RU2328734C1 - Method of antagonist active bacteria strain evaluation - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: pure bacteria cultures are released, with primary inoculation to rich nutrient medium like uriselect 4, it is followed with meat infusion agar reinoculation of culturs distinguished by morphological criteria and enzyme activity, studied for growth with test-strain Proteus vulgaris. Cultures not have been inhibited with creeping growth of movable strain Proteus vulgaris, are considered to be antagonistic relative to this type of microorganism.

EFFECT: method application provides mass search of extended specific spectrum of probiotic bacteria strains characterised by antagonistic activity.

3 cl, 1 tbl, 2 ex

Description

The invention relates to biotechnology, and in particular to methods for determining the antagonistic activity of probiotic strains.

A known method for determining the antagonistic activity of probiotics (RF patent No.2000118391, G01N 33/48 of 2002), including the effect of a probiotic on a bacterial test culture, characterized in that luminescent bacteria are used as a test culture, and the antagonistic activity of the probiotic is evaluated by a change in the intensity of bioluminescence of the bacteria of the test culture.

However, this method is intended to control the already developed probiotic preparations; it does not allow evaluating their antagonistic activity against representatives of opportunistic microflora.

The closest in technical essence to the claimed one, and taken as a prototype, is a method for controlling the antagonistic activity of drugs for the treatment and prevention of dysbiosis, based on the determination of delayed antagonism in a solid nutrient medium in relation to iso-, homo- and heterogeneous production strains of bacteria used in production preparations for the treatment and prevention of dysbiosis (patent RU 2177152 C2, publ. 12/20/2001).

The disadvantage of this method also lies in testing a limited number of cultures of probiotic strains and evaluating antagonistic activity against non-pathogenic microorganisms. Thus, this method is of limited use for the search for new probiotic strains of bacteria antagonistic with respect to opportunistic microflora, and is more designed to control the anatagonistic activity of existing probiotic strains.

The technical task of the proposed solution is the simultaneous determination of the ability to suppress creeping growth in relation to the representative of opportunistic microflora P.vulgaris, crops growing on meat and peptone agar, as well as expanding the range of morphological growth criteria of various isolates of primary seeded crops.

The problem is solved in that in the method for determining antagonistically active bacterial strains, including the isolation of pure bacterial cultures and studying their growth in the presence of a test strain, according to the invention, the primary sowing is performed on a rich nutrient medium such as uriselect 4, after which reseeding on meat-peptone agar is carried out cultures differing in morphological criteria and enzymatic activity, and P.vulgaris is used as a test strain.

The invention also lies in the fact that the sowing of the studied cultures and the test strain is carried out simultaneously.

The invention also lies in the fact that cultures of potential probiotic strains that were not suppressed by the creeping growth of the motile strain of Proteus vulgaris are considered antagonistic to this type of microorganism.

The method is as follows.

Material: bacterial source, potentially containing antagonistically active bacteria growing on meat and peptone agar, Petri dishes with UriSelect 4 nutrient medium, Petri dishes with meat and peptone agar, culture of the Proteus vulgaris test strain.

To isolate antagonistically active strains of bacteria cultured on meat and peptone agar, a suspension or wash is prepared from the test material, which is sown on a Petri dish with uricelect 4 medium in a way that allows obtaining single colonies.

After cultivation of crops from single colonies that differ from each other in color, shape, edges, sizes and other, visually distinguishable characteristics, crops are made on a cup with meat peptone agar, in the form of numbers or small strokes, simultaneously with the edge of the Petri dish, free from crops of the studied cultures, the culture of the test strain Proteus vulgaris is inoculated with an injection. A Petri dish with crops is cultivated at a temperature of 37 ° C until the culture of the test strain Proteus vulgaris overgrows with the entire surface of the Petri dish.

Cultures of potential probiotic strains that grew in the form of numbers or strokes and formed growth retardation zones and, accordingly, were not suppressed by the creeping growth of the motile strain of Proteus vulgaris, are considered antagonistic to this type of microorganism.

Examples are provided to illustrate the method.

Example 1. In order to verify the effectiveness of the search for cultures with antagonistic activity, a suspension is prepared from the litter of the livestock building, serial ten-fold dilutions of this suspension are plated on Petri dishes with uricelect medium 4. After incubation in an incubator, in a few limiting dilutions, single colonies of bacteria are determined, varying in color, shape, size, nature of the surface, etc. (total of 6 types). Meat peptone agar was made from each type of colony as separate numbers. At the same time, P.vulgaris culture was seeded. After cultivation in an incubator at 37 ° C for 24 hours, 2 cultures were found with distinct growth retardation zones.

Thus, in the above example, the possibility of simultaneous testing of at least 5 cultures on one sector of a 3-sector Petri dish (and, accordingly, at least 15-18 cultures on one 3-sector Petri dish, with the presence of 3 different chromogenic substrates in the composition of the nutrient medium, uricelect 4, used for the primary isolation of pure cultures, allowed us to identify 6 different cultures, while when cultivated on meat and peptone agar, the differences between the colonies were less pronounced and only 2 were visually distinguished type colonies).

Example 2. To compare the proposed method in terms of specificity with the common method of suspension of 2 cultures that have, and 2 cultures that do not have antagonistic activity, according to the results of our method, they are mixed with a suspension of bacteria of the test strain Proteus vulgaris in the same concentrations, pure crops are taken as a control.

After cultivation, serial dilutions of mixtures of cultures and pure cultures were seeded on urislect 4 medium and, after incubation in an incubator at 37 ° C, colonies of Proteus vulgaris and the studied cultures were counted.

Table

Culture 1 * Culture 2 * Culture 3 ** Culture 4 ** Test culture, CFU / ml 6.7 × 10 ⁶ 4.2 × 10 ⁷ 1.3 × 10 ⁴ 2.4 × 10 ² P.vulgaris, CFU / ml 2.5 × 10 ² 1.8 × 10 ³ 3.6 × 10 ⁵ 3.9 × 10 ⁵ Note: * - a culture with antagonistic activity against P.vulgaris, ** - a culture that does not have antagonistic activity against P.vulgaris.

As follows from the table, the claimed method is not inferior to the analogue in its ability to distinguish cultures of microorganisms with antagonistic activity.

Thus, the inventive method allows an efficient search for bacterial strains with antagonistic activity against such a representative of opportunistic microflora as P. vulgaris by testing large quantities of a wide range of microorganisms cultivated on meat peptone agar. In this regard, we can consider it appropriate to use the claimed method with the aim of developing probiotics.

Claims (3)

1. The method of determining antagonistically active strains of bacteria, including the selection of pure cultures of bacteria and the study of their growth in the presence of the test strain P.vulgaris, characterized in that the primary sowing is carried out on a rich nutrient medium such as uriselect 4, after which they are transferred to meat-peptone agar cultures differing in morphological criteria and enzymatic activity, and P.vulgaris is used as a test strain. 2. The method according to claim 1. characterized in that the sowing of the studied cultures and the strain test are performed simultaneously. 3. The method according to claim 1. characterized in that cultures of potential probiotic strains that were not suppressed by creeping growth of the motile strain of Proteus vulgaris are considered antagonistic to this type of microorganism.