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

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

 

The invention relates to a strain of microorganism for the restoration of microflora of the soil and the gastrointestinal tract (GIT) of animals that have antibacterial and antifungal activity, and to the preparative form based on it and can be used in biotechnology, veterinary medicine and plant protection to obtain drugs against bacterial and fungal infections of animals and plants, as well as for use as a microbiological fertilizers and probiotics to restore the microflora of the digestive tract of animals.

Registered and listed by the state chemical Commission of the Ministry of agriculture of the Russian Federation bacterial fungicides are not intended for use on berry crops of raspberry and black currant. Thus, the drug Integral (liquid-based strain 24D Bacillus subtilis) registered for use against diseases of grain crops (wheat, spring barley), vegetables (potatoes, tomatoes and cucumbers). From berry crops the drug is allowed to fight against Anthracnose of black currant.

Drug mitosporic-M (powder-based strain 24D Bacillus subtilis) 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. 27.04.1999 year).

Drug Backoff (stachiw the decomposing powder-based strain IPM 215 Bacillus subtilis) is designed to protect against diseases of vegetable and flower crops, grown in the open and protected ground, and black currant from powdery mildew.

Among microbiological fertilizers similar to the proposed drug is a drug Bactofection, which is based on soil microorganisms Bacillus mucilaginosus, designed to enrich the soil plant-digestible forms of phosphorus, as well as restore its fertility, increase the germination rate and yield of many crop species in open and protected ground. The authors of this fertilizer does not include specific data on the impact on indicative fertile soil groups of microorganisms and effects 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 you make as a symbiotic community, designed to increase the number of soil microorganisms and, consequently, fertility and productivity.

However, the above drugs have a narrow spectrum of action. A number of strains of bacteria of the genus Bacillus, which are antagonists of various pathogens of agricultural crops. The Bacillus subtilis strain B-116 exerts an antagonistic effect on the fungi Pythium and Rhysoctonia. The bacterial strain Bacillus subtilis ARRIAM N128 used for the teachings of the drug against fungal diseases of cotton (USSR Author's certificate No. 1717156, CL 01N 63/00, publ. 1992) [1].

However, these strains have limited action on individual fungal diseases of various crops.

Known bacterial strain Bacillus subtilis ARRIAM N131 used to obtain the drug against pathogens rot of apples and grapes during storage (USSR Author's certificate No. 1706504, CL 01N 63/00, 1992) [2].

However, this strain also has a limited action on individual fungal pathogens of agricultural crops.

The closest analogue (prototype) is a bacterial strain Bacillus subtilis VKPM In 7036, intended to receive the drug against fungal diseases of plants (Patent RF №2086128, IPC A01N 63/00, C12N 1/20, published 10.08.1997 year). The strain has been selected to suppress bacterial growth. With the best options conducted experiments on suppression of growth: Phytophthore infestans, Microsporium solani, Fusarium solani (the causative agent of dry rot).

The closest analogue (prototype) formulation is the drug Fitop obtained on the basis of the above-mentioned strain of Bacillus subtilis VKPM In 7036 in the following ratio, wt.% (Patent RF №2086128, IPC A01N 63/00, C12N 1/20, published 10.08.1997 g): the spores of the bacteria Bacillus subtilis VKPM In 7036 with title 7·1010spores/g (0,1-1,0); starch (0,2-3,0); sugar - rest. The residual is the moisture of the product is not more than (2-5) %. Drug Fitop produced and used in agriculture for more than 10 years.

However, the strain-prototype and the drug based on it have not wide enough spectrum of antagonistic activity, and was not analyzed for the ability to restore the microflora of the digestive tract of animals.

The technical result of the claimed technical solution is ensuring the recovery of soil microbiocenosis, the digestive tract of animals and expansion of the spectrum of antibacterial and antifungal activity.

This technical result is achieved by obtaining method of directed selection of four variants of the bacterial strains of Bacillus licheniformis: IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 for the restoration of microflora of the soil and the gastrointestinal tract of animals, which have bactericidal and fungicidal activity and deponirovanie from 18.03.2010, in the all-Russian Collection of Industrial Microorganisms (VKPM) FSUE gosniigenetika, respectively, under the number VKPM B-10561, 10562, 10563, and In-10564. (certificates of Deposit included).

This technical result is achieved by the fact that the drug is characterized by the fact that it contains a filler with a biomass of bacteria in 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 their mixture with the title of each bacterial strain is not less than 1·103CFU/g or 1·103 CFU/ml as filler it contains water or powdered sorbent. As a powdered sorbent contains a zeolite, or a polysaccharide, or a monosaccharide, or a disaccharide, or a mixture of polysaccharide with a monosaccharide or polysaccharide in a ratio of from 1:100 to 1:10. As it contains polysaccharide starch, monosaccharides - glucose, as the disaccharide is sucrose.

The original clones to obtain the claimed strains were isolated from healthy animals in the ecologically clean district of the Novosibirsk region (Zavyalovsky Bor):

a strain of Bacillus licheniformis: IC-831-1-2 isolated from faeces goose,

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

a strain of Bacillus licheniformis IC-833-1-2 isolated from faeces goose,

a strain of Bacillus licheniformis IC-834-1-2 isolated from chicken faeces.

The original clones were subjected to selection for suppression of bacterial and fungal growth. With the best clones was performed experiments on suppression of growth: enterobacteria, gram-positive cocci, neurontinbuy bacteria, fungi of the genus Candida, pathogens Fusarium plants, purple spot raspberry, Septoria raspberries.

Immobilization of bacteria spores on the starch provides additional mechanical protection, prevents aggregation of the dispute and provides a more uniform distribution in the mass of filler.

When applying the attachment of the preparation of the starch helps to speed up the germination of spores of bacteria, that increases the effectiveness of his actions.

The powder is formulated with sucrose (sugar) or glucose is not only a sorbent - filled, but also on the product stabilizing and preservative effect, creates better conditions for storage of bacteria spores.

