for the quality of their products, be able to quantitatively and qualitatively analyze and evaluate their performance.
In the regulatory and technical documentation, controlled quality indicators are divided into 3 groups: organoleptic, physico-chemical and microbiological.
Microbiological research methods establish the degree of contamination of the product with microorganisms and make it possible to identify upcoming changes in the quality of the product, its spoilage.
QMAFAnM (Mesophilic Aerobic and Facultative Anaerobic Microorganism Count) is the most commonly used test for microbial safety. This indicator is used everywhere to assess the quality of products, with the exception of those in the production of which special microbial cultures are used (for example, beer, kvass, dairy products and so on.). QMAFAnM includes various taxonomic groups of microorganisms – bacteria, yeasts, molds. Their total number indicates the sanitary and hygienic state of the product, the degree of its contamination with microflora.
Products containing a large number of bacteria, even non-pathogenic and not changing their organoleptic characteristics, cannot be considered complete. Significant content of viable bacterial cells in food products(with the exception of those in the production of which sourdough is used) indicates either an insufficiently effective heat treatment raw materials, or about poor washing of equipment, or about unsatisfactory storage conditions for the product. Increased bacterial contamination of the product also indicates its possible deterioration.
For the consumer, the QMAFAnM indicator characterizes the quality, freshness and safety of food products. At the same time, assessing the quality of a product only by this indicator has a number of disadvantages. Firstly, this is only a general, quantitative assessment of microorganisms, since the study does not take into account pathogenic, conditionally pathogenic, psychrophilic and thermophilic microorganisms. Secondly, the method is unacceptable for products containing technological and specific microflora.
The QMAFAnM indicator allows assessing the level of sanitary and hygienic conditions in the social sphere at work, it allows you to identify violations of the storage and transportation of the product.
The number of mesophilic aerobic and facultative anaerobic microorganisms ( KMAFAnM) or total bacterial contamination is one of the main indicators sanitary quality raw milk. It determines the ways of further processing of milk and affects its cost.
Sanitary-indicative microflora, by the amount of which one can indirectly judge the safety of products and the sanitary condition of the enterprise. A large number of QMAFAnM most often indicates violations of sanitary rules and the technological regime of production, as well as the timing and temperature regimes of storage, transportation and sale of food products
The number of mesophilic aerobic and facultative anaerobic microorganisms (QMAFAnM) is one of the main indicators of the sanitary state of meat. High bacterial load is a common cause food poisoning occurring in humans.
E. coli is an opportunistic bacterium (more than 100 species) that lives in the intestines of humans, animals and birds. They are highly resistant to adverse conditions and remain in water, soil, and on objects for a long time. They develop most intensively at a temperature of 37 ° C, but they can also multiply at room temperature. They die at +60 ° C in 15 minutes. Most types of E. coli are safe. However, some types of E. coli produce dangerous toxins during their life (mainly endotoxins), which can lead to poisoning. The most susceptible to this disease are young children, the elderly and debilitated people. This disease occurs in the form of varying severity of enteritis, enterocolitis in combination with a syndrome of general intoxication.
BGKP Bacteria of the Escherichia coli group (Escherichia coli, Enterococcus, Proteus, Clostridium perfringens, thermophilic, Salmonella).
This group includes more than 100 species of microorganisms that live in the intestines of humans, animals and birds. They are highly resistant to adverse conditions and can be stored for a long time in water, soil, and on objects.
Food poisoning can be caused by a product with a very high contamination (content) of these bacteria or a product in which there are individual representatives of this group that are unsafe for humans. Basically, the presence of BGKP indicates the general sanitary condition of production, including the cleanliness of equipment.
On the other hand, the detection of CGB in the product may indicate incorrect storage conditions.
Thus, it can be said that 3 (three) market players are the culprit for the presence and / or growth of this microorganism - the manufacturer, the carrier and the seller. Who is more to blame and who is less is not important from the point of view of the consumer.
From the point of view of the Law "On Protection of Consumer Rights", the extreme party closest to the consumer will be the point of sale, i.e. salesman.
The detection of bacteria of the genus Escherichia in food, water, soil, and equipment indicates fresh fecal contamination, which is of great sanitary and epidemiological significance.
Bacteria of the Escherichia coli group are neutralized by conventional pasteurization methods (65 - 75 ° C). At 60 ° C, E. coli dies after 15 minutes.
yeast A group of unicellular fungi.
