RU2460801C1 - Method for detecting sanitary indicator bacteria and starter culture in meat products - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves preparing an formulated enrichment medium, pH control, sterilisation, detection of sanitary indicator bacteria and starter cultures in meat products and their identification. The formulated medium is prepared by dissolving 10.00 g of peptone, 5.00 g of meat extract, 5.00 g of yeast extract, 1.00 g of Tween-80, 3.00 g of sodium phosphate, 0.10 g of magnesium sulphate, 0.01 g of ferrous sulphate in 950 ml of distilled water; the medium is sterilised at 1.1 atm; thereafter 20 g of glucose are dissolved in 50 ml of distilled water; the prepared 40% glucose solution is sterilised at 0.5 atm for 20 min, then added to the medium in the aseptic environment and mixed. While the sanitary indicator bacteria and starter cultures are recovered by sampling 25 g of a meat product and placed in a bottle containing 250 ml of the medium, then incubated at 37°C during 16 h; afterwards the bacteria are separated from the medium by centrifugation at 4000 rpm, washed from the residual nutrient medium in physiologic saline and centrifuged once more at 4000 rpm; the produced bacterial cells are applicable to recover genome Dna and identify by electrophoretic PCR analysis with using genus and species specific primers for each microorganism.

EFFECT: invention provides the complex recovery of both sanitary indicator bacteria, and starter cultures.

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Description

The invention relates to biotechnology and can be used in microbiology. The invention provides a comprehensive identification of sanitary-indicative bacteria (Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Salmonella typhimurium, Listeria monocytogenes), regulated by SanPiN 2.3.2.1078-01 and starter cultures of the genera Lactobacillus, Staphylococcus, Pediococcus in meat products. The nutrient medium contains peptone, meat extract, yeast extract, glucose, tween-80, sodium phosphate, magnesium sulfate, iron sulfate, distilled water. Identification is carried out by the method of PCR analysis with an electrophoretic detection scheme [2, 3, 7].

Ensuring food safety is an important part of the sanitary and epidemiological well-being of the population. Poor-quality food products, including meat products can lead to diseases of various etiologies. To prevent the risk of diseases caused by bacteria of the species Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Salmonella typhimurium, Listeria monocytogenes, which may be present in meat products, it is necessary to carry out their timely identification, including identification using an enrichment medium and identification based on molecular genetic research methods.

In the manufacture of fermented (uncooked and uncooked) meat products, to ensure safety, it is planned to add effective inhibitors to the sanitary-indicative microflora in the formulation - starter cultures belonging to the genera Lactobacillus, Staphylococcus and Pediococcus. To control the process of development of starter cultures, it is necessary to monitor them throughout the process. Therefore, just as for sanitary-indicative bacteria, a simple and affordable way to detect bacteria of the genera Lactobacillus, Staphylococcus and Pediococcus in meat products for their subsequent identification by molecular genetic methods is necessary.

It should be noted that the starter cultures of the genus Staphylococcus were isolated from meat samples and adapted to cultivation in a rich nutrient medium.

To solve these problems, it is necessary to create an effective way to identify sanitary-indicative microflora and starter crops in meat products.

There is a method of identifying sanitary indicative microflora in the nutrient medium Luria-Bertani [1, 4], which is characterized by ease of manufacture and is widely used at the enrichment stage in practical laboratories conducting molecular genetic studies. Prepare Luria-Bertani's medium according to the following recipe, g / l:

Peptone 10.00 Yeast extract 5.00 Sodium chloride 5.00 Final pH (at 25 ° C) 7.0 ± 0.2

The ingredients are dissolved in distilled water and sterilized at 1.1 atm (121 ° C) for 15 minutes. Bacteria are incubated at 37 ° C in an incubator for 18-24 hours.

The disadvantage of this method is the depleted composition of the environment of Luria-Bertani. Bacteria need carbon, nitrogen, sulfur, and phosphorus plus a small amount of alkali and alkaline earth metals, as well as trace trace elements, to synthesize cell components.

Peptone is a source of nitrogen and sulfur, yeast extract - vitamins of group B, sodium chloride - sodium ions for membrane transport and maintaining optimal osmotic pressure. However, these components are sufficient only to meet the minimum needs of sanitary bacteria.