In addition, the use of the proposed components of the drug greatly simplifies the technology of its receipt as on the stages of cultivation (in technology there is no use of expensive enzymes, does not require purification and concentration of the drug), and on the stages of getting it ready form (removal and redistribution of moisture combined with getting ready form of the 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 belong to the gram-positive facultative anaerobic rod-shaped bacteria. When growth on meat-peptone agar give a wrinkled colonies with abundant mucus formation and secretion pinkish-red pigment in the upper layer of the environment under the colony. Colonies are cream-pink. Cells are rod-shaped, oval endospores, are Central or paracentral, don't inflate the cell when sporoobrazovanie. The length of the cage (1,09-1,96) ám, width of 0.36 to 0.97) microns. Capsule form, emit large amounts of mucus. A gram-stained polozhitelnomu are thermophilic, multiply with (35-65°C, the optimal growth temperature (37-40)°C.

After 48 hours of growth at 40°C on a meat-peptone agar cultures have opaque colonies heterogeneous texture, with mucous inclusions and wavy edge, embedded in the surface of agar plates. the pH of the medium: minimum of 5.7; maximum of 8.0; the optimum of 6.5 to 7.0. Strains multiply at 40°C on a meat-peptone agar and meat-peptone broth.

Biochemical properties of the strains. They do not grow in anaerobic conditions, do not form acetylaminophenol of glucose in the reaction Voges-Proskauer, oxidized with the formation of acid, glucose, mannose, fructose, ribose, lyxose, cellobiose, trehalose, maltose, turanose. Hydrolyzing starch, urea and esculin. Not have lecithinase activity. Possess catalase activity. Tolerant to sodium chloride concentrations in the medium range (1-9) %.

The strains are not zoopathogenic and phytopathogenic.

The strains produce biologically active metabolites, including antibiotics, and enzymes for a wide spectrum of action, suppresses the growth of pathogenic and conditionally pathogenic bacterial and fungal microflora.

For long term storage of strains of spore mass is freeze-dried. For mass propagation of bacteria use meat-peptone broth, cultivation conduct the ri 40°C.

Example 1. The study of epatajnosti 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 laboratory of biotechnological control of APF "Research center".

The pathogenicity of each microorganism was evaluated by survival of infected animals, their appearance and behavior, Visavuori bacteria from the blood and organs at different times after infection, and the macroscopic picture of the internal organs at necropsy of animals at the end of the observation period.

The paper uses laboratory animals are of two types: non-linear white mouse weight (18-20) g in the amount of 40 individuals of both sexes and the rats of Wistar breed weight (180-200) g in the amount of 40 individuals of both sexes. The animals formed the experimental and control group of 20 animals each. For detection of bacteria in the organs of experimental animals the animals of experimental groups during the 60 day orally was administered daily culture liquid bacteria obtained by cultivation of the investigated strain on meat-peptone broth.

Animals of the experimental and control groups were scored according to the following scheme:

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

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

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

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

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

After an autopsy crops of organs (heart, lung, liver, spleen and blood were performed on meat-peptone and blood agars in Petri dishes method prints. Crops were incubated in a thermostat at a temperature of (37±1)°C for 24 hours. The results were considered according to the presence/absence of colonies on the surface of a nutrient medium in a Petri dish.

As a result of the research showed the following. During the experiment, is not registered with the death of the animals. Not noted any changes in their appearance and behavior. When macroscopic assessment of internal organs during all periods of conducting autopsies pathological changes were found.

Microbiological analysis showed that the internal organs and blood were sterile in experimental and control options in all periods of research.

Based on the lack of mortality and any changes in their appearance and General behavior during the 60-day observation, the negative results of microbiological analyses of heart, lung, liver, spleen and blood, 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 antagoni the political activity of strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2

Obtained through breeding strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 are characterized by a high antagonistic activity against pathogenic and conditionally pathogenic microorganisms.

Antagonistic activity against the test cultures was checked by the method of deferred antagonism. As the test strains used Staphylococcus aureus, Candida albicans, Klebsiella pneumoniae, Shigella flexneri, Shigella sonnei, Yersinia pseudotuberculosis, Serratia marcescens, Escherichia coli, Enterococcus spp.

Used test strains meet the following criteria: were S-shaped, had a typical morphological and enzymatic properties.

Study the antagonistic activity of the inventive strains of Bacillus licheniformis produced on meat-peptone agar.

Recipe meat-peptone agar 1 DM3:

1. peptone meat enzymatic - 10.0 g;

2. yeast extract 5.0 g;

3. sodium chloride 5.0 g;

4. the microbiological agar - 15.0 g;

5. distilled water to a total volume of 1 DM3.

After mixing, the medium was heated to dissolve the agar and filtered through a cotton-gauze filter flask with a capacity of 500 cm3300 cm3. Bulb with medium covered with a cotton-gauze plugs, was wrapped around the neck of parchment and sterilized in an autoclave at a pressure of 0.15 MPa for 40 minutes After the OST is for up to temperature (45±1)°C the medium was poured into sterile Petri dishes. For control of sterility cups dried medium was placed in a thermostat at a temperature of (37±1)°C for (24±2) hours. Non-sterile Cup was otbrakovyval.

Culture of pathogenic microorganisms mentioned above, were grown on Petri dishes for (18±2) hours on meat-peptone agar. In a sterile test tube was added 1 ml of saline and prepared suspensions of test strains with the concentration of microbial cells (5,0±1,0)×108CFU/cm3.

For testing from each batch were collected 3 samples 1, Further 0.5 g of the drug was dissolved in 0.5 cm3. The obtained suspension was sown by the method of strokes using a microbiological loop diameter Petri dishes with meat-peptone agar. Crops were incubated in a thermostat at a temperature of (37±1)°C for (48±2) hours. Then grown in the form of bar culture was podseval using a microbiological loop suspension of pathogens by the method of the perpendicular strokes.

