RU2440413C1 - Strain of bacteria bacillus licheniformis (its versions), possessing bactericidal and fungicidal activity, and preparation based on said strain - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to strain of microorganism, ensuring recovery of soil and animal gastrointestinal tract (GIT) microbiocenosis, possessing antibacterial and fungicidal activity, and to based on them preparation form and can be used in biotechnology, veterinary medicine and plant protection. Strains Bacillus licheniformis IC-831-1-2, Bacillus licheniformis IC-832-1-2, Bacillus licheniformis IC-833-1-2, Bacillus licheniformis IC-834-1-2 are obtained by method of directed selection and are deposited in All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika under registration numbers All-Russian collection of industrial microorganisms respectively: B-10561, B-10562, B-10563 and B-10564. Preparation is characterised by containing filling agent with bacteria biomass in spore form Bacillus licheniformis All-Russian collection of industrial microorganisms B-10561, or All-Russian collection of industrial microorganisms B-10562, or All-Russian collection of industrial microorganisms B-10563, or All-Russian collection of industrial microorganisms B-10564, or their mixture with titre of each bacteria strain not less than 1·10³ CFU/g or 1·10³ CFU/ml. As filling agent it contains water or powder-like sorbent.

EFFECT: invention makes it possible to increase efficiency of restoring soil, animal GIT microbiocenosis and extend spectrum of antibacterial and fungicidal activity.

9 cl, 9 tbl, 10 ex

Description

The invention relates to a strain of a microorganism that provides restoration of the microbiocenosis of the soil and the gastrointestinal tract (GIT) of animals with antibacterial and fungicidal activity, and to a formulation based on it and can be used in biotechnology, veterinary medicine and plant protection to obtain preparations against bacterial and fungal infections of animals and plants, as well as for use as microbiological fertilizers and probiotics for the restoration of microbiocenosis of the gastrointestinal tract of animals.

Bacterial fungicides registered and included in the List of the State Chemical Commission of the Ministry of Agriculture of the Russian Federation are not intended for use on berry crops - raspberries and black currants. So, the Integral preparation (a liquid based on Bacillus subtilis strain 24D) is registered for use against diseases of grain crops (wheat, spring barley), vegetables (potatoes, tomatoes and cucumbers). From berry crops, the drug is approved for the fight against black currant anthracnosis.

The drug Fitosporin-M (powder based on strain 24D of Bacillus subtilis) is registered for use against diseases of grain crops (wheat, barley), legumes (peas), vegetables (potatoes, cabbage, tomatoes, cucumbers), (RF patent No. 2129375, IPC A01N 63/00, C12N 1/20, publ. 04/27/1999).

The drug Bactofit (a wettable powder based on the IPM 215 Bacillus subtilis strain) is intended to protect against diseases of vegetable and flower crops grown in open and protected ground, as well as blackcurrant from powdery mildew.

Among microbiological fertilizers, the analogue of the claimed drug is the bactophosphine preparation, the basis of which is the soil microorganisms Bacillus mucilaginosus, designed to enrich the soil with phosphorus forms assimilated by plants, as well as restore its fertility, increase the germination and yield of many types of crops in open and protected ground. The authors of this fertilizer do not provide specific data on the effect on the group of microorganisms that are indicative of fertile soils, as well as the impact on crop yields.

Microbiological fertilizer Baikal Em-1 contains a consortium of representatives of various groups of microorganisms, including fungi, which, developing in the soil when introduced as a symbiotic community, are designed to increase the number of soil microflora and, accordingly, fertility and productivity.

However, the above drugs have a narrow spectrum of action. A number of bacterial strains of the genus Bacillus are known, which are antagonists of various pathogens of agricultural diseases. The strain Bacillus subtilis B-116 has an antagonistic effect on the fungi Pythium and Rhysoctonia. The bacterial strain Bacillus subtilis VNIISKHM N128 is used to obtain a preparation against fungal diseases of cotton (USSR Author's Certificate No. 1717156, class A01N 63/00, publ. 1992) [1].

However, these strains have a limited effect directed at individual fungal pathogens of various agricultural crops.

A known bacterial strain Bacillus subtilis VNIISKHM N131 used to obtain the drug against pathogens of rot of apples and grapes during storage (USSR Author's Certificate No. 1706504, class A01N 63/00, 1992) [2].

However, this strain also has a limited effect aimed at individual fungal pathogens of agricultural crops.

The closest analogue (prototype) is the bacterial strain Bacillus subtilis VKPM B 7036, designed to obtain a drug against fungal diseases of plants (RF Patent No. 2086128, IPC A01N 63/00, C12N 1/20, published on 08/10/1997). The strain was selected for inhibition of bacterial growth. With the best options, experiments were carried out to suppress growth: Phytophthore infestans, Microsporium solani, Fusarium solani (causative agent of dry rot).

The closest analogue (prototype) of the formulation is the Fitop preparation, obtained on the basis of the above mentioned strain of Bacillus subtilis VKPM B 7036 in the following ratio of components, wt.% (RF Patent No. 2086128, IPC A01N 63/00, C12N 1/20, published 10.08 .1997): spores of bacteria Bacillus subtilis VKPM B 7036 with a titer of 7 · 10 ¹⁰ spores / g (0.1-1.0); starch (0.2-3.0); sugar is the rest. The residual moisture content of the drug is not more than (2-5)%. The drug Fitop is produced and used in agriculture for more than 10 years.

However, the prototype strain and the drug based on it have a not wide enough spectrum of antagonistic activity, and it was also not investigated for the ability to restore the microbiocenosis of the gastrointestinal tract of animals.

The technical result of the claimed technical solutions is to ensure the restoration of soil microbiocenosis, gastrointestinal tract of animals and the expansion of the spectrum of antibacterial and fungicidal activity.

The specified technical result is achieved by obtaining by the method of directed selection of four variants of bacterial strains of Bacillus licheniformis: IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2, providing recovery microbiocenosis of the soil and gastrointestinal tract of animals with bactericidal and fungicidal activity and deposited on March 18, 2010 at the All-Russian Collection of Industrial Microorganisms (VKPM) FSUE GosNIIGenetika respectively under No. VKPM: V-10561, V-10562, V-10563 and B -10564. (certificates of deposit are attached).

The specified technical result is also achieved by the fact that in the preparation, characterized in that it contains a filler with biomass of bacteria in the spore form of Bacillus licheniformis IC-831-1-2, or IC-832-1-2, or IC-833-1- 2, or IC-834-1-2, or a mixture thereof with a titer of each bacterial strain of at least 1 · 10 ³ CFU / g or 1 · 10 ³ CFU / ml. As a filler, it contains water or a powdered sorbent. As a powdered sorbent, it contains a zeolite, or a polysaccharide, or a monosaccharide, or a disaccharide, or a mixture of a polysaccharide with a monosaccharide or polysaccharide in a ratio of 1: 100 to 1:10. It contains starch as a polysaccharide, glucose as a monosaccharide, and sucrose as a disaccharide.

The original clones for obtaining the claimed strains were isolated from healthy animals in an ecologically clean area of the Novosibirsk region (Zavyalovsky Bor):

- Bacillus licheniformis strain: IC-831-1-2 isolated from goose feces,

- strain Bacillus licheniformis IC-832-1-2 isolated from feces of a cow,

- strain Bacillus licheniformis IC-833-1-2 isolated from goose feces,

- Bacillus licheniformis strain IC-834-1-2 was isolated from chicken feces.

The original clones were selected for inhibition of bacterial and fungal growth. With the best clones, experiments were performed to suppress growth: enterobacteria, gram-positive cocci, non-fermentable bacteria, Candida fungi, pathogens of plant fusarium, purple raspberry spotting, raspberry septoria.