In the course of life, yeast metabolize food components, forming their own specific end products of metabolism. At the same time, the physical, chemical and, as a result, organoleptic properties of the products change - the product deteriorates. Yeast growths on foods are often visible to the naked eye as a surface coating (for example, on cheese or meat products) or manifest themselves by starting the fermentation process (in juices, syrups, and even in fairly liquid jams).
Yeasts of the genus Zygosaccharomyces have long been one of the most important agents of product spoilage. Food Industry. The fact that they can grow in the presence of high concentrations of sucrose, ethanol, acetic acid, benzoic acid and sulfur dioxide, which are the most important preservatives.
Some types of yeast are facultative and opportunistic pathogens, causing disease in people with weakened immune systems.
Yeasts of the genus Candida are components of the normal human microflora, however, with a general weakening of the body by injuries, burns, surgery, prolonged use of antibiotics, in early childhood and old age, etc., Candida fungi can develop massively, causing a disease - candidiasis.
Cryptococcus neoformans causes cryptococcosis.
The genus Malassezia in violation of the immune system cause pitiriasis (variegated lichen), folliculitis and seborrheic dermatitis.
mold
mold fungi are the cause of such pathological conditions of the body as allergies, bronchial asthma, dermatitis.
Common fungal mold can cause serious illness and even death in immunocompromised people. In such patients, mold (more specifically, fungal spores) can cause pulmonary aspergillosis.
The most dangerous mold is the fungus Aspergillus, a constant companion not only of humans, but also of birds, animals, and plants. It can be found everywhere: in soil, ventilation systems, food
The invention relates to microbiology, namely to the determination of food contamination. The method includes preparation of meat-peptone agar, pouring it into Petri dishes, sampling from food products, preparing a suspension from a sample of food products, preparing decimal dilutions of the test suspension and placing decimal dilutions of the test suspension in Petri dishes, cultivating and counting the number of colonies according to the formula: x=a n ×10, n is the degree of dilution. Moreover, to prepare decimal dilutions of the test suspension, a 0.6-0.8% solution of meat-peptone agar is used, while decimal dilutions of the test suspension are placed on membrane filters located on the surface of the meat-peptone agar in a Petri dish. The method is original in solution, easy to implement, informative, gives statistically significant results; allows you to significantly reduce the consumption of nutrient media, sterile bacteriological dishes and the time of analysis; allows to give a real quantitative assessment of the content of microorganisms that give confluent growth and form very small colonies, and also allows you to study intrapopulation processes using light microscopy. 1 ill., 1 tab.
The invention relates to the field of veterinary and sanitary examination, sanitation and microbiology, namely to the determination of food contamination and the sanitary and hygienic state of environmental objects.
The closest is the method for determining the number of microorganisms in sausages and meat products in water. A known method for determining the number of mesophilic aerobic and facultative anaerobic microorganisms in 1 g of the product is as follows: preparing a dilution solution and meat-peptone agar for seeding; analysis; accounting results. 1. Disadvantage existing method is that the sodium chloride solution (0.85%) used for sample dilution is non-buffered and isotonic only with respect to mammalian cells, and a large amount of nutrient medium, bacteriological dishes and labor costs are used for analysis. In addition, this method does not allow a real quantitative assessment of the content of microorganisms that give confluent growth and form very small (dew) colonies (Methods of General Bacteriology. Edited by F. Gerhard et al. M .: Mir, 1983, p. 442-512).
The objective of the invention is to reduce the amount of nutrient medium used, bacteriological dishes and the cost of working time by using a physiological solution of semi-liquid MPA instead of a 0.85% sodium chloride solution, followed by inoculation of a drop of diluted test suspension on the surface of the membrane filter.
Application this method is based on the fact that a physiological solution of semi-liquid meat-peptone agar (0.6-0.8%) is used as a physiological solution for dilution, consisting of 1 dm 3 of distilled water, 10 g of peptone, 5 g of sodium chloride, 0.3 g of anhydrous KH 2 PO 4 , 0.6 g of anhydrous NaH 2 PO 4 and 0.6-0.8 g of agar-agar; The pH of the medium is 7.0-7.2, drops of which are applied to the surface of membrane filters.