Analysis was performed after 8 hours incubation at (37±1)°With the largest zones of inhibition of growth of test strains. Control of crop pathogens served their parallel plating on Petri dishes with the same dense environment (meat-peptone agar), but without the study Association of the antagonist. As a result of the research found that a strain of Bacillus licheniformis IC-831-1-2 characterized by the following what ukazatelyami zones of growth inhibition (mm) test strains of pathogens (table 1).

Table 1.
Characteristics of the strain B. licheniformis IC-831-1-2 in terms of zones of growth inhibition of the test strains of pathogens
Name of test strainZone size (mm)
Staphylococcus aureus strain IC-1161-4-110
Staphylococcus aureus strain IC-1189-4-112
Staphylococcus aureus IC-1126-4-113
Staphylococcus aureus IC-1142-4-115
Staphylococcus aureus IC-1192-4-112
Staphylococcus aureus IC-1200-4-18
Staphylococcus aureus IC-1246-4-110
Staphylococcus aureus IC-1120-4-112
Staphylococcus aureus IC-1128-4-112
Staphylococcus aureus IC-1257-4-112
Staphylococcus aureus IC-1249-4-113
Staphylococcus aureus IC-1233-4-120
Staphylococcus aureus IC-1149-4-115
Staphylococcus aureus IC-1241-4-130
Staphylococcus aureus IC-1251-4-123
Staphylococcus aureus IC-1226-4-120
Staphylococcus aureus IC-1230-4-112
Staphylococcus aureus IC-1140-4-122
Staphylococcus aureus IC-1222-4-115
Staphylococcus aureus IC-1165-4-118
Staphylococcus aureus IC-1205-4-117
Staphylococcus haemoliticus IC-1256-4-220
Staphylococcus aureus IC-1232-4-114
Staphylococcus aureus IC-1167-4-112
Staphylococcus aureus IC-1131-4-17
Staphylococcus aureus IC-1259-4-17
Staphylococcus aureus IC-1243-4-17
Staphylococcus aureus IC-1274-4-130
Staphylococcus aureus IC-1231-4-112
Staphylococcus aureus IC-1233-4-115
Staphylococcus aureus IC-1234-4-110
Staphylococcus haemoliticus IC-1258-4-28
Candida albicans strain IC-1124-9-110
Candida albicans strain IC-1172-9-110
Candida albicans strain IC-1152-9-112
Candida albicans strain IC-1155-9-111
Candida albicans strain IC-1143-9-110
Candida albicans strain IC-1141-9-113
Candida albicans strain IC-1134-9-130

Continuation of table 1
Candida albicans IC-53-9-112
Candida albicans IC-1147-9-110
Candida albicans IC-1150-9-111
Candida albicans IC-1203-9-111
Candida albicans IC-1195-9-1 10
Candida krusei IC-1244-9-215
Candida albicans IC-1144-9-115
Candida albicans IC-1132-9-110
Candida albicans IC-1130-9-120
Candida albicans IC-1156-9-130
Candida albicans IC-1122-9-130
Candida albicans IC-26-9-110
Candida albicans IC-1150-9-130
Candida albicans IC-55-9-111
Candida albicans IC-1119-9-19
Candida albicans IC-1240-9-110
Candida albicans IC-1166-9-110
Candida albicans IC-1137-9-114
Candida albicans IC-1186-9-110
Klebsiella pneumoniae strain IC-27-10-16
Shigella flexneri IC-815-14-27
Shigella sonnei IC-47-14-15
Yersinia pseudotuberculosis strain IC-45-
13-18
Serratia marcescens IC-1170-1712
Escherichia coli IC-1184-5-110
Escherichia coli IC-1273-5-18
Escherichia coli IC-1429-5-13
Escherichia coli IC-1427-5-130
Escherichia coli IC-1151-5-17
Enterococcus spp. IC-1236-1615
Enterococcus faecium IC-1253-16-130
Enterococcus spp. IC-1163-1615
Enterococcus spp. IC-1164-1630
Enterococcus spp. IC-1423-1610
Enterococcus spp. IC-1168-1610
Enterococcus spp. IC-1175-1612

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

Table 2.
Characteristics of the strain B. licheniformis IC-832-1-2 in terms of zones of growth inhibition of the test strains of pathogens
Name of test strainZone size (mm)
Candida albicans strain IC-1124-9-130
Candida albicans strain IC-1172-9-130
Candida albicans strain IC-53-9-130
Candida albicans strain IC-1147-9-130
Candida albicans strain IC-1150-9-130
Candida albicans strain IC-1203-9-120
Candida albicans strain IC-1136-9-130
Candida albicans strain IC-1195-9-120
Candida krusei strain IC-1244-9-218
Candida albicans strain IC-1144-9-120
Candida albicans strain IC-1132-9-18
Candida albicans strain IC-1130-9-115
Candida albicans strain IC-1156-9-1 30
Candida albicans strain IC-1122-9-130
Candida albicans strain IC-26-9-18
Candida albicans strain IC-1150-9-130
Candida albicans strain IC-55-9-110
Candida albicans strain IC-1119-9-115
Candida albicans strain IC-1155-9-130
Candida albicans strain IC-1186-9-110
Candida albicans strain IC-1143-9-130
Candida albicans strain IC-1141-9-130
Candida albicans strain IC-1134-9-130
Pseudomonas aeruginosa strain IC-1210-7-112
Shigella flexneri strain IC-815-14-27
Yersinia pseudotuberculosis strain IC-45-13-15
Escherichia coli strain IC-1427-5-130
Enterococcus faecium strain IC 1253-16-130
Enterococcus spp. strain IC-1164-16Enterococcus spp. strain IC-1236-160
Staphylococcus aureus strain IC-1126-4-124
Staphylococcus aureus strain IC-1161-4-18
Staphylococcus aureus strain IC-1189-4-116
Staphylococcus aureus strain IC-1142-4-125
Staphylococcus aureus strain IC-1192-4-123
Staphylococcus aureus strain IC-1200-4-120
Staphylococcus aureus strain IC-1246-4-120
Staphylococcus aureus strain IC-1120-4-122
Staphylococcus aureus strain IC-1128-4-124
Staphylococcus aureus strain IC-1257-4-120
Staphylococcus aureus strain IC-1149-4-112