Immobilization of bacterial spores on starch provides them with additional mechanical protection, prevents aggregation of spores and provides a more even distribution in the mass of the filler.

When using the drug, starch helps accelerate the germination of bacterial spores, which increases its effectiveness.

Powder based on sucrose (sugar) or glucose is not only a sorbent - filler, but also has a stabilizing and preserving effect on the drug, creates milder conditions for storing bacterial spores.

In addition, the use of the claimed components of the drug greatly simplifies the technology of its preparation both at the stages of cultivation (the technology does not use expensive enzymes, cleaning and concentration of the drug is not required), and at the stages of obtaining its finished form (removal and redistribution of moisture combined with obtaining the finished form drug).

Cultural, morphological and biochemical properties of strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2.

The strains are gram-positive facultatively anaerobic rod-shaped bacteria. When growing on meat-peptone agar, they give a wrinkled form of colonies with abundant mucus formation and pinkish-red pigment in the upper layer of the medium under the colony. The colonies are creamy pink. The cells are rod-shaped, oval endospores, located centrally or paracentrally, do not inflate the cell during spore formation. Cell length (1.09-1.96) microns, width (0.36-0.97) microns. They do not form a capsule; a large amount of mucus is secreted. Gram stains positively. The strains are thermophilic, multiply at (35-65) ° C, optimal growth at a temperature of (37-40) ° C.

After 48 hours of growth at 40 ° C, on meat-peptone agar, cultures have opaque colonies of heterogeneous consistency, with mucous inclusions and a wavy edge, grown into the surface of the agar plate. pH of the medium: minimum 5.7; maximum 8.0; optimal 6.5-7.0. The strains reproduce at 40 ° C on meat-peptone agar and meat-peptone broth.

Biochemical properties of strains. They do not grow under anaerobic conditions, do not form acetylmethylcarbinol from glucose in the Voges-Proskauer reaction, and ferment with glucose, mannose, fructose, ribose, lyxose, cellobiose, trehalose, maltose, and turanose to ferment. Hydrolyze starch, urea and esculin. Do not have lecithinase activity. Possess catalase activity. Tolerant to the concentration of sodium chloride in the nutrient medium in the range of (1-9)%.

Strains are not zoopathogenic and phytopathogenic.

Strains produce biologically active metabolites, including antibiotics and broad-spectrum enzymes that inhibit the growth of pathogenic and conditionally pathogenic bacterial and fungal microflora.

With long-term storage of the strains, the spore mass is freeze-dried. For the mass reproduction of bacteria using meat-peptone broth, cultivation is carried out at a temperature of 40 ° C.

Example 1. The study of the pathogenicity of strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2.

The study was conducted in the biotechnological control laboratory of NPF Research Center.

The pathogenicity of each microorganism was assessed by the survival rate of infected animals, their appearance and behavior, the sowing of bacteria from the blood and organs at various times after infection, as well as the macroscopic picture of the internal organs when opening the animals at the end of the observation period.

Two types of laboratory animals were used in the work: nonlinear white mice weighing (18-20) g in the amount of 40 individuals of both sexes and Wistar rats weighing (180-200) g in the amount of 40 individuals of both sexes. From animals formed an experimental and control group of 20 animals each. To detect the penetration of bacteria into the organs of experimental animals, animals from the experimental groups were orally administered a daily bacterial culture fluid for 60 days obtained by culturing the studied strain on meat and peptone broth.

Animals from the experimental and control groups were killed according to the following scheme:

1. day 5 - 4 individuals from each group (control and experimental);

2. day 15 - 4 individuals from each group (control and experimental);

3. day 30 - 4 individuals from each group (control and experimental);

4. day 45 - 4 individuals from each group (control and experimental);

5. day 60 - 4 individuals from each group (control and experimental).

After an autopsy, sowing from organs (heart, lung, liver, spleen) and blood was carried out on meat-peptone and blood agar in Petri dishes by imprinting. Crops were incubated in a thermostat at a temperature of (37 ± 1) ° C for 24 hours. The results were taken into account by the presence / absence of colonies on the surface of the nutrient medium in the Petri dish.

As a result of the studies established the following. No animal deaths were recorded during the experiment. No changes in their appearance and behavior were noted. A macroscopic evaluation of internal organs at all periods of autopsy revealed no pathological changes.

Microbiological analysis showed that the internal organs and blood were sterile in the experimental and control versions at all periods of the study.

Based on the absence of animal death and any changes in their appearance and general behavior, during a 60-day observation, negative results of microbiological analysis of the heart, lungs, liver, spleen and blood, it was concluded that strains of Bacillus licheniformis IC-831-1 -2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 are non-pathogenic.

Example 2. The study of the antagonistic activity of strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2

The strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 obtained by selection are characterized by high antagonistic activity against pathogenic and conditionally pathogenic microorganisms.

Antagonistic activity against test cultures was tested by delayed antagonism. Staphylococcus aureus, Candida albicans, Klebsiella pneumonia, Shigella flexneri, Shigella sonnei, Yersinia pseudotuberculosis, Serratia marcescens, Escherichia coli, Enterococcus spp. Were used as test strains.

Used test strains met the following requirements: they were in the S-form, had typical morphological and enzymatic properties.

The study of the antagonistic activity of the claimed strains of Bacillus licheniformis was performed on meat-peptone agar.

The formulation of meat peptone agar at 1 DM ³ :

1. meat enzymatic peptone - 10.0 g;

2. yeast extract - 5.0 g;

3. sodium chloride - 5.0 g;

4. microbiological agar - 15.0 g;

5. distilled water - up to a total volume of 1 dm ³ .

After mixing the components, the medium was heated until the agar was completely dissolved and filtered through a cotton-gauze filter into flasks with a capacity of 500 cm ³ and 300 cm ³ each. The flasks with the medium were closed with cotton-gauze plugs, wrapped the neck with parchment and sterilized in an autoclave at a pressure of 0.15 MPa for 40 minutes. After cooling to a temperature of (45 ± 1) ° C, the medium was poured into sterile Petri dishes. To control sterility, plates with dried medium were placed in a thermostat at a temperature of (37 ± 1) ° С for (24 ± 2) hours. Non-sterile cups were rejected.

The cultures of pathogenic microorganisms mentioned above were grown on Petri dishes for (18 ± 2) hours on meat-peptone agar. 1 ml of physiological saline was poured into sterile tubes and suspensions of test strains were prepared with a concentration of microbial cells (5.0 ± 1.0) × 10 ⁸ CFU / cm ³ .

For testing, 3 samples of 1 g were taken from each batch of the drug. Next, 0.5 g of the drug was diluted in 0.5 cm ³ . The resulting suspension was plated using a microbiological loop along the diameter of a Petri dish with meat-peptone agar. Crops were incubated in a thermostat at a temperature of (37 ± 1) ° С for (48 ± 2) hours. Then, a suspension of pathogens was seeded to the culture grown as a stroke using a microbiological loop by the method of perpendicular strokes.

The results were taken into account after 8 hours of incubation at (37 ± 1) ° С according to the size of the zones of growth inhibition of test strains. The control of the growth of pathogen cultures was their parallel seeding on Petri dishes with the same dense medium (meat-peptone agar), but without the studied association of the antagonist. As a result of the studies, it was found that the strain of Bacillus licheniformis IC-831-1-2 is characterized by the following indicators of growth retardation zones (mm) of the test strains of pathogens (table 1).