The closest analogue of the proposed method for identifying sanitary indicative microflora and starter cultures is a method for detecting starter cultures on MRS medium [5, 6]. Prepare MRS medium according to the following recipe, g / l:

Proteozopeptone 10.00 Meat extract 10.00 Yeast extract 5.00 Glucose 20.00 Twin 80 1.00 Ammonium Citrate 2.00 Sodium Acetate 5.00 Magnesium sulfate 0.10 Manganese Sulfate 0.05 Sodium hydrogen phosphate 2.00 Final pH (at 25 ° C) 6.5 ± 0.2

The ingredients are dissolved in distilled water and sterilized at 1.1 atm (121 ° C) for 15 minutes. Bacteria are incubated at 37 ° C in an incubator for 18-24 hours.

The MRS medium contains, along with peptone and yeast extract, a number of other ingredients necessary for the growth of starter cultures. The composition of the meat extract includes such components as nitrogen, organic substances, insoluble and coagulating protein, albumose, mineral and other substances. Glucose is a source of carbon, tween-80 - fatty acids. Sodium hydrogen phosphate is a source of phosphorus, which occurs naturally in the form of phosphates. Magnesium sulfate is a source of alkaline earth metals, and manganese sulfate is a trace element.

The disadvantages of this method include the fact that to create selectivity for starter cultures in the environment, sodium acetate and ammonium citrate are used, which does not allow it to be used to identify sanitary-indicative bacteria. In addition, the use of these sterilization modes can lead to caramelization of glucose, a decrease in its content in the nutrient medium and a shortage as a carbon source for the growth and development of bacteria.

A common disadvantage of the above methods is that they do not allow the simultaneous identification of sanitary-indicative microflora and starter crops in meat products.

The objective of the invention is directed to the development of a method for the simultaneous detection of sanitary-indicative bacteria and starter cultures in meat products for their subsequent identification by the NDP method with an electrophoretic detection scheme.

The problem is solved in the proposed method for the detection of sanitary indicative bacteria and starter cultures in meat products, including preparation of enrichment medium according to the recipe, pH control, sterilization, detection of sanitary indicative bacteria and starter cultures in meat products and their identification, in which according to the invention for preparation of a prescription medium in 950 ml of distilled water, 10.00 g of peptone, 5.00 g of meat extract, 5.00 g of yeast extract, 1.00 g of tween-80, 3.00 g of sodium phosphate, 0.10 g of magnesium are dissolved I sulfate, 0.01 g of iron sulfate, the medium is sterilized at 1.1 atm for 15 minutes, then 20 g of glucose is dissolved in 50 ml of distilled water, the resulting 40% glucose solution is sterilized at 0.5 atm for 20 minutes Then, under aseptic conditions, they are added to the medium and mixed, and to identify sanitary indicative bacteria and starter cultures, 25 g of a sample of meat product is taken under sterile conditions and placed in a flask containing 250 ml of medium, then incubated at 37 ° C for 16 h, after which the bacteria are separated from the centrifugation medium at 4000 rpm, wash off the residual nutrient medium with saline and centrifuge again at 4000 rpm, genomic DNA is isolated from the obtained bacterial cells and identified by PCR analysis with an electrophoretic detection scheme using genus and species-specific primers for each the desired microorganism [2, 3, 7].

The technical result in the implementation of the invention is the integrated identification of sanitary indicative bacteria and starter cultures in meat products.

The sensitivity of the medium was determined by the growth of test strains: Staphylococcus aureus subsp. aureus 209P (VKPM B-6646), Escherichia coli K 12 C 600 (VKPM B-1010), Proteus vulgaris ATCC 13315 (VKPM B-1667), Salmonella typhimurium LT2 (VKPM B-3533), Listeria monocytogenes 766 (GISK named L A.A. Tarasevich), Lactobacillus curvatus 1 (VKPM B-8889), Lactobacillus plantarum 21 (VKPM B-8654), Pediococcus acidilactici 34 (VKPM B-8887), Pediococcus pentosaceus 28 (VKPM B-888ococn stapaph VKPM B-8953), Staphylococcus xylosus 45 (VKPM B-8945). Comparative characteristics of the sensitivity of the claimed environment and environments of the prototypes are presented in table 1.