Continued table 2
Staphylococcus aureus strain IC-1251-4-110
Staphylococcus aureus strain IC-1226-4-1
Staphylococcus aureus strain IC-1140-4-111
Staphylococcus aureus strain IC-1165-4-115
Staphylococcus aureus strain IC-1205-4-112
Staphylococcus haemoliticus strain IC-1256-4-215
Staphylococcus aureus strain IC-1241-4-130
Staphylococcus aureus strain IC-1230-4-110
Staphylococcus aureus strain IC-1232-4-111
Staphylococcus aureus strain IC-1167-4-130

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

Table 3.
Characteristics of the strain B. licheniformis IC-833-1-2 in terms of zones of growth inhibition of the test strains of pathogens
Name of test strainZone size (mm)
Candida albicans strain IC-1124-9-130
Candida albicans strain IC-1172-9-1 30
Candida albicans strain IC-1143-9-130
Candida albicans strain IC-53-9-130
Candida albicans strain IC-1147-9-130
Candida albicans strain IC-1150-9-130
Candida albicans strain IC-1203-9-130
Candida albicans strain IC-1195-9-18
Candida krusei strain IC-1244-9-26
Candida albicans strain IC-1144-9-110
Candida albicans strain IC-1132-9-110
Candida albicans strain IC-1130-9-120
Candida albicans strain IC-1156-9-130
Candida albicans strain IC-1133-9-18
Candida albicans strain IC-1122-9-130
Candida albicans strain IC-26-9-110
Candida albicans strain IC-1150-9-130
Candida albicans strain IC-55-9-130
Candida albicans strain IC-1119-9-130
Candida albicans strain IC-1139-9-130
Candida albicans strain IC-1141-9-130
Candida albicans strain IC-1240-9-110
Candida albicans strain IC-1152-9-130
Candida albicans strain IC-1155-9-130
Klebsiella pneumoniae strain IC-43-10-16

Continuation of table 3
Klebsiella pneumoniae strain IC-31-10-19
Shigella flexneri strain IC-815-14-212
Shigella sonnei strain IC-1216-14-130
Serratia marcescens strain IC-817-17-130
Yersinia pseudotuberculosis strain IC-45-13-130
Staphylococcus aureus strain IC 25-4-17
Staphylococcus aureus strain IC-1161-4-126
Staphylococcus aureus strain IC-1189-4-115
Staphylococcus aureus strain IC-1126-4-118
Staphylococcus aureus strain IC-1142-4-116
Staphylococcus aureus strain IC-1192-4-120
Staphylococcus aureus strain IC-1200-4-120
Staphylococcus aureus strain IC-1246-4-116
Staphylococcus aureus strain IC-1233-4-130
Staphylococcus aureus strain IC-1120-4-115
Staphylococcus aureus strain IC-1140-4-118
Staphylococcus aureus strain IC-1241-4-130
Staphylococcus aureus strain IC-1232-4-120
Staphylococcus aureus strain IC-1167-4-130
Staphylococcus aureus strain IC-1274-4-130
Staphylococcus aureus strain IC-1231-4-17
Staphylococcus aureus strain IC-1234-4-18
Staphylococcus haemoliticus strain IC-1258-4-2 5
Staphylococcus aureus strain IC-1230-4-112
Staphylococcus aureus strain IC-1222-4-110
Staphylococcus aureus strain IC-1165-4-120
Staphylococcus aureus strain IC-1205-4-124
Staphylococcus haemoliticus strain IC-1256-4-29
Staphylococcus aureus strain IC-1128-4-117
Staphylococcus aureus strain IC-1251-4-118
Staphylococcus aureus strain IC-1226-4-120
Staphylococcus aureus strain IC-1249-4-120
Staphylococcus aureus strain IC-1257-4-116
Escherichia coli strain IC-1153-5-18
Escherichia coli strain IC-34-5-15
Escherichia coli strain IC-1148-5-17
Escherichia coli strain IC 36-5-16
Escherichia coli strain IC-1427-5-120
Escherichia coli strain IC-1151-5-1 6
Enterococcus spp. strain IC-1236-1616
Enterococcus faecium strain IC 1253-16-130
Enterococcus spp. strain IC-1164-1630
Enterococcus spp. strain IC-1423-168
Enterococcus spp. strain IC-1168-168
Enterococcus spp. strain IC-1175-1610

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

td align="center"> 16
Table 4.
Characteristics of the strain B. licheniformis IC-834-1-2 in terms of zones of growth inhibition of the test strains of pathogens
Name of test strainZone size (mm)
Staphylococcus aureus strain IC-1161-4-114
Candida albicans strain IC-1124-9-118
Candida albicans strain IC-1172-9-19
Candida albicans strain IC-1152-9-1 12
Candida albicans strain IC-1155-9-114
Candida albicans strain IC-1143-9-112
Candida albicans strain IC-1141-9-115
Candida albicans strain IC-1134-9-130
Escherichia coli strain IC-1250-5-15
Shigella flexneri strain IC-815-14-29
Candida albicans strain IC-53-9-112
Candida albicans strain IC-1147-9-130
Candida albicans strain IC-1150-9-130
Candida albicans strain IC-1203-9-115
Salmonella enteritidis strain IC-1159-6-15
Candida albicans strain IC-1136-9-17
Candida albicans strain IC-1195-9-110
Candida krusei strain IC-1244-9-214
Candida albicans strain IC-1144-9-115
Candida albicans strain IC-1132-9-11
Candida albicans strain IC-1130-9-113
Candida albicans strain IC-1156-9-130
Candida albicans strain IC-1122-9-130
Candida albicans strain IC-1150-9-130
Candida albicans strain IC-55-9-130
Candida albicans strain IC-1119-9-130
Staphylococcus aureus strain IC-1189-4-120
Serratia marcescens strain IC-817-17-116
Yersinia pseudotuberculosis strain IC-45-13-116
Staphylococcus aureus strain IC-1126-4-117
Staphylococcus aureus strain IC-1142-4-117
Staphylococcus aureus strain IC-1192-4-115
Staphylococcus aureus strain IC-1200-4-115
Staphylococcus aureus strain IC-1246-4-115
Staphylococcus aureus strain IC-1120-4-116
Staphylococcus aureus strain IC-1128-4-1
Staphylococcus aureus strain IC-1257-4-115
Escherichia coli strain IC-1153-5-110

