RU2351655C1 - Method of probiotic analysis for antagonistic activity concerning lyophilised aerobiotic bacteria biomass in relation to pathogenic mycobacteria - Google Patents

Abstract

FIELD: chemistry, biochemistry.

SUBSTANCE: analysed sampled probiotic is introduced to the medium containing: L-asparagine 0.4 g, twice-substituted potassium phosphate 0.05 g, magnesium sulphate 0.05 g, citric acid 0.2 g, ammoniacal ferric citrate 0.005 g, sodium pyruvate 0.05 g, glycerine 4.0 g, agar 3.0 g, humivit 10 ml, aqueous solution of vitellus (1:1) 10.0 ml and distilled water to 100.0 ml in amount 16-32% of medium volume. Then test tubes are heated up in boiling-water bath during 5 min with slanting the medium. Indicator strain culture is seeded on slant surface and cultivated. Antagonistic activity is estimated by ratio of indicator strain colony number grown on the medium with adding casein broth in amount 20%, to indicator strain colony number grown on the medium after probiotic is added and heated up.

EFFECT: improved efficiency of direct antagonism method.

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Description

The invention relates to microbiology, relates to a method for evaluating the antagonistic activity of probiotics based on lyophilized biomass of aerobic bacteria and can be used to select probiotic preparations for medical and veterinary use.

One of the indicators of the specific activity of probiotics and production strains for their manufacture is the presence of antagonistic action against pathogenic and conditionally pathogenic microflora.

A known method of delayed antagonism, when the sowing of indicator strains in a solid nutrient medium is carried out after a certain period after sowing the test culture [1].

The antagonistic activity of probiotics and bacterial strains can be determined in vitro by regulated methods, including co-cultivation with indicator cultures in liquid or 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 added in an amount of 16-32% of the total volume of the medium, after which the tubes are heated in a boiling water bath for 5 minutes, 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), the number of colonies of the indicator strain grown on the medium in accordance with the invention after introducing a probiotic into it (experiment).

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 a probiotic in an amount of 16-32% of the total volume of the medium, the tube is heated in a boiling water bath for 5 minutes, after which the medium is mowed, and then the indicator strain is inoculated onto the surface of the pigtail, followed by lactation 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 a culture medium.

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. The yolk is mixed in a sterile flask and distilled water in a 1: 1 ratio, and 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 of dark brown to black color 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 probarin drug Okarin (ImBio, Nizhny Novgorod), which is a lyophilized biomass of live microbial cultures - representatives of the normal intestinal microflora of a healthy person, including three strains of E. coli and fecal enterococcus.

The invention is illustrated by examples.

Example 1. Determination of the antagonistic activity of ocarin (ImBio, Nizhny Novgorod) in relation to 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 biotic inoculated in the amount of 20% of the total volume of the medium (0.6 ml) was inoculated, the tube was heated in a boiling water bath for 5 minutes, after which the medium was mowed, and then on the pigtail surface was seeded with an indicator strain followed by cultivation at a temperature (37 ° C) and temporary (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 casein broth in an amount of 20% of the total medium volume (0.6 ml). A quantitative assessment was carried out by the ratio of the number of indicator strain colonies grown on the medium in accordance with the invention with the addition of casein broth (control) to the number of indicator strain colonies grown on the medium in accordance with the invention after introducing a probiotic into it followed by heating (experiment).

The results are presented in table 1.

Table 1 Assessment of antagonistic activity of ocarin in relation to pathogenic mycobacteria Probiotic drug Growth Blocking Index * M.avium GISK M.tuberculosis Academia M.bovis Vallee M.bovis Ravenal Ocarin in an amount of 20% of the total volume of the medium 9 7 8 9 Note: 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 introducing a probiotic into it, followed by heating (experiment).

Example 2. The effectiveness of the method for evaluating the antagonistic activity of ocarin (ImBio, Nizhny Novgorod) in relation to the reference strains M.tuberculosis (pc. Academia), M.avium (pc. HISC), 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 ocarin 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 4.0 2 one one one 8.0 3 2 2 2 16,0 9 7 8 9 32,0 9 8 8 9 Note: 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 introducing a probiotic into it, followed by heating (experiment).

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

Table 3 Antagonistic activity of ocarin in relation to 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 4.0 3 2 2 8.0 5 2 2 16,0 9 7 7 32,0 9 8 7 Note: 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 introducing a probiotic into it, followed by heating (experiment).

Studies have confirmed the possibility of evaluating the antagonistic activity of probiotics based on lyophilized biomass of aerobic 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 42-3947.12.07.2000

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 determining the antagonistic activity of probiotics based on lyophilized biomass of aerobic bacteria in relation to pathogenic mycobacteria, including the introduction of a probiotic test sample in an amount of 16-32% 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
heating in a boiling water bath for 5 min, mowing the medium, sowing the culture of the indicator strain on the surface of the jamb, culturing at a temperature and temporary optimum, followed by a quantitative assessment of the antagonistic activity relative to the number of colonies of the indicator strain grown on the medium with the addition of casein broth in an amount of 20 %, the number of colonies of the indicator strain grown on the medium after the introduction of a probiotic and heating.