The above data indicate that the claimed environment is more sensitive and effective, because on it, growth of all test strains was observed, and on MRS medium, only starter cultures. In the Luria-Bertani environment, there was no growth in starter cultures, but there was an active growth in sanitary-indicative bacteria.

The possibility of practical use of the proposed method for identifying sanitary indicative bacteria and starter cultures in raw meat and finished fermented products was studied (Table 2).

The objects of study were chilled pork (the longest muscle of the back) and 2 samples of fermented meat product. In the first case, fermentation was carried out by the bacteria Lactobacillus plantarum 21 (VKPM B-8654), Staphylococcus carnosus 108 (VKPM B-8953) (sample No. 1), in the second - Lactobacillus curvatus 1 (VKPM B-8889), Staphylococcus carnosus 108 (VKPM Bn-8889) -8953), Pediococcus pentosaceus 28 (VKPM B-8888) (sample No. 2).

Using the experimental and Luria-Bertani media, Escherichia coli and Staphylococcus aureus were detected in chilled pork. When using the MRS medium as an enrichment medium, these bacteria were not detected, because sodium acetate and ammonium citrate inhibited their growth. Other sanitary indicative and starter cultures were not found in the chilled pork under study.

In the studied fermented meat products, Lactobacillus spp starter cultures were found. and Staphylococcus carnosus for sample No. 1 and Lactobacillus spp., Staphylococcus carnosus and Pediococcus spp. for sample No. 2 using the proposed environment and the environment MRS. The low-component composition of the Luria-Bertani medium did not allow these bacteria to be detected. The presence of sanitary indicative bacteria in the finished fermented products was not detected. Identification of bacteria detected both on the claimed medium and on prototype media was carried out using PCR analysis with an electrophoretic detection scheme using rhodo- and species-specific primers for each desired microorganism type [2, 3, 7].

The proposed method is as follows.

The first step is to prepare the medium.

10.00 g of peptone are dissolved in 950 ml of distilled water; 5.00 g of meat extract; 5.00 g of yeast extract; 1.00 g of Tween-80; 3.00 g of sodium phosphate; 0.10 g of magnesium sulfate; 0.01 g of iron sulfate; control the pH with a pH meter (pH 7.0 ± 0.2), sterilize in an autoclave at 1.1 atm (121 ° C) for 15 minutes. In order to exclude caramelization of glucose during sterilization, a 40% glucose solution is separately prepared: 20 g of glucose is dissolved in 50 ml of distilled water, sterilized at 0.5 atm (112 ° C) for 20 minutes. Then, under aseptic conditions, 50 ml of glucose solution is added to the medium and mixed. The medium can be stored in the refrigerator for up to 2 months.

Then the second stage is carried out - the identification of sanitary-indicative bacteria and starter cultures in meat products on a nutrient medium.

From raw materials and prepared meat products, including fermented, 25 g of the sample is taken and placed in a flask containing 250 ml of medium, then incubated at 37 ° C for 16 hours. Bacteria are separated from the medium by centrifugation at 4000 rpm. Then the bacteria are washed from the remains of the nutrient medium with physiological saline and again centrifuged at 4000 rpm. Genomic DNA is isolated from the obtained bacterial cells, which is identified by PCR analysis with an electrophoretic detection scheme using rhodo- and species-specific primers for each desired microorganism [2, 3, 7].

When preparing a DNA template sample (DNA isolation), the AxyPrep Bacterial Genomic DNA Miniprep Kit by Axygen Scientific, Inc. was used. USA. Primers for PCR analysis were selected using literature data, and ready-made amplification kits were used for Litech, Russia. During the PCR reaction, a set of reagents for PCR amplification of DNA “GenPak PCR Core” from Isogen, Moscow was used. The PCR reaction was carried out in a volume of 25 μl in standard plastic tubes with a volume of 0.5 ml (Sarstedt, Germany) on a Tertsik thermocycler (DNA technology, Russia). To visualize amplicons, 10 μl of the PCR product was separated by electrophoresis in 2% agarose gel, followed by staining with ethidium bromide (0.5 μg / ml). The results were recorded using a Vitran photo-recording system. The size of the fragments was determined in comparison with the “100 bp DNA marker M 15”, from 100-1000 bp