12
Continuation of table 4
Escherichia coli strain IC-1184-5-15
Escherichia coli strain IC-1273-5-15
Escherichia coli strain IC-1193-5-15
Escherichia coli strain IC-1116-5-15
Escherichia coli strain IC-1424-5-15
Escherichia coli strain IC-1238-5-15
Escherichia coli strain IC 36-5-17
Enterococcus spp. strain IC-1163-168
Staphylococcus aureus strain IC-1249-4-119
Enterococcus spp. strain IC-1236-1610
Staphylococcus aureus strain IC-1233-4-110
Staphylococcus aureus strain IC-1149-4-18
Escherichia coli strain IC-1430-5-17
Staphylococcus aureus strain IC-1251-4-110
Staphylococcus aureus strain IC-1226-4-110
Escherichia coli strain IC-1427-5-130
Enterococcus faecium strain IC 1253-16-110
Staphylococcus aureus strain IC-1140-4-120
Staphylococcus aureus strain IC-1222-4-118
Staphylococcus aureus strain IC-1165-4-120
Staphylococcus aureus strain IC-1205-4-118
Staphylococcus aureus strain IC-1256-4-220
Candida albicans strain IC-1133-9-18
Staphylococcus aureus strain IC-1241-4-110
Staphylococcus aureus strain IC-1230-4-130
Enterococcus spp. strain IC-1164-1630
Staphylococcus aureus strain IC-1232-4-120
Staphylococcus aureus strain IC-1167-4-122
Staphylococcus aureus strain IC-1131-4-110
Staphylococcus aureus strain IC-1259-4-110
Staphylococcus aureus strain IC-1243-4-110
Staphylococcus aureus strain IC-1179-4-19
Staphylococcus aureus strain IC-1125-4-18
Staphylococcus aureus strain IC-1274-4-130
Staphylococcus aureus strain IC-1231-4-120
Candida albicans strain IC-1154-9-122
Staphylococcus aureus strain IC-1229-4-18
Candida albicans strain IC-1202-9-16
Staphylococcus aureus strain IC-1233-4-18
Staphylococcus aureus strain IC-1234-4-110
Staphylococcus aureus strain IC-1258-4-28
Enterococcus app. strain IC-1423-1610
Enterococcus spp. strain IC-1168-1610
Enterococcus spp. strain IC-1175-16

Thus, the examined strains of bacteria are antagonistic activity against a wide spectrum of pathogenic and conditionally pathogenic microorganisms.

Example 3. Methods of culturing bacteria of Bacillus licheniformis to obtain microbial biomass

To obtain biomass dispute bacteria strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 cultivated in a liquid nutrient medium of the following composition:

1. peptone meat enzymatic - 10.0 g;

2. yeast extract 5.0 g;

3. sodium chloride 5.0 g;

4. distilled water to a total volume of 1 DM3.

pH (7,2±0,2).

1 DM3fermentation of a nutrient medium required 10 cm3the corresponding strain of Bacillus licheniformis with title 108CFU/cm3. The process of growing biomass was carried out in a fermenter for (48±2) hours. At the specified method of cultivation was awarded the title dispute is not higher (5-10)×1010CFU/cm3.

Example 4. Getting ready forms of the drug on the basis of bacterial strains B. licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2

First cook the mixture is collected and tested for quality raw biomass with filler. As filler can be used water (for processing plants spray application method) with a title dispute is not less than 1·103CFU/ml or powdered sorbent, e.g. the R zeolite. As the sorbent is also used powdered sugar and starch in the ratio of biomass, sugar and starch is 1:5:5, mix thoroughly. In the mixing process occurs immobilization of bacteria spores on the starch particles. Later in the mixture injected sugar or glucose powder at the rate of 1 part of a mixture of 10 parts of powder. The result is a concentrate of the drug with a title dispute is not less than 1×1010CFU/g Then it is mixed with sugar or glucose powder and starch in the same proportion, to obtain a finished product with a title dispute is not less than 1·103-106CFU/g

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

Example 5. The compounds of preparations

5.1. Composition 1.

An aqueous solution of biomass spores of the bacteria Bacillus licheniformis IC-831-1-2 with a title dispute is not less than 1·103CFU/ml of This solution was stored in a dark place for no more than 1-2 days.

5.2. Part 2.

The mixture of biomass spores of the bacteria Bacillus licheniformis IC-831-1-2 with the zeolite with a title dispute is not less than 1·106CFU/g

5.3. Part 3.

The mixture of biomass spores of the bacteria Bacillus licheniformis IC-831-1-2 with a title dispute is not less than 1·106CFU/g with a mixture of starch and powder on the basis of sucrose in the ratio of 1:100.

5.4. Part 4.

The mixture of biomass spores of the bacteria Bacillus licheniformis IC-832-1-2 with a title dispute is not less than 1·106CFU/g with a mixture of starch and powder on the basis of sucrose in the ratio of 1:10.

5.5. Part 5.

The mixture of biomass spores of the bacteria Bacillus licheniformis IC-833-1-2 with a title dispute is not less than 1·106CFU/g with a mixture of starch and powder on the basis of sucrose in the ratio of 1:50.

5.6. Part 6.

The mixture of biomass spores of the bacteria Bacillus licheniformis IC-834-1-2 with a title dispute is not less than 1·106CFU/g with a mixture of starch and powder on the basis of sucrose in the ratio of 1:10.