Table 1. Characterization of strain B. licheniformis IC-831-1-2 in terms of growth retardation zones of pathogen test strains The name of the test strain Zone Size (mm) Staphylococcus aureus strain IC-1161-4-1 10 Staphylococcus aureus strain IC-1189-4-1 12 Staphylococcus aureus IC-1126-4-1 13 Staphylococcus aureus IC-1142-4-1 fifteen Staphylococcus aureus IC-1192-4-1 12 Staphylococcus aureus IC-1200-4-1 8 Staphylococcus aureus IC-1246-4-1 10 Staphylococcus aureus IC-1120-4-1 12 Staphylococcus aureus IC-1128-4-1 12 Staphylococcus aureus IC-1257-4-1 12 Staphylococcus aureus IC-1249-4-1 13 Staphylococcus aureus IC-1233-4-1 twenty Staphylococcus aureus IC-1149-4-1 fifteen Staphylococcus aureus IC-1241-4-1 thirty Staphylococcus aureus IC-1251-4-1 23 Staphylococcus aureus IC-1226-4-1 twenty Staphylococcus aureus IC-1230-4-1 12 Staphylococcus aureus IC-1140-4-1 22 Staphylococcus aureus IC-1222-4-1 fifteen Staphylococcus aureus IC-1165-4-1 eighteen Staphylococcus aureus IC-1205-4-1 17 Staphylococcus haemoliticus IC-1256-4-2 twenty Staphylococcus aureus IC-1232-4-1 fourteen Staphylococcus aureus IC-1167-4-1 12 Staphylococcus aureus IC-1131-4-1 7 Staphylococcus aureus IC-1259-4-1 7 Staphylococcus aureus IC-1243-4-1 7 Staphylococcus aureus IC-1274-4-1 thirty Staphylococcus aureus IC-1231-4-1 12 Staphylococcus aureus IC-1233-4-1 fifteen Staphylococcus aureus IC-1234-4-1 10 Staphylococcus haemoliticus IC-1258-4-2 8 Candida albicans strain IC-1124-9-1 10 Candida albicans strain IC-1172-9-1 10 Candida albicans strain IC-1152-9-1 12 Candida albicans strain IC-1155-9-1 eleven Candida albicans strain IC-1143-9-1 10 Candida albicans strain IC-1141-9-1 13 Candida albicans strain IC-1134-9-1 thirty

Continuation of table 1 Candida albicans IC-53-9-1 12 Candida albicans IC-1147-9-1 10 Candida albicans IC-1150-9-1 eleven Candida albicans IC-1203-9-1 eleven Candida albicans IC-1195-9-1 10 Candida krusei IC-1244-9-2 fifteen Candida albicans IC-1144-9-1 fifteen Candida albicans IC-1132-9-1 10 Candida albicans IC-1130-9-1 twenty Candida albicans IC-1156-9-1 thirty Candida albicans IC-1122-9-1 thirty Candida albicans IC-26-9-1 10 Candida albicans IC-1150-9-1 thirty Candida albicans IC-55-9-1 eleven Candida albicans IC-1119-9-1 9 Candida albicans IC-1240-9-1 10 Candida albicans IC-1166-9-1 10 Candida albicans IC-1137-9-1 fourteen Candida albicans IC-1186-9-1 10 Klebsiella pneumonia strain IC-27-10-1 6 Shigella flexneri IC-815-14-2 7 Shigella sonnei IC-47-14-1 5 Yersinia pseudotuberculosis strain IC-45- 13-1 8 Serratia marcescens IC-1170-17 12 Escherichia coli IC-1184-5-1 10 Escherichia coli IC-1273-5-1 8 Escherichia coli IC-1429-5-1 3 Escherichia coli IC-1427-5-1 thirty Escherichia coli IC-1151-5-1 7 Enterococcus spp. IC-1236-16 fifteen Enterococcus faecium IC-1253-16-1 thirty Enterococcus spp. IC-1163-16 fifteen Enterococcus spp. IC-1164-16 thirty Enterococcus spp. IC-1423-16 10 Enterococcus spp. IC-1168-16 10 Enterococcus spp. IC-1175-16 12

Strain B. licheniformis IC-832-1-4 is characterized by the following indicators of zones (mm) of growth retardation of test strains of pathogens (table 2).

Table 2. Characterization of strain B. licheniformis IC-832-1-2 in terms of growth retardation zones of pathogen test strains The name of the test strain Zone Size (mm) Candida albicans strain IC-1124-9-1 thirty Candida albicans strain IC-1172-9-1 thirty Candida albicans strain IC-53-9-1 thirty Candida albicans strain IC-1147-9-1 thirty Candida albicans strain IC-1150-9-1 thirty Candida albicans strain IC-1203-9-1 twenty Candida albicans strain IC-1136-9-1 thirty Candida albicans strain IC-1195-9-1 twenty Candida krusei strain IC-1244-9-2 eighteen Candida albicans strain IC-1144-9-1 twenty Candida albicans strain IC-1132-9-1 8 Candida albicans strain IC-1130-9-1 fifteen Candida albicans strain IC-1156-9-1 thirty Candida albicans strain IC-1122-9-1 thirty Candida albicans strain IC-26-9-1 8 Candida albicans strain IC-1150-9-1 thirty Candida albicans strain IC-55-9-1 10 Candida albicans strain IC-1119-9-1 fifteen Candida albicans strain IC-1155-9-1 thirty Candida albicans strain IC-1186-9-1 10 Candida albicans strain IC-1143-9-1 thirty Candida albicans strain IC-1141-9-1 thirty Candida albicans strain IC-1134-9-1 thirty Pseudomonas aeruginosa strain IC-1210-7-1 12 Shigella flexneri strain IC-815-14-2 7 Yersinia pseudotuberculosis strain IC-45-13-1 5 Escherichia coli strain IC-1427-5-1 thirty Enterococcus faecium strain IC 1253-16-1 thirty Enterococcus spp. strain IC-1164-16 thirty Enterococcus spp. strain IC-1236-16 0 Staphylococcus aureus strain IC-1126-4-1 24 Staphylococcus aureus strain IC-1161-4-1 8 Staphylococcus aureus strain IC-1189-4-1 16 Staphylococcus aureus strain IC-1142-4-1 25 Staphylococcus aureus strain IC-1192-4-1 23 Staphylococcus aureus strain IC-1200-4-1 twenty Staphylococcus aureus strain IC-1246-4-1 twenty Staphylococcus aureus strain IC-1120-4-1 22 Staphylococcus aureus strain IC-1128-4-1 24 Staphylococcus aureus strain IC-1257-4-1 twenty Staphylococcus aureus strain IC-1149-4-1 12

Continuation of Table 2 Staphylococcus aureus strain IC-1251-4-1 10 Staphylococcus aureus strain IC-1226-4-1 16 Staphylococcus aureus strain IC-1140-4-1 eleven Staphylococcus aureus strain IC-1165-4-1 fifteen Staphylococcus aureus strain IC-1205-4-1 12 Staphylococcus haemoliticus strain IC-1256-4-2 fifteen Staphylococcus aureus strain IC-1241-4-1 thirty Staphylococcus aureus strain IC-1230-4-1 10 Staphylococcus aureus strain IC-1232-4-1 eleven Staphylococcus aureus strain IC-1167-4-1 thirty

Strain B. licheniformis IC-833-1-2 is characterized by the following indicators of growth retardation zones (mm) of test strains of pathogens (table 3).