Use as a solution for dilution (0.6-0.8% meat-peptone semi-liquid agar) followed by inoculation of a drop of the diluted test suspension on a membrane filter is original in solution, easy to implement, informative, gives statistically significant results; allows you to significantly reduce the consumption of nutrient media, sterile bacteriological dishes and the time of analysis; allows to give a real quantitative assessment of the content of microorganisms that give confluent growth and form very small (dew) colonies, and also allows you to study intrapopulation processes using light microscopy.
For analysis, food samples are taken in accordance with applicable regulatory documents(GOST 18963-73. Drinking water. Methods of sanitary and bacteriological analysis. M., 1986; GOST 9958-81. Sausage products and meat products. M., 1982; GOST 7702.2.1-95. Poultry meat, offal and semi-finished products birds. M., 1994).
To prepare a suspension, a sample of food products is placed in a sterile flask (glass) of a homogenizer and a 0.85% sodium chloride solution is added in a fourfold amount. Homogenization is carried out in an electric mixer. First, the material is crushed into pieces at a slow speed of rotation of the knives, then at 15000-20000 rpm for 2.5 minutes. It is allowed, in the absence of a homogenizer, to prepare the test suspension in a sterile porcelain mortar by grinding 20 g of the product with 2-3 g of sterile sand, gradually adding 80 ml of sterile saline. For inoculation on nutrient media, a suspension is taken with a sterile graduated pipette after 15 minutes of exposure at room temperature. 1 ml of suspension contains 0.2 g of the product.
Meat-peptone agar is poured into glass or plastic Petri dishes (9 cm in diameter) and after the agar has cooled down, 5-6 membrane filters are placed on its surface with sterile tweezers. The diagram shows the main steps for determining the number of mesophilic aerobic and facultative anaerobic microorganisms by the proposed method.
0.6-0.8% physiological solution of semi-liquid MPA is poured into 9 cm 3 in sterile test tubes. Then, in 9 cm 3 physiological solution of semi-liquid MPA, decimal dilutions of the studied suspension are prepared. To do this, 1 cm 3 of the test suspension is added to the first tube with 9 cm 3 of semi-liquid agar, 1 cm 3 of the test suspension is thoroughly mixed from the first tube, transferred to the second, etc. 0.1 ml (1 drop) of the diluted culture is applied to a membrane filter located on the MPA in a cup. In one cup, you can put 5-6 drops of agar with different culture dilutions. Drops of agar with a diluted culture harden in 10-15 minutes. After that, Petri dishes are cultivated upside down in a thermostat at 37°C for 48 hours. To determine the number of viable bacterial cells, colonies are counted in drops of agar.
To determine the number of mesophilic aerobic and facultative anaerobic microorganisms, the number of grown colonies is multiplied by the degree of dilution of the culture according to the formula:
where x is the number of mesophilic aerobic and facultative anaerobic microorganisms,
a - the number of grown colonies,
n is the degree of dilution.
To quantify the content of microorganisms that give confluent growth and form very small (dew) colonies, as well as to study intrapopulation processes using light microscopy, colonies grown on membrane filters are fixed in vapors of 25% glutaraldehyde for 30-40 minutes. Then the membrane filter is placed on the surface of a glass slide and a few drops of propylene oxide are applied to it. The membrane filter becomes transparent and even very small (dew) colonies can be read under a microscope or a magnifying glass and, if necessary, microphotography can be taken.
The method is illustrated in the following specific examples of implementation (see table).