5.7. Part 7.

The mixture of biomass spores of the bacteria Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2, IC-834-1-2 in the ratio of 1:1:1:1 with a title dispute each type of bacteria is not less than 1·103CFU/g with a mixture of starch and powder-based glucose in the ratio 1:20.

Example 6. Data storage the dry form of the drug on the basis of strains of 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 was carried out relevant studies.

Within 48 months of storage the dry form of the drug at the temperature of (30±0,5)°With the title and antagonistic activity of the drug has not changed, which confirms the high stability of drugs in spore form, which provides a significant simplification of their application in veterinary medicine and agriculture.

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.

For testing strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 about the ability in order to eurovet pathogenic fungi used method agar blocks. The suspension of the drug with a titer of 1×106CFU/cm3made in meat-peptone agar, cooled to a temperature (36±1)°C. For cultivation of pathogenic fungi used:

environment of čapek for Didymella applanata;

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

Inoculated studied strain of the medium was poured into Petri dishes and on the frozen surface, in the centre was placed a block of 10 mm diameter, cut from the colony of phytopathogen.

Analysis was performed with a frequency of 1 every 3 days. The activity of the strain was taken into account by changing the diameter of the colony of the fungus in comparison with control (medium without any strain). On the basis of the obtained data was determined inhibitory activity, which was calculated by the formula

,

where

Dtothe diameter of colonies in the control variant,

Daboutthe diameter of the colony in a test version.

The studies found the following indicators of inhibitory activity of the strain Bacillus licheniformis IC-831-1-2 on the 7th day:

against Didymella applanata of 12.7%;

against Fusarium moniliformes - 62%;

against Fusarium oxysporum - 70%;

against Fusarium sporotrichiella - 19,2%;

with respect to Alterneria alternata to 10.5%.

The studies found the following indicators inhibitory activity of strains of Bacillus lichenformis IC-832-1-2 on the 7th day:

against Didymella applanata and 17.2%;

against Botrytis cinerea - 21%;

against Fusarium moniliformes - 43%;

against Fusarium sporotrichiella of 17.2%;

with respect to Alterneria alternata 38.7 percent.

The studies found the following indicators of inhibitory activity of the strain Bacillus licheniformis IC-833-1-2 on the 7th day:

against Botrytis cinerea - 38%;

against Fusarium oxysporum - 62%;

against Fusarium moniliformes - 12%;

with respect to Alterneria alternata - 42%.

The studies found the following indicators of inhibitory activity of the strain Bacillus licheniformis IC-834-1-2 on the 7th day:

against Didymella applanata - 67%;

against Botrytis cinerea - 67%;

against Fusarium moniliformes - 44%;

against Fusarium oxysporum - 67%;

against Fusarium sporotrichiella - 36%;

with respect to Alterneria alternata - 11%.

Thus, the inventive strains of Bacillus licheniformis show antagonistic activity against the above fungal phytopathogens.

Example 8. Data of field trials to study the effects of drugs on the basis of strains of Bacillus licheniformis IC-831-1-2, IC-832-1-2, IC-833-1-2 and IC-834-1-2 on the incidence of raspberry purple blotch and currant neighbour

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 antifungal activity against diseases of raspberries and currants in the open ground used for industrial plantations mA the ins varieties Zorenka Altai and currant varieties Sophia. As a chemical standard used drug Topaz in a concentration of 0.1%. Processing of data was performed with the suspension of preparations with a titer of 106spores/ml.

Processing landings were conducted in July and June. The processing time was determined when the first symptoms of disease.

Diagram of field experience:

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

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

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

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

Topaz 0,1%;

control.

Experiment was repeated four times, square plots of 10 m2the fluid flow - 4 DM3on option. Placement of randomized plots. Schemes on raspberry and currant similar.

The defeat leaves currant neighbour was taken into account according to the following scale:

0 - points - healthy leaves;

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

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

3 points - severe disease (busy spots from 26 to 50% of the leaf blade surface);

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

The degree of damage to the raspberry canes were assessed by a special 4-point scale for purple spot raspberry:

0 points - healthy stalk;

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

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

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

4 points - the surface of the affected area is deformed, the plot runs around the stem.

Disease progression was determined by the following formula:

where

R is the index of the disease, %;

a number of plants relevant score, PCs;

b - point scale;

N is the total number of plant units;

K - highest score scale used.

Prevalence was calculated by the formula

where

R is the index of the prevalence of the disease, %;

and the number of plants in the control units;

b - the number of plants in the experiment, pieces

Biological efficacy of these preparations was determined by the formula

where

a - the average prevalence in the control, %;

b - the average infestation in the treated area, %.

The results of field experiments are presented in tables.

Table 5.
The influence of the proposed products on the basis of strains of Bacillus licheniformis to defeat raspberry purple spots.
OptionPrevalence, %Development %Biological efficiency, %
Control75,025,0-
The strain B. lich. IC 831-1-234,18,566,0
The strain B. lich. IC-832-1-243,210,856,8
The strain B. lich. IC 833-1-240,8to 12.061,4
The strain B. lich. IC 834-1-237,59,062,7
Topaz 0,1%38,610,856,8
NDS054,0

Table 6.
The influence of the proposed products on the basis of strains of Bacillus licheniformis to defeat raspberry purple spots.
OptionPrevalence, %Development %Biological efficiency, %
Control95,035,0-
The strain B. lich. IC 831-1-232,58,176,9
The strain B. lich. IC-832-1-235,0the 15.655,4
The strain B. lich. IC 833-1-237,013,059,5
The strain B. lich. 1C 834-1-244,011,275,9
Topaz 0,1%50,012,564,3
NDS057,0

Table 7.
The influence of the proposed products on the basis of strains of Bacillus licheniformis to defeat currant neighbour.
OptionBiological efficiency, %
Time records
June 27July 4July 11
B. licheniformis IC 831-1-280,661,3of 54.8

B. stearothermophilus IC-832-1-482,359,7of 54.8
Century stearothermophilus IC 833-1-479,552,059,1
Century stearothermophilus IC 834-1-476,665,053,1
Topaz 0,1%64,556,556,5
Note 1.(*)biological effectivenessaccutane the gains of the disease.
Note 2. Systemic fungicidal and insecticidal drug Topaz effectively treats berry bushes and cucumbers from powdery mildew and flower plants from rust. It is valid for 10 days and not washed off by the rain.