Table 3. Characterization of B. licheniformis strain IC-833-1-2 in terms of growth retardation zones of pathogen test strains The name of the test strain Zone Size (mm) Candida albicans strain IC-1124-9-1 thirty Candida albicans strain IC-1172-9-1 thirty Candida albicans strain IC-1143-9-1 thirty Candida albicans strain IC-53-9-1 thirty Candida albicans strain IC-1147-9-1 thirty Candida albicans strain IC-1150-9-1 thirty Candida albicans strain IC-1203-9-1 thirty Candida albicans strain IC-1195-9-1 8 Candida krusei strain IC-1244-9-2 6 Candida albicans strain IC-1144-9-1 10 Candida albicans strain IC-1132-9-1 10 Candida albicans strain IC-1130-9-1 twenty Candida albicans strain IC-1156-9-1 thirty Candida albicans strain IC-1133-9-1 8 Candida albicans strain IC-1122-9-1 thirty Candida albicans strain IC-26-9-1 10 Candida albicans strain IC-1150-9-1 thirty Candida albicans strain IC-55-9-1 thirty Candida albicans strain IC-1119-9-1 thirty Candida albicans strain IC-1139-9-1 thirty Candida albicans strain IC-1141-9-1 thirty Candida albicans strain IC-1240-9-1 10 Candida albicans strain IC-1152-9-1 thirty Candida albicans strain IC-1155-9-1 thirty Klebsiella pneumonia strain IC-43-10-1 6

Continuation of Table 3 Klebsiella pneumonia strain IC-31-10-1 9 Shigella flexneri strain IC-815-14-2 12 Shigella sonnei strain IC-1216-14-1 thirty Serratia marcescens strain IC-817-17-1 thirty Yersinia pseudotuberculosis strain IC-45-13-1 thirty Staphylococcus aureus strain IC-25-4-1 7 Staphylococcus aureus strain IC-1161-4-1 26 Staphylococcus aureus strain IC-1189-4-1 fifteen Staphylococcus aureus strain IC-1126-4-1 eighteen Staphylococcus aureus strain IC-1142-4-1 16 Staphylococcus aureus strain IC-1192-4-1 twenty Staphylococcus aureus strain IC-1200-4-1 twenty Staphylococcus aureus strain IC-1246-4-1 16 Staphylococcus aureus strain IC-1233-4-1 thirty Staphylococcus aureus strain IC-1120-4-1 fifteen Staphylococcus aureus strain IC-1140-4-1 eighteen Staphylococcus aureus strain IC-1241-4-1 thirty Staphylococcus aureus strain IC-1232-4-1 twenty Staphylococcus aureus strain IC-1167-4-1 thirty Staphylococcus aureus strain IC-1274-4-1 thirty Staphylococcus aureus strain IC-1231-4-1 7 Staphylococcus aureus strain IC-1234-4-1 8 Staphylococcus haemoliticus strain IC-1258-4-2 5 Staphylococcus aureus strain IC-1230-4-1 12 Staphylococcus aureus strain IC-1222-4-1 10 Staphylococcus aureus strain IC-1165-4-1 twenty Staphylococcus aureus strain IC-1205-4-1 24 Staphylococcus haemoliticus strain IC-1256-4-2 9 Staphylococcus aureus strain IC-1128-4-1 17 Staphylococcus aureus strain IC-1251-4-1 eighteen Staphylococcus aureus strain IC-1226-4-1 twenty Staphylococcus aureus strain IC-1249-4-1 twenty Staphylococcus aureus strain IC-1257-4-1 16 Escherichia coli strain IC-1153-5-1 8 Escherichia coli strain IC-34-5-1 5 Escherichia coli strain IC-1148-5-1 7 Escherichia coli strain IC-36-5-1 6 Escherichia coli strain IC-1427-5-1 twenty Escherichia coli strain IC-1151-5-1 6 Enterococcus spp. strain IC-1236-16 16 Enterococcus faecium strain IC 1253-16-1 thirty Enterococcus spp. strain IC-1164-16 thirty Enterococcus spp. strain IC-1423-16 8 Enterococcus spp. strain IC-1168-16 8 Enterococcus spp. strain IC-1175-16 10

The strain B. licheniformis IC-834-1-2 is characterized by the following indicators of growth retardation zones (mm) of test strains of pathogens (table 4).

Table 4. Characterization of strain B. licheniformis IC-834-1-2 in terms of growth retardation zones of test pathogen strains The name of the test strain Zone Size (mm) Staphylococcus aureus strain IC-1161-4-1 fourteen Candida albicans strain IC-1124-9-1 eighteen Candida albicans strain IC-1172-9-1 9 Candida albicans strain IC-1152-9-1 12 Candida albicans strain IC-1155-9-1 fourteen Candida albicans strain IC-1143-9-1 12 Candida albicans strain IC-1141-9-1 fifteen Candida albicans strain IC-1134-9-1 thirty Escherichia coli strain IC-1250-5-1 5 Shigella flexneri strain IC-815-14-2 9 Candida albicans strain IC-53-9-1 12 Candida albicans strain IC-1147-9-1 thirty Candida albicans strain IC-1150-9-1 thirty Candida albicans strain IC-1203-9-1 fifteen Salmonella enteritidis strain IC-1159-6-1 5 Candida albicans strain IC-1136-9-1 7 Candida albicans strain IC-1195-9-1 10 Candida krusei strain IC-1244-9-2 fourteen Candida albicans strain IC-1144-9-1 fifteen Candida albicans strain IC-1132-9-1 12 Candida albicans strain IC-1130-9-1 13 Candida albicans strain IC-1156-9-1 thirty Candida albicans strain IC-1122-9-1 thirty Candida albicans strain IC-1150-9-1 thirty Candida albicans strain IC-55-9-1 thirty Candida albicans strain IC-1119-9-1 thirty Staphylococcus aureus strain IC-1189-4-1 twenty Serratia marcescens strain IC-817-17-1 16 Yersinia pseudotuberculosis strain IC-45-13-1 16 Staphylococcus aureus strain IC-1126-4-1 17 Staphylococcus aureus strain IC-1142-4-1 17 Staphylococcus aureus strain IC-1192-4-1 fifteen Staphylococcus aureus strain IC-1200-4-1 fifteen Staphylococcus aureus strain IC-1246-4-1 fifteen Staphylococcus aureus strain IC-1120-4-1 16 Staphylococcus aureus strain IC-1128-4-1 16 Staphylococcus aureus strain IC-1257-4-1 fifteen Escherichia coli strain IC-1153-5-1 10

Continuation of table 4 Escherichia coli strain IC-1184-5-1 5 Escherichia coli strain IC-1273-5-1 5 Escherichia coli strain IC-1193-5-1 5 Escherichia coli strain IC-1116-5-1 5 Escherichia coli strain IC-1424-5-1 5 Escherichia coli strain IC-1238-5-1 5 Escherichia coli strain IC-36-5-1 7 Enterococcus spp. strain IC-1163-16 8 Staphylococcus aureus strain IC-1249-4-1 19 Enterococcus spp. strain IC-1236-16 10 Staphylococcus aureus strain IC-1233-4-1 10 Staphylococcus aureus strain IC-1149-4-1 8 Escherichia coli strain IC-1430-5-1 7 Staphylococcus aureus strain IC-1251-4-1 10 Staphylococcus aureus strain IC-1226-4-1 10 Escherichia coli strain IC-1427-5-1 thirty Enterococcus faecium strain IC 1253-16-1 10 Staphylococcus aureus strain IC-1140-4-1 twenty Staphylococcus aureus strain IC-1222-4-1 eighteen Staphylococcus aureus strain IC-1165-4-1 twenty Staphylococcus aureus strain IC-1205-4-1 eighteen Staphylococcus aureus strain IC-1256-4-2 twenty Candida albicans strain IC-1133-9-1 8 Staphylococcus aureus strain IC-1241-4-1 10 Staphylococcus aureus strain IC-1230-4-1 thirty Enterococcus spp. strain IC-1164-16 thirty Staphylococcus aureus strain IC-1232-4-1 twenty Staphylococcus aureus strain IC-1167-4-1 22 Staphylococcus aureus strain IC-1131-4-1 10 Staphylococcus aureus strain IC-1259-4-1 10 Staphylococcus aureus strain IC-1243-4-1 10 Staphylococcus aureus strain IC-1179-4-1 9 Staphylococcus aureus strain IC-1125-4-1 8 Staphylococcus aureus strain IC-1274-4-1 thirty Staphylococcus aureus strain IC-1231-4-1 twenty Candida albicans strain IC-1154-9-1 22 Staphylococcus aureus strain IC-1229-4-1 8 Candida albicans strain IC-1202-9-1 6 Staphylococcus aureus strain IC-1233-4-1 8 Staphylococcus aureus strain IC-1234-4-1 10 Staphylococcus aureus strain IC-1258-4-2 8 Enterococcus app. strain IC-1423-16 10 Enterococcus spp. strain IC-1168-16 10 Enterococcus spp. strain IC-1175-16 12

Thus, the studied bacterial strains exhibit antagonistic activity against a wide range of pathogenic and conditionally pathogenic microorganisms.

