RU2350648C1 - Method of estimation of probiotics opposing activity on basis of lyophilised biomass of anaerobic bacteria in relation to pathogenic micobacteria - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: for joint cultivation at a temperature and time optimum for the bacteria use the medium containing: L-asparagin 0.4 g, disubstituted potassium phosphate 0.05 g, sulfurous magnesium 0.05 g, lemon acid 0.2 g, citric ammoniacal iron 0.005 g, sodium pyruvate 0.05 g, glycerinum 4.0 g, agar 3.0 g, humivite 10.0 ml, aqueous solution of egg yolk (1:1) 10.0 ml, distilled water to 100.0 ml. Investigated assay of the probiotic is applied in the medium in amount of 8-16% from volume of medium. Crops of culture of the display strain are conducted on an agar slope surface. Opposing activity is estimated under the relation of quantity of colonies of the display strain which has grown on medium with addition of Blaurock medium in amount of 10% from the volume of medium, to quantity of colonies of the display strain which has grown on medium after placing of the probiotic into it.

EFFECT: rising of efficiency of the method of direct antagonism.

3 tbl, 3 ex

Description

A method for evaluating the antagonistic activity of probiotics based on lyophilized biomass of anaerobic bacteria in relation to pathogenic mycobacteria

The invention relates to microbiology, relates to a method for evaluating the antagonistic activity of probiotics based on lyophilized biomass of anaerobic bacteria, in particular bifidobacteria, in relation to pathogenic mycobacteria, and can be used to select probiotic preparations for medical and veterinary use, effective for mycobacterioses.

A known method of controlling the antagonistic activity of industrial strains of Bifidobacterium bifidum (pieces No. 1, 791 and LVA-3) in relation to conditionally pathogenic indicator bacteria by the method of delayed antagonism, including rehydration of the lyophilized strain, its cultivation on the surface of the liquid nutrient medium MPC-5 in anaerobic conditions with subseeding to the grown culture of liquid culture of any one indicator strain / 1 /.

The antagonistic activity of probiotics and bacterial strains can be determined in vitro by regulated methods, including rehydration of the lyophilized strain and co-culture with indicator cultures in a liquid or in solid nutrient medium / 2 /. The determination of the antagonistic activity of probiotics in relation to indicator strains by the method of direct antagonism is considered by us as the preferred analogue.

A common disadvantage of the known methods is their inefficiency in determining the antagonistic activity of probiotics in relation to mycobacteria due to the peculiarities of the biology of the latter, concerning the growth rate and requirements for nutrient media, which complicates co-cultivation with other microorganisms.

The purpose of the invention is improving efficiency.

This goal is achieved by using for joint cultivation of a medium containing L-asparagine, potassium disubstituted phosphate, magnesium sulfate, citric acid, ammonium citrate, sodium pyruvate, glycerin, agar, humivit, an aqueous solution of chicken yolk (1: 1) and distilled water in the following ratio of components:

L-asparagine 0.4 g disubstituted potassium phosphate 0.05 g magnesium sulfate 0.05 g lemon acid 0.2 g ammonium citrate 0.005 g pyruvate sodium 0.05 g glycerol 4.0 g agar 3.0 g gumivit 10.0 ml aqueous solution of chicken yolk (1: 1) 10.0 ml distilled water up to 100.0 ml

moreover, the test material is introduced in an amount of 8-16% of the total volume of the medium, and antagonistic activity is estimated by the ratio of the number of colonies of the indicator strain grown on the medium in accordance with the invention with the addition of a culture medium (control) to the number of colonies of the indicator strain grown on the medium in accordance with the invention after the introduction of a probiotic (experience).

The method is as follows.

The vial with a dry probiotic is opened and rehydrated with 0.9% sodium chloride solution. In a sterile test tube with a medium containing L-asparagine, potassium disubstituted potassium, magnesium sulfate, citric acid, ammonium citric acid, sodium pyruvate, glycerin, agar, humivite, an aqueous solution of chicken yolk (1: 1) ) and distilled water, inoculate the probiotic in an amount of 8-16% of the total volume of the medium, after which the medium is mowed, the indicator strain is inoculated onto the surface of the pigtail, followed by cultivation at a temperature (37 ° C) and temporary (30 days) optimum. Control - kosyachki molten and cooled to 70 ° C medium containing L-asparagine, potassium disubstituted potassium, magnesium sulfate, citric acid, ammonium citrate, sodium pyruvate, glycerin, agar, humivite, an aqueous solution of chicken yolk (1: 1) and distilled water, with the addition of the medium of cultivation of the probiotic strain in the amount of 8-16%.