Thus, the processing of raspberry plantations drugs on the basis of strain Century licheniformis IC 831-1-2 in the field experiment were reduced: the development of the disease is almost 3 times the prevalence is more than 2 times. The yield increase was 0.3 t/ha Treatment plants currant strain Century licheniformis IC 831-1-2 in a field experience reduced disease development in 2.2 times. Biological efficiency was (54,8-80,6) % depending on the duration of the account.

Processing of raspberry plantations drug-based strain Century stearothermophilus IC 832-1-4 in the field experiment were reduced: the development of the disease is almost 3 times the prevalence is more than 2 times. The yield increase was 0.3 t/ha Treatment plants currant strain Century stearothermophilus IC 832-1-4 in a field experience reduced disease development in 2.2 times. Biological efficiency was (54,8-82,3) % depending on the duration of the account.

Processing of raspberry plantations drug-based strain Century stearothermophilus IC 833-1-4 in the field experiment were reduced: the development of the disease is almost 3 times the prevalence is more than 2 times. Increase productivity which was 0.3 t/ha Processing plants currant strain Century stearothermophilus IC 833-1-4 in a field experience reduced disease development in 2.2 times. Biological efficiency was (52,0-of 79.5) % depending on the duration of the account.

Processing of raspberry plantations drug-based strain Century stearothermophilus IC 834-1-4 in the field experiment were reduced: the development of the disease is almost 3 times the prevalence is more than 2 times. The yield increase was 0.3 t/ha Treatment plants currant strain Century stearothermophilus IC 834-1-4 in a field experience reduced disease development in 2.2 times. Biological efficiency was (53,1-76,6) % depending on the duration of the account.

Example 9. Studies of the claimed preparation in the form of microbiological fertilizer

In the following table 8 summarizes the research of the inventive preparations in the form of microbial fertilizers on cultivar of spring wheat Bagansky - 95.

The seeds were processed on : machine PS-10, with normal flow of the working solution of 10 litres per tonne of seed. The consumption of drugs was 10 ml per tonne. The titer of the preparation - 1×108CFU/ml Feeding was carried out by spraying boom sprayer with fluid flow 50 l/ha (consumption of drugs 10 ml/ha).

In the field testing of the drug Fitap and experimental drug based on the strain Century stearothermophilus IC-834-1-4 installed led the group of content groups of microorganisms - indicators of soil fertility: the enrichment of microbial cenosis soil microorganisms of different ecological-trophic groups (total bacterial count), the increase in 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 during application of the bacterial preparation and security elements of mineral nutrition and is typical for soils with intensive processes of nitrification. Increased the number of true denitrification and ammonification microorganisms, as well as the number of autochthonous microflora, which carries out the destruction of humus compounds. The humification index to the harvesting significantly increased in the variant with making Filipa by 19.5% in the variant with application of the drug on the basis of strain Century stearothermophilus IC-834-1-4 16.6% compared to control.

Thus, the drug-based strain Century stearothermophilus IC-834-1-4 not only positively affect the dynamics of changes in the quantitative composition of soil microorganisms, but is more effective compared to its counterpart - drug Fitop based on strain B. subtilis VKPM B-7036.

Table 8.
The study drugs in the form of the microbe is ideological fertilizer.
Version of experienceExemplary group of soil microorganisms, CFU/gDeviation from control
1Total bacterial count
1.1Controlof 98.2×105
1.2Treatment of seeds strain163×10564,8
Bacillus stearothermophilus
IC-834-1-4
1.3Seed treatment medication132,1×10533,9
Fitop
1.4Control40,13×105
1.5Feeding vegetating86×10545,87
plant strain Century
stearothermophilus
IC-834-1-4
1.6Feeding vegetating32,07×105of 8.06
plant drug Fitop
2Bacteria of the genus Bacillus
2.1Control11,3×104
2.2Treatment of seeds strain17,7×1046,4
Bacillus stearothermophilus
IC-834-1-4
2.3Seed treatment medication12,5×1041,2
Fitop
2.4Control13,5×104
2.5Feeding vegetating23,1×1049,6
plant strain Century
stearothermophilus
IC-834-1-4
2.6Feeding vegetating15,1×1041,6
plant drug Fitop
2.7controlof 14.3×104
2.8Seed treatment and fertilizer21,3×1047
vegetative plants
strain Century stearothermophilus
IC-834-1-4
2.9Seed treatment and fertilizerof 16.7×1042,4
vegetative plants

0,9×104
drug Fitop
3the stienen denitrification
3.1Control33,5×105
3.2Treatment of seeds strain
Bacillus stearothermophilus
IC-834-1-455,2×10521,7
3.3Seed treatment medication
Fitop34,9×1051,4
3.4Control36,6×105
3.5Seed treatment and fertilizer
vegetative plants
strain Century stearothermophilus
IC-834-1-4to 58.1×105a 21.5
3.6Seed treatment and fertilizer27,3×1059,3
vegetative plants
drug Fitop
4Nitrogen-fixing (ammonification) microorganisms
4.1Control37,5×105
4.2Treatment of seeds strain
Bacillus stearothermophilus
IC-834-1-433,6×105
4.3Seed treatment medication
Fitop56.7 x 10519,2
5Autochthonous microflora
5.1Controlof 3.8×104
5.2Treatment of seeds strain
Bacillus stearothermophilus
IC-834-1-4of 2.3×104of-1.5
5.3Seed treatment medication
Fitop6,3×1042,5
5.4Control
5.5Feeding vegetating0,5×104
plant strain Century
stearothermophilus
IC-834-1-4
5.6Feeding vegetating
plant drug Fitopof 2.3×1041,4
5.7Control3,9×104
5.8Seed treatment and fertilizer
vegetative plants
drug Fitop0,8×104-3,1
5.9Seed treatment and fertilizer
vegetative plants
strain Century stearothermophilus
IC-834-1-4of 5.4×1041,5