Example 3. The method of cultivation of bacteria Bacillus licheniformis to obtain microbial biomass

To obtain biomass of bacterial spores, strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 were cultured in a liquid nutrient medium of the following composition:

1. meat enzymatic peptone - 10.0 g;

2. yeast extract - 5.0 g;

3. sodium chloride - 5.0 g;

4. distilled water - up to a total volume of 1 dm ³ .

pH (7.2 ± 0.2).

For 1 dm ^{3 of} fermentation medium, 10 cm ^{3 of the} corresponding Bacillus licheniformis strain with a titer of 10 ⁸ CFU / cm ³ is required. The biomass growing process was carried out in a fermenter for (48 ± 2) hours. With this cultivation method, a spore titer of not higher than (5-10) × 10 ¹⁰ CFU / cm ^{3 was} obtained.

Example 4. Obtaining the finished form of the drug based on the bacteria of the strains B. licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2

First, a mixture is prepared of the collected and tested raw biomass with a filler tested in terms of quality. As a filler, water (for processing plants by spraying) with a spore titer of at least 1 · 10 ³ CFU / ml or a powdery sorbent, for example zeolite, can be used. Powdered sugar and starch in the ratio of biomass, powder and starch 1: 5: 5 are also used as a sorbent, they are thoroughly mixed. During the mixing process, bacterial spores are immobilized onto starch particles. Next, sugar or glucose powder is added to the mixture at the rate of 1 part of the mixture per 10 parts of powder. The result is a drug concentrate with a spore titer of at least 1 × 10 ¹⁰ CFU / g. Then it is mixed with sugar or glucose powder and starch in such a proportion to obtain a finished product with a spore titer of at least 1 · 10 ³ -10 ⁶ CFU / g.

The residual moisture content of the drug is not more than 5%.

Example 5. The composition of the drugs

5.1. Composition 1.

An aqueous solution of biomass spores of bacteria Bacillus licheniformis IC-831-1-2 with a spore titer of at least 1 · 10 ³ CFU / ml. This solution is stored in a dark place for no more than 1-2 days.

5.2. Composition 2.

A mixture of biomass spores of bacteria Bacillus licheniformis IC-831-1-2 with a zeolite with a spore titer of at least 1 · 10 ⁶ CFU / g.

5.3. Composition 3.

A mixture of biomass spores of bacteria Bacillus licheniformis IC-831-1-2 with a spore titer of at least 1 · 10 ⁶ CFU / g with a mixture of starch and sucrose-based powder in a ratio of 1: 100.

5.4. Composition 4.

A mixture of biomass spores of bacteria Bacillus licheniformis IC-832-1-2 with a spore titer of at least 1 · 10 ⁶ CFU / g with a mixture of starch and sucrose-based powder in a ratio of 1:10.

5.5. Composition 5.

A mixture of biomass spores of bacteria Bacillus licheniformis IC-833-1-2 with a spore titer of at least 1 · 10 ⁶ CFU / g with a mixture of starch and sucrose-based powder in a ratio of 1:50.

5.6. Composition 6.

A mixture of biomass spores of bacteria Bacillus licheniformis IC-834-1-2 with a spore titer of at least 1 · 10 ⁶ CFU / g with a mixture of starch and sucrose-based powder in a ratio of 1:10.

5.7. Composition 7.

A mixture of biomass spores of bacteria Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2, IC-834-1-2 in a ratio of 1: 1: 1: 1 with each spore titer bacteria species at least 1 · 10 ³ CFU / g with a mixture of starch and glucose-based powder in a ratio of 1:20.

Example 6. Data on the storage of the dry form of the drug based on strains B. licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2

To establish the shelf life and storage conditions of the drug, relevant studies were conducted.

Within 48 months of storage of the dry form of the drug at a temperature of (30 ± 0.5) ° С, the titer and antagonistic activity of the drug did not change, which confirms the high thermal stability of the preparations in spore form, which significantly simplifies the technology of their use in veterinary medicine and plant growing.

Example 7. Data on the inhibitory activity of strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 against phytopathogens.

To test the strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 with respect to their ability to inhibit phytopathogens, the agar block method was used. A suspension of the drug with a titer of 1 × 10 ⁶ CFU / cm ^{3 was} introduced into meat-peptone agar, cooled to a temperature of (36 ± 1) ° C. For the growth of phytopathogens used:

- Chapek Wednesday for Didymella applanata;

potato glucose agar for Fusarium oxysporum, Botrytis cinerea, Alterneria alternaya, Fusarium moniliformes, Fusarium sporotrichiella, Rhizoctonia solani.

The medium inoculated with the studied strain was poured into Petri dishes and onto a frozen surface, a block 10 mm in diameter, cut from a phytopathogen colony, was placed in the center.

Analysis was carried out with a frequency of 1 time in 3 days. The activity of the strain was taken into account by changing the diameter of the fungus colony in comparison with the control (medium without introducing the strain). Based on the data obtained, the inhibitory activity was determined, which was calculated by the formula

,

,

Where

D _to - the diameter of the colony in the control version,

D _about - the diameter of the colony in the experimental version.

As a result of the studies, the following indicators of the inhibitory activity of the strain Bacillus licheniformis IC-831-1-2 on the 7th day were established:

- in relation to Didymella applanata - 12.7%;

- in relation to Fusarium moniliformes - 62%;

- in relation to Fusarium oxysporum - 70%;

- in relation to Fusarium sporotrichiella - 19.2%;

- in relation to Alterneria alternata - 10.5%.

As a result of the studies, the following indicators of the inhibitory activity of strains of Bacillus licheniformis IC-832-1-2 on the 7th day were established:

- in relation to Didymella applanata - 17.2%;

- in relation to Botrytis cinerea - 21%;

- in relation to Fusarium moniliformes - 43%;

- in relation to Fusarium sporotrichiella - 17.2%;

- in relation to Alterneria alternata - 38.7%.

As a result of the studies, the following indicators of the inhibitory activity of the strain Bacillus licheniformis IC-833-1-2 on the 7th day were established:

- in relation to Botrytis cinerea - 38%;

- in relation to Fusarium oxysporum - 62%;

- in relation to Fusarium moniliformes - 12%;

- regarding Alterneria alternata - 42%.

As a result of the studies, the following indicators of the inhibitory activity of the strain Bacillus licheniformis IC-834-1-2 on the 7th day were established:

- in relation to Didymella applanata - 67%;

- in relation to Botrytis cinerea - 67%;

- in relation to Fusarium moniliformes - 44%;

- in relation to Fusarium oxysporum - 67%;

- in relation to Fusarium sporotrichiella - 36%;

- in relation to Alterneria alternata - 11%.