Symbols: method 1 - the closest analogue
method 2 - suggested
Example 1. Determination of the number of mesophilic aerobic and facultative anaerobic microorganisms in boiled sausage. Determining the number of mesophilic aerobic and facultative anaerobic microorganisms was carried out in two ways: method 1 (prototype) - For analysis, meat-peptone agar is poured into glass or plastic Petri dishes (diameter 9 cm). Sampling of food products was carried out in accordance with the current regulatory documents (GOST 9958-81. Sausage products and meat products. M., 1982). To prepare a suspension, a weighed portion of food products was placed in a sterile flask (glass) of a homogenizer and a 0.85% sodium chloride solution was added in a fourfold amount. Homogenization was carried out in an electric mixer. First, the material was crushed into pieces at a slow speed of rotation of the knives, then at 15000-20000 rpm for 2.5 minutes. For inoculation on nutrient media, a suspension was taken with a sterile graduated pipette after 15 min of exposure at room temperature. 1 ml of suspension contains 0.2 g of the product. Prepared 3 dilutions of the investigated suspension in physiological sodium chloride solution: physiological sodium chloride solution is poured into 9 cm 3 in sterile test tubes. Then, in 9 cm 3 physiological sodium chloride solution, decimal dilutions of the studied suspension are prepared. To do this, 1 cm 3 of the test suspension is added to the first test tube with 9 cm 3 of sodium chloride, from the first test tube, after thoroughly mixing 1 cm 3 of the test suspension, transferred to the second, etc. and then from each dilution, 0.1 ml was applied to a Petri dish (total 3 dishes). After that, Petri dishes were cultured upside down in a thermostat at 37°C for 48 hours. To determine the number of viable bacterial cells, colonies were counted in agar drops. To determine the number of mesophilic aerobic and facultative anaerobic microorganisms, the number of grown colonies was multiplied by the degree of dilution of the culture according to the formula:
where x is the number of mesophilic aerobic and facultative anaerobic microorganisms,
a - the number of grown colonies,
n - degree of dilution,
Method 2 (proposed) includes the preparation of a dilution solution (0.6-0.8% physiological solution of semi-liquid MPA 0.6-0.8% physiological solution of semi-liquid MPA) and meat-peptone agar for seeding; analysis; accounting results.
For analysis, meat-peptone agar is poured into glass or plastic Petri dishes (9 cm in diameter), after the agar has cooled down, up to 6 membrane filters are placed on its surface with sterile tweezers. Sampling of food products was carried out in accordance with the current regulatory documents (GOST 9958-81. Sausage products and meat products. M., 1982). To prepare a suspension, a weighed portion of food products was placed in a sterile flask (glass) of a homogenizer and a 0.85% sodium chloride solution was added in a fourfold amount. Homogenization was carried out in an electric mixer. First, the material was crushed into pieces at a slow speed of rotation of the knives, then at 15000-20000 rpm for 2.5 minutes. For inoculation on nutrient media, a suspension was taken with a sterile graduated pipette after 15 min of exposure at room temperature. 1 ml of suspension contains 0.2 g of the product. Prepared 3 dilutions of the investigated suspension in a physiological solution of MPA: 0.6-0.8% physiological solution of semi-liquid MPA is poured into 9 cm 3 in sterile test tubes. Then, in 9 cm 3 of physiological solution of semi-liquid MPA, decimal dilutions of the studied suspension are prepared. To do this, 1 cm 3 of the test suspension is added to the first tube with 9 cm 3 of semi-liquid agar, 1 cm 3 of the test suspension is thoroughly mixed from the first tube, transferred to the second, etc. and then from each dilution, 0.1 ml was applied to the surface of the membrane filter located on the MPA in a Petri dish. Moreover, 3 dilutions were placed in one Petri dish. After that, Petri dishes were cultured upside down in a thermostat at 37°C for 48 hours. To determine the number of viable bacterial cells, colonies were counted in agar drops. To determine the number of mesophilic aerobic and facultative anaerobic microorganisms, the number of grown colonies was multiplied by the degree of dilution of the culture according to the formula:
where x is the number of mesophilic aerobic and facultative anaerobic microorganisms,
a - the number of grown colonies,
n is the degree of dilution.
The number of mesophilic aerobic and facultative anaerobic microorganisms, determined by method 1 - (9×10 2) and by method 2 - (10×10 2), did not differ significantly.
