Method for determining adhesive properties of bacteria of enterococcus family by means of caco-2 cell line

FIELD: biotechnologies.

SUBSTANCE: method involves preparation of bacterial suspension, separation of the obtained biomass of bacteria by centrifugation, dilution of the obtained biomass in physiological solution, preparation of monolayer of CaCo-2 cells, introduction of bacterial culture, cultivation of cells, flushing with physical solution, removal of monolayer with bacterial cells and counting of the amount of bacterial cells related to 1000 cells of CaCo-2; with that, bacteria refer to highly-adhesive ones if the number of bonded cells is 1010 to 3000, to average-adhesive ones of 210 to 1000, and to low-adhesive ones of 0 to 200.

EFFECT: improvement of the method.

 

The invention relates to the field of Microbiology and biotechnology and can be used in food biotechnology. The method is used to determine the adhesive properties of bacteria using the cell line of SASO-2 (epileptologie cells adenocarcinoma colon man in the monolayer.

A known method of determining the adhesion of microbes to the epithelial cells of the intestine of rats or mice, are described in the THROES 4.2.2602-10. 4.2. Control methods. Biological and microbiological factors. System pre-clinical studies the safety of drugs. The selection, validation and storage of industrial strains used in the production of probiotics. Methodical instructions. For staging techniques adhesion use daily culture strains. The daily culture of the strain grown on stubble adequate nutrient medium wash phosphate buffered saline (SFR) and then washed twice SFR with a pH of 7.2 by centrifugation (1500 rpm, 10 min). In addition, culture can be grown on solid medium, covered with cellophane, at 37°C for 16-18 hours Preparing a suspension containing 2×109CFU/ml Adhesiveness of the strains studied in the in vitro system in the epithelial cells of the intestine and colon of rats Fischer. Rats or mice hammer carbon dioxide from the intestines secrete pieces size ×5 mm, carefully washed from mucus and content in SFR. Epithelial cells derived from pieces of the intestinal mucosa on the vibrator model runway.

Cells are washed twice with cooled SFR (pH of 7.2) by centrifugation at 800 rpm for 10 min. After determining the concentration of suspended cells with the camera Goryaeva under a light microscope (bring it up to 2×106cells/ml of Preparing a suspension of the tested culture concentration of 2×109CFU/ml To 0.5 ml suspension of epithelial cells are 0.5 ml of bacterial suspension culture. The cell is a bacterial mixture incubated in a thermostat at (37±1)°C for 30 min, shaking occasionally. The mixture is then washed three times SFR from unattached bacteria at 600 rpm for 10 minutes All manipulations carried out in the cold. To the precipitate add 1-2 drops SFR and prepare smears on the glass. Drugs record 96° alcohol and stained by Romanovsky-Giemsa. Under light microscope, count the average number of bacteria adhering to 25 enterocytes or colonocytes. When assessing the adhesion of each strain of microorganism experience repeated at least 3 times. Calculate the average adhesion (SPA), the number of microbes on 1 cell - microbe/cell 10 fields of view, taking into account the results of all experiments.

The level of adhesion of certain bacteria strains conditionally separated into four degrees:

- non-adhesive (When A = 0);

- slaboagressivnoy (SPA = 1-5);

- sredneagressivnyh (SPA = 5-10);

- high adhesive (SPA above 10).

The described method is a placeholder.

The disadvantage of the above method is that this method uses animal cells (cells of rats and mice). Testing on animals used for food borne pathogens has long been known. But currently, the development of cell science is allowed to conduct research directly on the epithelial cells of the person. It plays an important role, especially in the field of food biotechnology.

The technical result is to increase the accuracy of detection of the adhesive activity of probiotic bacteria of the genus Enterococcus was developed by way of definition on the epithelial cells of the colon of a person line SASO-2 monolayer.

The task of the invention is directed to the development of a method for determining the adhesion properties of the probiotic bacteria of the genus

Enterococus. The method uses a cell line SASO-2 monolayer, which is obtained from the colon of a person.

The problem is solved developed a method for determining the adhesive properties of bacteria of the genus Enterococus using cell lines SASO-2, including the preparation of bacterial suspensions: the cultivation of bacterial strains on the corresponding liquid medium for 18 h; branch is derived biomass from the culture medium by centrifugation, for this purpose, 20 ml of culture suspension of each strain centrifuged at minus 4°C with a frequency of 4000 rpm for 15 min; the breeding of obtained biomass in saline to a concentration of 4×107CFU/cm3; preparation of monolayer cells SASO-2: growing monolayer cells SASO-2 in culture medium DMEM with 15% serum, fetal bovine, 5% of the antibiotic penicillin-streptomycin, 6-hole tablet for 5 days; removing the formed monolayer nutrient laboratory aspirator; rinsing the monolayer cells twice with saline solution; determination of adhesive properties of bacteria in each well of the tablet to form a monolayer of cells SASO-2 contribute 0.5 ml of bacterial culture and 2 ml of DMEM medium with 15% serum fetal bovine; cultured for 1 h at 37 ° °C; remove nutrient medium laboratory aspirator and washed the monolayer cells twice with saline solution, removing the monolayer with related bacterial cells carry out the solution of Versene 0.02% and trypsin 0,25% in the ratio of 3:1 in each well of a 6-hole tablet add 1 ml of solution for counting related bacterial cells use dilution method for growing cells on a corresponding dense environment, the adhesive properties of microorganisms is determined by the number of the bacteria, contacting 1000 cells SASO-2: from 1010 to 3000 bacterial cells - high adhesive strain; from 210 to 1000 - sredneetazhnye; from 0 to 200 - riskadvisory.