Thus, the claimed strains of Bacillus licheniformis exhibit antagonistic activity in relation to the above listed fungal phytopathogens.

Example 8. Data from field trials to study the effect of drugs based on strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 on the defeat of raspberry purple spotting and currant septoria

For testing strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 to determine the presence of fungicidal activity against raspberry and currant diseases in the open industrial plantings of raspberries of the Zorenka Altai variety and currants of the Sofia variety were used on the ground. As a chemical standard used the drug Topaz in a concentration of 0.1%. The treatment was carried out with a suspension of preparations with a titer of 10 ⁶ spores / ml.

Processing of plantings was carried out in July and June. The timing of treatment was determined when the first symptoms of the disease appeared.

Field experience diagram:

- Bacillus licheniformis strain IC-831-1-2;

- Bacillus licheniformis strain IC-832-1-2;

- Bacillus licheniformis strain IC-833-1-2;

- Bacillus licheniformis strain IC-834-1-2;

- Topaz 0.1%;

- the control.

The experiment was repeated four times, the plot area was 10 m ² , the flow rate of the working fluid was 4 dm ³ per option. The allocation of plots is randomized. The patterns on raspberries and currants are similar.

Damage to currant leaves with septoria was taken into account on the following scale:

0 - points - healthy leaves;

1 point - poor development of the disease (occupied by spots from 0 to 10% of the surface of the leaf blade);

2 points - the average development of the disease (occupied by spots from 11 to 25% of the surface of the leaf blade);

3 points - a strong development of the disease (occupied by spots from 26 to 50% of the surface of the leaf blade);

4 points - a very strong development of the disease (more than 50% of the surface of the leaf blade is occupied by spots).

The degree of damage to raspberry shoots was evaluated on a special 4-point scale for purple raspberry spotting:

0 points - a healthy stem;

1 point - the surface of the affected area is smooth, thickening of the cortex is possible, the site is located on one side of the stem;

2 points - the surface of the affected area is smooth, bark cracks are possible, the site is located on both sides of the stem;

3 points - the surface of the affected area is deformed, the area is located on both sides of the stem;

4 points - the surface of the affected area is deformed, the area rings the stem.

The development of the disease was determined by the following formula:

Where

R is the disease development index,%;

a - the number of plants of the corresponding score, pcs .;

b - scale point;

N is the total number of plants, pcs .;

K is the highest score of the scale used.

Prevalence was calculated by the formula

где

Where

R is the prevalence index of the disease,%;

a - the number of plants in the control, pcs .;

b is the number of plants in the experiment, pcs.

The biological effectiveness of biological products was determined by the formula

Where

a is an indicator of the average prevalence in the control,%;

b - the average rate of damage in the treated area,%.

The results of field experiments are presented in tables.

Table 5. The effect of the claimed drugs based on strains of Bacillus licheniformis on the defeat of raspberries with purple spotting. Option Prevalence,% Development,% Biological efficiency,% The control 75.0 25.0 - Strain B. lich. IC 831-1-2 34.1 8.5 66.0 Strain B. lich. IC-832-1-2 43,2 10.8 56.8 Strain B. lich. IC 833-1-2 40.8 12.0 61,4 Strain B. lich. IC 834-1-2 37.5 9.0 62.7 Topaz 0.1% 38.6 10.8 56.8 NDS ₀₅ 4.0

Table 6. The effect of the claimed drugs based on strains of Bacillus licheniformis on the defeat of raspberries with purple spotting. Option Prevalence,% Development,% Biological efficiency,% The control 95.0 35.0 - Strain B. lich. IC 831-1-2 32,5 8.1 76.9 Strain B. lich. IC-832-1-2 35.0 15.6 55,4 Strain B. lich. IC 833-1-2 37.0 13.0 59.5 Strain B. lich. 1C 834-1-2 44.0 11,2 75.9 Topaz 0.1% 50,0 12.5 64.3 NDS ₀₅ 7.0

Table 7. The effect of the claimed drugs based on strains of Bacillus licheniformis on the defeat of currants with Septoria. Option Biological efficiency,% Accounting Dates 27th of June 4th of July July 11th B. licheniformis IC 831-1-2 80.6 61.3 54.8

B. stearothermophilus IC-832-1-4 82.3 59.7 54.8 B. stearothermophilus IC 833-1-4 79.5 52.0 59.1 B. stearothermophilus IC 834-1-4 76.6 65.0 53.1 Topaz 0.1% 64.5 56.5 56.5 Note 1. ^(*) - biological effectiveness is calculated according to the growth rate of the disease. Note 2. The systemic fungicidal and insecticidal preparation Topaz effectively treats berry shrubs and cucumbers from powdery mildew, and flowering plants from rust. It acts for 10 days and is not washed off by rain.

Thus, the treatment of raspberry plantings with preparations based on strain B. licheniformis IC 831-1-2 in a field experiment reduced: the development of the disease by almost 3 times, the prevalence by more than 2 times. The yield increase was 0.3 t / ha. Treatment of currant plantings with strain B. licheniformis IC 831-1-2 in a field experiment reduced the development of the disease by 2.2 times. Biological efficiency was (54.8-80.6)%, depending on the duration of the accounting.

The treatment of raspberry plantings with a preparation based on strain B. stearothermophilus IC 832-1-4 in a field experiment reduced: the development of the disease by almost 3 times, the prevalence by more than 2 times. The yield increase was 0.3 t / ha. Treatment of currant plantings with strain B. stearothermophilus IC 832-1-4 in a field experiment reduced the development of the disease by 2.2 times. Biological efficiency was (54.8-82.3)%, depending on the duration of the accounting.

The treatment of raspberry plantings with a preparation based on strain B. stearothermophilus IC 833-1-4 in a field experiment reduced: the development of the disease by almost 3 times, the prevalence by more than 2 times. The yield increase was 0.3 t / ha. Treatment of currant plantings with strain B. stearothermophilus IC 833-1-4 in a field experiment reduced the development of the disease by 2.2 times. Biological effectiveness was (52.0-79.5)%, depending on the duration of the accounting.

The treatment of raspberry plantings with a preparation based on strain B. stearothermophilus IC 834-1-4 in a field experiment reduced: the development of the disease by almost 3 times, the prevalence by more than 2 times. The yield increase was 0.3 t / ha. Treatment of currant plantings with strain B. stearothermophilus IC 834-1-4 in a field experiment reduced the development of the disease by 2.2 times. Biological effectiveness was (53.1-76.6)%, depending on the duration of the accounting.

Example 9. Research of the claimed drug in the form of microbiological fertilizer

The following table 8 shows the study of the claimed drugs in the form of microbiological fertilizers on a variety of spring wheat Baganskaya - 95.

Seeds were processed using a PS-10 pickling machine, with a working solution consumption rate of 10 l per ton of seeds. The consumption of drugs was 10 ml per ton. The titer of the drug is 1 × 10 ⁸ CFU / ml. Top dressing was carried out by spraying with a boom sprayer with a flow rate of 50 l / ha (flow rate - 10 ml / ha).

As a result of field tests of the Fitop preparation and an experimental preparation based on the B. stearothermophilus strain IC-834-1-4, an increase in the content of groups of microorganisms - indicators of soil fertility was established: enrichment of the microbial cenosis of the soil with microorganisms of various ecological and trophic groups (total microbial number), increase the number of spore-forming bacteria of the genus Bacillus, the content of which is an important indicator of a favorable trophic regime of the soil with the introduction of a bacterial preparation and availability e mineral elements and characteristic of soils with intensive processes of nitrification. The number of true denitrifiers and ammonifying microorganisms has increased, as well as the number of autochthonous microflora, which carries out the destruction of humic compounds. The humification index at the time of harvesting significantly increased in the variant with the introduction of Fitopa by 19.5% and in the variant with the introduction of the drug based on the strain B. stearothermophilus IC-834-1-4 by 16.6% compared with the control.