Example 2. Determination of the number of mesophilic aerobic and facultative anaerobic microorganisms in meat. Determining the number of mesophilic aerobic and facultative anaerobic microorganisms was carried out in two ways: method 1 (prototype) - For analysis, meat-peptone agar is poured into glass or plastic Petri dishes (diameter 9 cm). Sampling of food products was carried out in accordance with the current regulatory documents (GOST 9958-81. Sausage products and meat products. M., 1982). To prepare a suspension, a weighed portion of food products was placed in a sterile flask (glass) of a homogenizer and a 0.85% sodium chloride solution was added in a fourfold amount. Homogenization was carried out in an electric mixer. First, the material was crushed into pieces at a slow speed of rotation of the knives, then at 15000-20000 rpm for 2.5 minutes. For inoculation on nutrient media, a suspension was taken with a sterile graduated pipette after 15 min of exposure at room temperature. 1 ml of suspension contains 0.2 g of the product. Prepared 6 dilutions of the investigated suspension in physiological sodium chloride solution: physiological sodium chloride solution is poured into 9 cm 3 sterile test tubes. Then, in 9 cm 3 physiological sodium chloride solution, decimal dilutions of the studied suspension are prepared. To do this, 1 cm 3 of the test suspension is added to the first test tube with 9 cm 3 of sodium chloride, from the first test tube, after thoroughly mixing 1 cm 3 of the test suspension, transferred to the second, etc. and then from each dilution, 0.1 ml was applied to a Petri dish (6 dishes in total). After that, Petri dishes were cultured upside down in a thermostat at 37°C for 48 hours. To determine the number of viable bacterial cells, colonies were counted in agar drops. To determine the number of mesophilic aerobic and facultative anaerobic microorganisms, the number of grown colonies was multiplied by the degree of dilution of the culture according to the formula:
where x is the number of mesophilic aerobic and facultative anaerobic microorganisms,
a - the number of grown colonies,
n is the degree of dilution.
Method 2 (proposed), including the preparation of a dilution solution (0.6-0.8% physiological solution of semi-liquid MPA and 0.6-0.8% physiological solution of semi-liquid MPA) and meat-peptone agar for seeding; analysis; accounting results.
For analysis, meat-peptone agar is poured into glass or plastic Petri dishes (9 cm in diameter), and after the agar has cooled down, 5-6 membrane filters are placed on its surface with sterile tweezers. Sampling of food products was carried out in accordance with the current regulatory documents (GOST 9958-81. Sausage products and meat products. M., 1982). To prepare a suspension, a weighed portion of food products was placed in a sterile flask (glass) of a homogenizer and a 0.85% sodium chloride solution was added in a fourfold amount. Homogenization was carried out in an electric mixer. First, the material was crushed into pieces at a slow speed of rotation of the knives, then at 15000-20000 rpm for 2.5 minutes. For inoculation on nutrient media, a suspension was taken with a sterile graduated pipette after 15 min of exposure at room temperature. 1 ml of suspension contains 0.2 g of the product. Prepared 6 dilutions of the investigated suspension in a physiological solution of MPA: 0.6-0.8% physiological solution of semi-liquid MPA is poured into 9 cm 3 in sterile test tubes. Then, in 9 cm 3 physiological solution of semi-liquid MPA, decimal dilutions of the studied suspension are prepared. To do this, 1 cm 3 of the test suspension is added to the first tube with 9 cm 3 of semi-liquid agar, 1 cm 3 of the test suspension is thoroughly mixed from the first tube, transferred to the second, etc. and then from each dilution, 0.1 ml was applied to the surface of the membrane filter located on the MPA in a Petri dish. Moreover, 6 dilutions were placed in two Petri dishes. After that, Petri dishes were cultured upside down in a thermostat at 37°C for 48 hours. To determine the number of viable bacterial cells, colonies were counted in agar drops. To determine the number of mesophilic aerobic and facultative anaerobic microorganisms, the number of grown colonies was multiplied by the degree of dilution of the culture according to the formula:
where x is the number of mesophilic aerobic and facultative anaerobic microorganisms,
a - the number of grown colonies,
n is the degree of dilution.
After cultivation in Petri dishes at 37°C for 48 hours, the number of mesophilic aerobic and facultative anaerobic microorganisms determined by method 1 - (8×10 5) and by method 2 - (7×10 5) did not differ significantly.
From the above examples, it can be seen that when comparing the two methods, the number of CFU determined by the proposed method did not differ significantly from that when determined by the generally accepted method. At the same time, the developed method has a number of advantages. So, to determine the number of viable cells for five types of samples were: according to the existing one - 98 minutes; according to the proposed method - 48 min. The cost of the nutrient medium amounted to the prototype - 420 ml; according to the proposed method - 135 ml. The number of Petri dishes was according to the prototype - 28 pieces; according to the proposed method - 9 pieces.
Number of mesophilic aerobic and facultative anaerobic microorganisms (QMAFAnM)
Determination of the number of mesophilic aerobic and facultative anaerobic microorganisms (KMAFAnM or total microbial number, TMC) refers to the assessment of the number of a group of sanitary indicative microorganisms. The composition of QMAFAnM includes various taxonomic groups of microorganisms - bacteria, yeasts, mold fungi. Their total number indicates the sanitary and hygienic state of the product, the degree of its contamination with microflora. Optimum temperature for the growth of QMAFAnM 35-37 o C (under aerobic conditions); the temperature limit of their growth is within 20-45 ° C. Mesophilic microorganisms live in the body of warm-blooded animals, and also survive in soil, water, and air.