The method of determining the adhesion properties of the probiotic strains of bacteria of the genus Enterococus using lines of epithelial human cells based on the interaction of bacteria with a cell wall epithelial cells of colon carcinoma human colon, SASO-2 monolayer, and the counting of bound peroxidase bacteria monolayer.

The advantages of the proposed method are that the method uses directly the human cell SASO-2 to determine the adhesive properties of bacteria, which is one of the important properties of probiotic strains used in the food industry.

The method allows to study analyzed the properties directly on the epithelial cells of the human, which provides a basis for extrapolating the results to humans in General. With this method cells are used in the form of a monolayer, it allows to determine the amount of adhesion of bacteria with greater accuracy.

To calculate related bacterial cells were used dilution method for growing cells in a dense environment MRS. Adhesive properties of microorganisms is determined by the number of bacteria that communicates with 1000 cells SASO-2: from 1010 to 3000 smear the territorial cells high adhesive strain; from 210 to 1000 - sredneetazhnye; from 0 to 200 - riskadvisory. The gap between the results given by the error in the count.

A specific example of the proposed method

Determination of adhesive properties of strains of Enterococcus thailandicus PBC-2 (VKPM B-10684) and Enterococcus faecalis 55 (VKPM B-8652).

Carry out the preparation of bacterial suspensions: cultivated strains of Enterococcus thailandicus PBC-2 and Enterococcus faecalis 55 on the respective liquid medium for 18 h; separate the produced biomass from the culture medium by centrifugation, for this purpose, 20 ml of culture suspension of each strain centrifuged at minus 4°C with a frequency of 4000 rpm for 15 min, diluted obtained biomass in saline to a concentration of 4×107CFU/cm3. Growing monolayer cells SASO-2 in culture medium DMEM with 15% serum, fetal bovine, 5% of the antibiotic penicillin-streptomycin, 6-hole tablet for 5 days; then remove from the formed monolayer culture medium laboratory aspirator; washed the monolayer cells twice with saline. To determine the adhesive properties of bacteria in each well of the tablet to form a monolayer of cells SASO-2 contribute 0.5 ml of bacterial suspension and 2 ml of DMEM medium with 15% serum fetal bovine; cultivate the for 1 h at 37°C; remove culture medium laboratory aspirator and washed the monolayer cells twice with saline. Removing the monolayer with related bacterial cells carry out the solution of Versene 0.02% and trypsin 0,25% in the ratio of 3:1 in each well of a 6-hole tablet add 1 ml of solution.

To calculate related bacterial cells use dilution method for growing cells on a corresponding dense environment, the adhesive properties of microorganisms was determined by the number of bacteria by contacting 1000 cells SASO-2: from 1010 to 3000 bacterial cells - high adhesive strain; from 210 to 1000 - sredneetazhnye; from 0 to 200 - riskadvisory.

The results showed that with 1000 cells SASO-2 contacts 2×103the cells of E. thailandicus PBC-2, 109×103the cells of E. faecalis 55. This explores what it Is. thailandicus PBC-2 and E. faecalis 55 are characterized by high adhesive properties.

Grown on tablets monolayer cells SASO-2 can be considered as a model of the intestinal epithelium, therefore the use of this method improves the accuracy of detection of the adhesive activity of probiotic microorganisms used in the food industry.

The method for determining the adhesive properties of bacteria of the genus Enterococcus using cell lines SASO-2, including the preparation of bacterial suspension, the Department received b is MASSY at 4000 rpm for 15 min, breeding the obtained biomass in saline to a concentration of 4×107CFU/cm3growing monolayer cells SASO-2, removing the formed monolayer nutrient laboratory aspirator, washing of the monolayer cells twice with saline solution and determining the adhesive properties of bacteria in each well of the tablet to form a monolayer of cells SASO-2 contribute 0.5 ml of bacterial culture and 2 ml of DMEM medium with 15% serum, fetal bovine, cultured for 1 h at 37°C, remove the culture medium laboratory aspirator and washed the monolayer cells twice with saline solution, removing the monolayer with related bacterial cells carry out the solution of Versene 0.02% and trypsin 0,25% in the ratio 3:1, to count related bacterial cells use dilution method for growing cells on a corresponding dense MRS adhesive properties of bacteria was determined by the number of bacteria by contacting 1000 cells SASO-2: 0-200 - riskadvisory strain, from 210 to 1000 - sredneetazhnye, from 1010 to 3000 bacterial cells - high adhesive.



 

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