Thus, the preparation based on the B. stearothermophilus strain IC-834-1-4 not only positively affects the dynamics of changes in the numerical composition of soil microorganisms, but is also more effective compared to its analogue, the Fitop preparation based on the B. subtilis strain VKPM B -7036.

Table 8. The study of drugs in the form of microbiological fertilizers. Experience option Indicative group of soil microflora, CFU / g Deviation from control one Total microbial number 1.1 The control 98.2 × 10 ⁵ 1.2 Strain treatment of seeds 163 × 10 ⁵ 64.8 Bacillus stearothermophilus IC-834-1-4 1.3 Seed treatment 132.1 × 10 ⁵ 33.9 Fitop 1.4 The control 40.13 × 10 ⁵ 1.5 Fertilizing vegetative 86 × 10 ⁵ 45.87 plants strain B. stearothermophilus IC-834-1-4 1.6 Fertilizing vegetative 32.07 × 10 ⁵ 8.06 plants with Fitop 2 Bacillus bacteria 2.1 The control 11.3 × 10 ⁴ 2.2 Strain treatment of seeds 17.7 × 10 ⁴ 6.4 Bacillus stearothermophilus IC-834-1-4 2.3 Seed treatment 12.5 × 10 ⁴ 1,2 Fitop 2.4 The control 13.5 × 10 ⁴ 2.5 Fertilizing vegetative 23.1 × 10 ⁴ 9.6 plants strain B. stearothermophilus IC-834-1-4 2.6 Fertilizing vegetative 15.1 × 10 ⁴ 1,6 plants with Fitop 2.7 the control 14.3 × 10 ⁴ 2.8 Seed Processing and Feeding 21.3 × 10 ⁴ 7 vegetative plants strain B. stearothermophilus IC-834-1-4 2.9 Seed Processing and Feeding 16.7 × 10 ⁴ 2,4 vegetative plants

Fitop 3 True Denitrifiers 3.1 The control 33.5 × 10 ⁵ 3.2 Strain treatment of seeds Bacillus stearothermophilus IC-834-1-4 55.2 × 10 ⁵ 21.7 3.3 Seed treatment Fitop 34.9 × 10 ⁵ 1.4 3.4 The control 36.6 × 10 ⁵ 3.5 Seed Processing and Feeding vegetative plants strain B. stearothermophilus IC-834-1-4 58.1 × 10 ⁵ 21.5 3.6 Seed Processing and Feeding 27.3 × 10 ⁵ 9.3 vegetative plants Fitop four Nitrogen-fixing (ammonifying) microorganisms 4.1 The control 37.5 × 10 ⁵ 4.2 Strain treatment of seeds Bacillus stearothermophilus IC-834-1-4 33.6 × 10 ⁵ 4.3 Seed treatment Fitop 56.7 × 10 ⁵ 19.2 5 Indigenous Microflora 5.1 The control 3.8 × 10 ⁴ 5.2 Strain treatment of seeds Bacillus stearothermophilus IC-834-1-4 2.3 × 10 ⁴ -1.5 5.3 Seed treatment Fitop 6.3 × 10 ⁴ 2.5 5.4 The control 0.9 × 10 ⁴ 5.5 Fertilizing vegetative 0.5 × 10 ⁴ plants strain B. stearothermophilus IC-834-1-4 5.6 Fertilizing vegetative plants with Fitop 2.3 × 10 ⁴ 1.4 5.7 The control 3.9 × 10 ⁴ 5.8 Seed Processing and Feeding vegetative plants Fitop 0.8 × 10 ⁴ -3.1 5.9 Seed Processing and Feeding vegetative plants strain B. stearothermophilus IC-834-1-4 5.4 × 10 ⁴ 1,5

Example 10. Data on the restoration of microflora of the gastrointestinal tract of animals using the inventive preparation

Studies are associated with determining the dynamics of changes in the quantitative composition of the representative groups of intestinal microflora during the entire period of removal of strains from the animal organism. The material for the work was:

1. Healthy outbred mice of both sexes from the nursery FGUN SSC VB "Vector". The animals were kept at a temperature of (25 ± 1) ° С and 12-hour illumination. Granules for feeding laboratory mice and rats were used as food. Animals were divided into the following groups:

- group 1 - control;

- group 2 - the experiment, a commercially available preparation of Vetom 3 (the active principle is the Bacillus subtilis strain VKPM B 7048);

- group 3 - experiment, experimental model of Vetom 13.1 (the active principle is the strain Bacillus stearothermophilus IC 831-1-4);

- group 4 - experience, experimental model of Vetom 14.1 (the active principle is the strain Bacillus stearothermophilus IC 832-1-4);

- group 5 - experience, experimental model of Vetom 15.1 (the active principle is the strain Bacillus stearothermophilus IC 833-1-4);

- group 6 - experiment, experimental model of Vetom 16.1 (the active principle is the strain Bacillus stearothermophilus IC 834-1-4).

Each group contained 5 animals.

2. In the experiments, preparations were used in the form of a culture fluid of the used strains with a titer of at least 1 × 10 ⁸ KOE / ml. The preparations of experimental animals were fed according to the following scheme:

- 0 day - water;

- 1 - 10 day - preparations corresponding to the test variants diluted with tap water up to a calculated titer of at least 1x10 ⁶ CFU / ml. Drinkers with drugs were changed daily.

- 11 - 50 day - water.

1. To quantitatively take into account the dynamics of strain isolation, the method of sequential dilution of animal feces was used, followed by plating of 100 μl of a suspension from each dilution of meat-peptone agar preheated at 100 ° C for 10 minutes in Petri dishes.