The obtained results prove that the claimed method allows to simplify the enrichment stage by eliminating the use of two media for separate cultivation of sanitary-indicative bacteria of the species Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Salmonella typhimurium, Listeria monocytogenes and starter cultures of the genera Lactobacillus, Staphylococcus Ped products, expand the range of enrichment media, as well as reduce the material costs associated with the preparation of two types of media. The use of molecular genetic methods during bacteriological analysis will significantly reduce the duration of the analysis, as well as more accurately assess the sanitary-hygienic picture of meat products.

Table 1 The properties of the enrichment medium in comparison with the prototype environments Indicator crops Growth on the claimed enrichment medium Growth in the Luria-Bertani environment Growth on MRS Staphylococcus aureus subsp. aureus 209P (VKPM B-6646) + + - Escherichia coli K 12 C 600 (VKPM B-1010) + + - Proteus vulgaris ATCC 13315 (VKPM B-1667) + + - Salmonella typhimurium LT2 (VKPM B-3533) + + - Listeria monocytogenes 766 (GISK named after L.A. Tarasevich) + + - Lactobacillus curvatus 1 (VKPM B-8889) + - + Lactobacillus plantarum 21 (VKPM B-8654) + - + Pediococcus acidilactici 34 (VKPM B-8887) + - + Pediococcus pentosaceus 28 (VKPM B-8888) + - + Staphylococcus carnosus 108 (VKPM B-8953) + - + Staphylococcus xylosus 45 (VKPM B-8945) + - +

table 2 Testing of enrichment medium on chilled pork and finished fermented meat products in comparison with prototype environments Highlighted Crops The inventive enrichment environment Wednesday Luria-Bertani MRS environment Chilled pork (the longest muscle of the back) Staphylococcus aureus + + - Escherichia coli + + - Proteus vulgaris - - - Salmonella typhimurium - - - Listeria monocytogenes - - - Staphylococcus carnosus - - - Lactobacillus spp. - - - Pediococcus spp. - - - Fermented meat product (sample No. 1) Staphylococcus aureus - - - Escherichia coli - - - Proteus vulgaris - - - Salmonella typhimurium - - - Listeria monocytogenes - - - Staphylococcus carnosus + - + Lactobacillus spp. + - + Pediococcus spp. - - - Fermented meat product (sample No. 2) Staphylococcus aureus - - - Escherichia coli - - - Proteus vulgaris - - - Salmonella typhimurium - - - Listeria monocytogenes - - - Staphylococcus carnosus + - + Lactobacillus spp. + - + Pediococcus spp. + - +

Claims (1)

A method for identifying sanitary-indicative bacteria and starter cultures in meat products, including preparing an enrichment medium according to the recipe, pH control, sterilization, identifying sanitary-indicative bacteria and starter cultures in meat products and their identification, characterized in that for preparing a medium for prescription in 9.00 ml of distilled water are dissolved 10.00 g peptone, 5.00 g meat extract, 5.00 g yeast extract, 1.00 g tween-80, 3.00 g sodium phosphate, 0.10 g magnesium sulfate, 0.01 g iron sulfate, sterilization medium comfort at 1.1 atm for 15 minutes, then 20.00 g of glucose is dissolved in 50 ml of distilled water, the resulting 40% glucose solution is sterilized at 0.5 atm for 20 minutes, then added to the medium under aseptic conditions and mix, and to isolate sanitary-indicative bacteria and starter cultures, 25 g of meat product sample is taken under sterile conditions and placed in a flask containing 250 ml of medium, then incubated at 37 ° C for 16 hours, after which the bacteria are separated from the medium by centrifugation at 4000 rpm, wash off residual nutrient food with physiological saline and centrifuged again at 4000 rpm, genomic DNA is isolated from the obtained bacterial cells and identified by PCR analysis with an electrophoretic detection scheme using genus and species-specific primers for each desired microorganism.