To prepare the medium, 30.0 ml of distilled water is poured into a chemically pure sterile flask with a capacity of 200 ml, heated in a water bath to 75-85 ° C. In water, 0.2 g of citric acid, 0.4 g of L-asparagine, 0.05 g of magnesium sulfate, 0.05 g of disubstituted potassium phosphate, 0.005 g of ammonium citrate, 0.05 g of sodium pyruvate, 4.0 are successively dissolved. g of glycerin, 10.0 ml of humivit and 3.0 g of agar. Each component is added after complete dissolution of the previous one. After introducing all the components, the pH is adjusted to 7.1-7.2 with 10% sodium hydroxide solution, the total volume is adjusted to 90.0 ml with sterile distilled water. The solution is autoclaved for 30 minutes at 1 atm. Fresh chicken eggs are washed well in running water, wiped with 96 ° ethanol, broken under sterile conditions, and the protein is separated from the yolk. In a sterile flask, the yolk is mixed with distilled water in a ratio of 1: 1, 10.0 ml of the resulting solution is added to the molten agar medium and mixed.

Gumivit - an alkaline hydrolyzate of lowland peat varieties (manufactured by Agroprom LLC), is a highly dispersed sol of sodium salts of a complex of humic acids - humic, humatemeanic and fulvic acids / 3 /. The solution of salts of humic acids consists of long fragmentary chains with various functional groups, the most important of which are carboxylic (COOH) and phenolic (OH), capable of forming chelate complexes with trace elements, which ensures their high exchange capacity and transport to the cell of microorganisms / 4, 5 /. Gumivit is a liquid from dark brown to black with a specific sweetish odor, due to which the finished medium has an intensely brown color, against which even small colonies are clearly visible in the initial growth phase.

The studies were carried out using the probiotic preparation bifidobacterin (ImBio, Nizhny Novgorod), which is a lyophilized biomass of live bifidobacteria Bifidobacterium bifidum.

The invention is illustrated by examples.

Example 1. Evaluation of the antagonistic activity of bifidobacterin (ImBio, Nizhny Novgorod) in relation to the reference strains M.tuberculosis (pc. Academia), M.avium (pc. HIS), M.bovis (pc. Vallee, Ravenal)

The ampoule with a dry probiotic was opened and rehydrated with 0.9% sodium chloride solution with a temperature of 15-20 ° C at the rate of 1 ml per dose of the drug. The duration of rehydration with vigorous shaking was 2-5 minutes. In a sterile test tube with 3.0 ml of molten and cooled to 70 ° C medium containing 0.4 g of L-asparagine, 0.05 g of potassium phosphate disubstituted, 0.05 g of magnesium sulfate, 0.2 g of citric acid, 0.005 g ammonium citrate, 0.05 g sodium pyruvate, 4.0 g glycerol, 3.0 g agar, 10.0 ml humivit, 10.0 ml aqueous solution of chicken yolk (1: 1) and distilled water (up to 100, 0 ml), the probiotic was inoculated in an amount of 10% of the total volume of the medium (0.3 ml), after which the medium was mowed, the indicator strain was sown on the surface of the pigtail after uyuschim culturing at temperature (37 ° C) and time (30 days) optimum. Control - kosyachok (3.0 ml) of molten and cooled to 70 ° С medium containing 0.4 g of L-asparagine, 0.05 g of potassium phosphate disubstituted, 0.05 g of magnesium sulfate, 0.2 g of citric acid, 0.005 g of ammonium citrate, 0.05 g of sodium pyruvate, 4.0 g of glycerol, 3.0 g of agar, 10.0 ml of humivit, 10.0 ml of an aqueous solution of chicken yolk (1: 1) and distilled water (up to 100 , 0 ml) with the addition of Blaurock medium in the amount of 10% of the total medium volume (0.3 ml). Antagonistic activity was evaluated by the ratio of the number of colonies of the indicator strain grown on the medium in accordance with the invention with the addition of casein broth (control) to the number of colonies of the indicator strain grown on the medium in accordance with the invention after the introduction of a probiotic followed by heating (experiment). The results are presented in table 1.

Table 1
Evaluation of the antagonistic activity of bifidobacterin in relation to pathogenic mycobacteria The number of probiotic,% of the total volume of medium Growth Blocking Index * M.avium GISK M.tuberculosis Academia M.bovis Vallee M.bovis Ravenal 10.0 10 10 6 10 Note: The growth blocking index is the ratio of the number of colonies of the indicator strain grown on the medium in accordance with the invention with the addition of Blaurock medium (control) to the number of colonies of the indicator strain grown on the medium in accordance with the invention after introducing the probiotic into it (experiment).