Table 9. Data on the restoration of microflora of the gastrointestinal tract of animals Date strain E. coli Enterococcus Lactobacillus Pathogenic, including Proteus Bifidobacterium one 2 four 5 6 7 0 day The control 7 × 10 ⁴ 1 × 10 ⁴ 3 × 10 ⁶ 10 ³ 10 ⁸ 7048 2.8 × 10 ⁵ 1 × 10 ⁴ 5.3 × 10 ⁷ 10 ³ 10 ⁸ IC-831-1-4 5 × 10 ⁵ 1 × 10 ⁴ 7 × 10 ⁶ 10 ³ 10 ⁸ IC-832-1-4 2 × 10 ⁵ 1 × 10 ⁴ 7 × 10 ⁶ 10 ³ 10 ⁸ IC-833-1-4 1 × 10 ⁵ 1 × 10 ⁴ 1 × 10 ⁶ 10 ³ 10 ⁸ IC-834-1-4 3 × 10 ⁵ 1 × 10 ⁴ 2.2 × 10 ⁶ 10 ³ 10 ⁸ 10 day The control 5 × 10 ⁵ 1.1 × 10 ⁴ 3.4 × 10 ⁷ 10 ⁴ 10 ⁸ 7048 2.2 × 10 ⁷ 1 × 10 ⁵ 3 × 10 ⁷ 10 ³ 10 ⁸ IC-831-1-4 2.7 × 10 ⁶ 3 × 10 ⁵ 3.8 × 10 ⁶ 10 ³ 10 ⁸ IC-832-1-4 2 × 10 ⁷ 2 × 10 ⁶ 5 × 10 ⁷ 10 ³ 10 ⁸ IC-833-1-4 2.6 × 10 ⁸ 1 × 10 ⁵ 2.2 × 10 ⁸ 10 ³ 10 ⁹ IC-834-1-4 1.8 × 10 ⁷ 3.7 × 10 ⁶ 2 × 10 ⁷ 10 ³ 10 ⁹ 20 day The control 1 × 10 ⁵ 1 × 10 ⁴ 1 × 10 ⁵ 10 ⁴ 10 ⁸ 7048 4 × 10 ⁷ 1 × 10 ⁴ 1 × 10 ⁶ 10 ⁴ 10 ⁹ IC-831-1-4 1 × 10 ⁷ 1.2 × 10 ⁷ 3 × 10 ⁸ 10 ² 10 ⁹ IC-832-1-4 1 × 10 ⁷ 1 × 10 ⁶ 6 × 10 ⁸ 10 ³ 10 ⁹ IC-833-1-4 2 × 10 ⁸ 5 × 10 ⁷ 1 × 10 ⁸ 10 ² 10 ⁹ IC-834-1-4 2 × 10 ⁸ 1 × 10 ⁷ 1 × 10 ⁹ 10 ² 10 ⁹ 30 day The control 3 × 10 ⁴ 1.2 × 10 ⁴ 1.0 × 10 ⁶ 10 ⁴ 10 ⁷ 7048 7.7 × 10 ⁷ 5 × 10 ⁷ 1.5 × 10 ⁸ 10 ⁴ 10 ⁹ IC-831-1-4 5 × 10 ⁷ 4.6 × 10 ⁷ 2.6 × 10 ⁸ 10 ² 10 ⁹ IC-832-1-4 3.6 × 10 ⁸ 5.1 × 10 ⁷ 3.9 × 10 ⁹ 10 ² 10 ¹⁰ IC-833-1-4 5 × 10 ⁸ 8 × 10 ⁸ 6 × 10 ⁸ 10 ² 10 ¹⁰ IC-834-1-4 2.0 × 10 ⁸ 4.6 × 10 ⁸ 2.0 × 10 ⁹ 10 ² 10 ¹⁰ 40 day The control 1,0 × 10 ⁵ 6 × 10 ⁴ 3 × 10 ⁵ 10 ⁴ 10 ⁸ 7048 2.1 × 10 ⁷ 3 × 10 ⁷ 1 × 10 ⁷ 10 ⁴ 10 ⁹ IC-831-1-4 7.5 × 10 ⁷ 2 × 10 ⁷ 1.3 × 10 ⁹ 10 ² 10 ⁹ IC-832-1-4 9.5 × 10 ⁹ 1 × 10 ⁸ 2 × 10 ¹⁰ 10 ² 10 ¹⁰ IC-833-1-4 6.3 × 10 ⁸ 3 × 10 ⁹ 6.2 × 10 ⁹ 10 ² 10 ¹⁰ IC-834-1-4 1 × 10 ⁹ 1 × 10 ⁹ 5 × 10 ⁹ 10 ² 10 ¹⁰ 50 day

The control 2 × 10 ⁵ 2.4 × 10 ⁴ 6 × 10 ⁶ 10 ⁴ 10 ⁸ 7048 6 × 10 ⁷ 2.0 × 10 ⁷ 2 × 10 ⁸ 10 ⁴ 10 ⁹ IC-831-1-4 1 × 10 ⁸ 2 × 10 ⁸ 1.5 × 10 ⁹ 10 ² 10 ⁹ IC-832-1-4 1.4 × 10 ⁹ 1.0 × 10 ⁸ 3 × 10 ¹⁰ 10 ² 10 ¹⁰ IC-833-1-4 2.0 × 10 ⁸ 3.9 × 10 ¹⁰ 1.0 × 10 ¹⁰ 0 10 ¹¹ IC-834-1-4 1 × 10 ⁹ 3 × 10 ⁹ 2.3 × 10 ¹⁰ 0 10 ¹¹

2. To quantify the dynamics of changes in the intestinal microflora, daily sowing was performed from feces of mice of all experimental groups according to the standard dysbacteriosis screening scheme, including:

- determination of bacteria of the group of Escherichia coli (BGKP) with normal enzymatic activity on Endo medium and Levin medium;

- enterococci on polymyxin agar;

- lactobacilli on Blikfeldt's medium;

- opportunistic microorganisms in the environment of Ploskirev and yolk-salt agar;

- bifidobacteria on thioglycol medium.

Table 9 shows the data on the restoration of microflora of the gastrointestinal tract of animals. The analysis of table 9 shows the high efficiency of restoration of the intestinal microflora of animals compared with the control and the commercial drug probiotic Vetom 3 (B. subtilis strain VKPM B 7048).

Indications for use (recommendatory). Preparations based on strains of Bacillus stearothermophilus IC-831-1-4, IC-833-1-4, IC-834-1-4 are recommended for the prevention and treatment of dysbacterioses of agricultural and wild birds, including chickens, ducks, geese, turkeys, guinea fowl and others, increasing the natural resistance of the bird organism, eliminating immunodeficiency caused by infectious (viruses, bacteria, protozoa, intracellular) and non-infectious (poor-quality feed, crowded content, stress due to non-compliance with temperature conditions of keeping) factors, increase with livestock safety, increase egg production, obtain additional broiler weight gain, reduce feed conversion, reduce poultry rearing time. Prevention and treatment of dysbacteriosis of all types of productive farm animals, treatment of infectious diseases of bacterial and protozoal etiology, prevention of viral diseases and immunodeficiency conditions, increase the safety of young animals in the early periods of development, increase productivity, and obtain additional weight gain.

Preparations based on the strains of Bacillus stearothermophilus IC-832-1-4 are recommended for the prevention and treatment of cattle dysbiosis, the treatment of infectious diseases of bacterial and protozoal etiology, the prevention of viral diseases and immunodeficiency (primary and secondary) conditions, and the preservation of young cattle in early periods of development, increasing the productivity of dairy cattle breeding, obtaining additional gains in young cattle breeding, improving the quality and grade of milk and meat, etc. dleniya broodstock fertility.

Claims (9)

1. The bacterial strain Bacillus licheniformis IC-831-1-2, having
bactericidal and fungicidal activity and deposited in the All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika under registration number VKPM V-10561. 2. The bacterial strain Bacillus licheniformis IC-832-1-2, with bactericidal and fungicidal activity and deposited in the All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika under registration number VKPM B-10562. 3. The bacterial strain Bacillus licheniformis IC-833-1-2, with bactericidal and fungicidal activity and deposited in the All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika under registration number VKPM B-10563. 4. The bacterial strain Bacillus licheniformis IC-834-1-2, with bactericidal and fungicidal activity and deposited in the All-Russian Collection of Industrial Microorganisms FSUE GosNIIGenetika under registration number VKPM B-10564. 5. A drug with bactericidal and fungicidal activity, characterized by the content of a filler with biomass of bacteria in the spore form of Bacillus licheniformis VKPM B-10561, or VKPM B-10562, or VKPM B-10563, or VKPM B-10564, or a mixture thereof with each titer bacterial strain of at least 1 · 10 ³ CFU / g or 1 · 10 ³ CFU / ml 6. The drug according to claim 5, characterized in that it contains water as a filler. 7. The drug according to claim 5, characterized in that as a filler it contains a powdered sorbent. 8. The preparation according to claim 7, characterized in that it contains a zeolite, or a polysaccharide, or a monosaccharide, or a disaccharide, or a mixture of a polysaccharide with a monosaccharide or polysaccharide in a ratio of 1: 100 to 1:10 as a powdery sorbent. 9. The drug according to claim 8, characterized in that it contains starch as a polysaccharide, glucose as a monosaccharide, and sucrose as a disaccharide.