The QMAFAnM indicator characterizes the total content of microorganisms in the product. Its control at all technological stages makes it possible to trace how “clean” the raw material goes to production, how the degree of its “purity” changes after heat treatment, and whether the product undergoes re-contamination after heat treatment, during packaging and storage. The QMAFAnM indicator is estimated by the number of mesophilic aerobic and facultative anaerobic microorganisms that have grown in the form of visible colonies on a dense nutrient medium after incubation at 37 ° C for 24-48 hours.
QMAFAnM is the most widely used microbial safety test. This indicator is used everywhere to assess the quality of products, with the exception of those in the production of which special microbial cultures are used (for example, beer, kvass, fermented milk products, etc.). The value of the QMAFAnM indicator depends on many factors. The most important are the mode of heat treatment of the product, temperature regime during its transportation, storage and sale, product humidity and relative air humidity, presence of oxygen, acidity of the product, etc. An increase in QMAFAnM indicates the multiplication of microorganisms, which may include pathogens and microorganisms that cause spoilage of the product (for example, molds).
Although the total number of QMAFAnM bacteria cannot directly indicate the presence or absence of pathogenic bacteria in food products, this indicator is quite widely used, for example, in the dairy industry. The indicator QMAFAnM (OMCH) characterizes the sanitary and hygienic regimes of production and storage conditions for dairy products. Products containing a large number of bacteria, even non-pathogenic and not changing their organoleptic characteristics, cannot be considered complete. A significant content of viable bacterial cells in food products (with the exception of those in the production of which sourdough is used) indicates either insufficiently effective heat treatment of raw materials, or poor equipment washing, or unsatisfactory storage conditions for the product. Increased bacterial contamination of the product also indicates its possible deterioration.
For the consumer, the QMAFAnM (OMCH) indicator characterizes the quality, freshness and safety of food products. At the same time, assessing the quality of a product only by this indicator has a number of disadvantages. Firstly, this is only a general, quantitative assessment of microorganisms, since the study does not take into account pathogenic, conditionally pathogenic, psychrophilic and thermophilic microorganisms. Secondly, the method is unacceptable for products containing technological and specific microflora.
The QMAFAnM indicator also allows assessing the level of sanitary and hygienic conditions in the social sphere at work, it allows you to identify violations of the storage and transportation of the product.
Detection methods
Classic method
The method for determining QMAFAnM by inoculation into agar nutrient media is based on the inoculation of the product or its dilution into a nutrient medium, incubation of the inoculations, and counting of all grown colonies.
There is also a method for determining the MNP (most probable number) QMAFAnM. It is based on inoculation of the product and/or dilutions of a test portion of the product into a liquid nutrient medium, incubation of the inoculations, taking into account visible signs of microorganism growth, subculture (if necessary) of the culture liquid on agar nutrient media to confirm the growth of microorganisms, counting their number using the MPN table.
Alternative (fast track) methods
For accelerated determination of QMAFAnM in the test sample, it is recommended to use 3M TM Petrifilm Aerobic Count Plate (AC). Petrifilm 3M TM Petrifilm Aerobic Count Plate (AC) contains ready-made nutrient medium, gel (freezing at room temperature) and a tetrazolium indicator, which facilitates the counting of colonies on petrifilm.
Regulations
Codex Alimentarius. Food hygiene. Basic texts. Recommended international technical norms and rules. General principles food hygiene. 2003.
General characteristics of the food product according to QMAFAnM
|
Group of microbial contamination |
CFU / g (cm 3) |
Product condition |
|
10 3 ÷ 10 4 , ≤ 10 5 |
Fresh, good quality, shelf stable |
|
|
> 10 5 ÷ 10 6 |
Manufactured or stored in violation of technological or sanitary-hygienic regimes |
|
|
> 10 6 ÷ 10 7 |
Potentially dangerous as a source of pathogenic microorganisms and their toxins |
|
|
> 10 7 ÷ 10 8 |
Damaged, which is confirmed visually (discoloration, odor, mold appearance) |
Indicative microbiological values of some products