Example 2. The effectiveness of the method for evaluating the antagonistic activity of bifidobacterin (ImBio, Nizhny Novgorod) in relation to the reference strains M.tuberculosis (pc. Academia), M.avium (pc. HIS), M. bovis (pc. Vallee, Ravenal depending on the amount of probiotic

Scheme for evaluating antagonistic activity, as in example 1. The results are presented in table 2.

table 2
Antagonistic activity of bifidobacterin against pathogenic mycobacteria The number of probiotic,% of the total volume of the medium Growth Blocking Index * M.avium GISK M.tuberculosis Academia M.bovis Vallee M.bovis Ravenal 2.0 one one 2 one 4.0 10 5 2 10 8.0 10 10 four 10 16,0 10 10 10 10 32,0 10 10 10 10 Note: The growth blocking index is the ratio of the number of colonies of the indicator strain grown on the medium in accordance with the invention with the addition of Blaurock medium (control) to the number of colonies of the indicator strain grown on the medium in accordance with the invention after introducing the probiotic into it (experiment).

Example 3. The effectiveness of the method for evaluating the antagonistic activity of bifidobacterin (ImBio, Nizhny Novgorod) in relation to the clinical strains M.bovis (No. 2 and 3) and M.avium (No. 4) isolated from lymph nodes of cattle with confirmed diagnosed with tuberculosis, depending on the amount of probiotic. The scheme for determining antagonistic activity, as in example 1. The results are presented in table 3.

Table 3
Antagonistic activity of bifidobacterin against pathogenic mycobacteria The number of probiotic,% of the total volume of medium Growth Blocking Index * M.avium No. 4 M.bovis Number 2 Number 3 2.0 one one one 4.0 3 2 10 8.0 5 10 10 16,0 10 10 10 32,0 10 10 10 Note: The growth blocking index is the ratio of the number of colonies of the indicator strain grown on the medium in accordance with the invention with the addition of Blaurock medium (control) to the number of colonies of the indicator strain grown on the medium in accordance with the invention after introducing the probiotic into it (experiment).

Studies have confirmed the possibility of evaluating the antagonistic activity of probiotics based on lyophilized biomass of anaerobic bacteria by the method in accordance with the invention. The results indicate that the antagonistic activity of probiotics is comparable with reference strains and clinical isolates obtained from cattle with a confirmed diagnosis of tuberculosis. The application of the method assumes the presence of parameters for the value of the growth blocking index and concentration of the test material for each drug. The method is simple to implement and can be used as the main or additional test in determining the antagonistic activity of probiotics based on bacteria and their metabolites.

Information sources

1. Bifidobacterin is dry. FS 42-3947. 07/12/2000.

2. Laboratory research methods in the clinic. Handbook Ed. Prof. V.V. Menshikova. - M .: Medicine, 1987, S.341-343.

3. Grishin G.I., Slinina K.N. // Practitioner. - 2004. - No. 3-4. - S.80-82.

4. Levinsky B.V. All about humates. - Irkutsk: IP Markov S.E., 1999 .-- 198 p.

5. Sorokina N.F. Physiology of the activity of peat oxidation products // New processes and products of peat processing. - Minsk: Nauka, 1982. - S.109-115.

Claims (1)

A method for evaluating the antagonistic activity of probiotics based on lyophilized biomass of anaerobic bacteria in relation to pathogenic mycobacteria, including the introduction of a probiotic test sample in an amount of 8-16% of the volume of medium containing L-asparagine, potassium phosphoric disubstituted, sulfuric acid magnesium, citric acid , citric acid ammonia iron, sodium pyruvate, glycerin, agar, humivit, an aqueous solution of chicken yolk (1: 1) and distilled water in the following ratio of components:
L-asparagine 0.4 g potassium phosphoric disubstituted 0.05 g magnesium sulfate 0.05 g lemon acid 0.2 g citric acid ammonia iron 0.005 g pyruvate sodium 0.05 g glycerol 4.0 g agar 3.0 g gumivit 10.0 ml aqueous solution of chicken yolk (1: 1) 10.0 ml distilled water up to 100.0 ml
subsequent mowing of the medium, sowing the culture of the indicator strain on the surface of the jamb, cultivation at a temperature and temporary optimum, while the antagonistic activity is estimated by the ratio of the number of colonies of the indicator strain grown on the medium with the addition of Blaurock medium in the amount of 10% of the volume of the medium to the number of indicator colonies strain grown on the medium after the introduction of a probiotic.