Example 10. Data recovery microflora of the gastrointestinal tract of animals with the use of the proposed drug

Research concerned with identifying the dynamics of changes in the quantitative composition of the exemplary groups of the intestinal microflora during the whole breeding season, strains of animals. The material served as:

1. Healthy outbred mice of both sexes from nursery fsri SRC VB "Vector". Animals were kept at a temperature of (25±1)°C and 12-hour lighting. As the feed used pellets for feeding laboratory rats and mice. Animals were divided into following groups:

- Group1 - control;

- group 2 - experience produced the drug Council 3 (active principle of a strain of Bacillus subtilis VKPM In 7048);

- group 3 - experience, an experimental model of drug Council 13.1 (active principle of a strain of Bacillus stearothermophilus IC 831-1-4);

- group 4 - experience, an experimental model of drug Council 14.1 (active principle of a strain of Bacillus stearothermophilus IC 832-1-4);

- group 5 - experience, an experimental model of drug Council 15.1 (active principle of a strain of Bacillus stearothermophilus IC 833-1-4);

group 6 - experience, an experimental model of drug Council 16.1 (active principle of a strain of Bacillus stearothermophilus IC 834-1-4).

Each group contained 5 animals.

2. In experiments were used drugs in the culture fluid used strains titer of at least 1×108CFU/ml Preparations of the experimental animals were drinking according to the following scheme:

- 0 day - water;

- 1 - day 10 - appropriate variants of experiment preparations, diluted with tap water to the calculated titer not less than 1x106CFU/ml Drinkers with drugs were replaced daily.

- 11 - 50 day is water.

1. For quantitative estimation of the dynamics of the selection of strains used the method of serial dilutions of faeces of animals with subsequent seeding of 100 ál of pre-warmed at 100°C for 10 minutes suspension from each dilution on meat-peptone agar in net assets is key Petri.

Table 9.
Data recovery microflora of the digestive tract of animals
Date, strainE. coliEnterococcusLactobacillusPathogenic, including ProteusBifidobacterium
124567
0 day
Control7×1041×1043×106103108
70482,8×1051×1045,3×107103108
IC-831-1-45×1051×1047×106103 108
IC-832-1-42×1051×1047×106103108
IC-833-1-41×1051×1041×106103108
IC-834-1-43×1051×104of 2.2×106103108
day 10
Control5×105of 1.1×104of 3.4×107104108
7048of 2.2×1071×1053×107103108
IC-831-1-4of 2.7×1063×105 of 3.8×106103108
IC-832-1-42×1072×1065×107103108
IC-833-1-4of 2.6×1081×105of 2.2×108103109
IC-834-1-41,8×1073.7 x 1062×107103109
20 day
Control1×1051×1041×105104108
70484×1071×1041×106104109
IC-831-1-4 1×107of 1.2×1073×108102109
IC-832-1-41×1071×1066×108103109
IC-833-1-42×1085×1071×108102109
IC-834-1-42×1081×1071×109102109
30 day
Control3×104of 1.2×1041,0×106104107
7048of 7.7×1075×107of 1.5×108104 109
IC-831-1-45×107of 4.6×107of 2.6×108102109
IC-832-1-4of 3.6×108of 5.1×1073,9×1091021010
IC-833-1-45×1088×1086×1081021010
IC-834-1-4of 2.0×108of 4.6×108of 2.0×1091021010
40 day
Control1,0×1056×1043×105104108
70482,1×1073×107 1×107104109
IC-831-1-47,5×1072×107of 1.3×109102109
IC-832-1-49,5×1091×1082×10101021010
IC-833-1-46,3×1083×1096,2×1091021010
IC-834-1-41×1091×1095×1091021010
50 day

Control2×1052.4 x 1046×106104108/sup>
70486×107of 2.0×1072×108104109
IC-831-1-41×1082×108of 1.5×109102109
IC-832-1-4of 1.4×1091,0×1083×10101021010
IC-833-1-4of 2.0×1083,9×10101,0×101001011
IC-834-1-41×1093×109of 2.3×101001011

2. For quantitative estimation of dynamics of changes of the intestinal microflora daily produced sowing from the feces of mice in all experimental groups according to the standard scheme of examination for disback Eris, including:

- define a group of bacteria Escherichia coli (bgcp) with normal enzymatic activity on the environment Endo and environment Levin;

- enterococci on polymyxinb agar;

- Lactobacillus on the environment Blichfeldt;

- conditionally pathogenic microorganisms in the environment Ploskirev and yolk-salt agar;

- bifidobacteria on thioglycolic environment.

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

Indications for use (recommendation). Preparations on the basis of strains of Bacillus stearothermophilus IC-831-1-4, IC-833-1-4, IC-834-1-4 recommended for the prevention and treatment of dysbacteriosis agricultural and wild birds, including chickens, ducks, geese, turkeys, Guinea fowls and other, increasing the natural resistance of the organism of birds, eliminating immunodeficiency caused by infectious (viruses, bacteria, protozoa, intracellular) and noncommunicable (low-quality forage, kuchenne content, the stress resulting from temperature regimes content) factors that increase the livability, increase egg production, obtaining additional is sustained fashion gain of broilers, reduction of feed conversion, reduce time poultry production. Prevention and treatment of dysbacteriosis of all types of productive farm animals, treatment of infectious diseases bacterial and protozoal etiology, prevention of viral diseases and immunodeficiencies, to increase the security of youngsters in the early periods of development, improved productivity, more gain.

Preparations on the basis of strains of Bacillus stearothermophilus IC-832-1-4 recommended for the prevention and treatment of dysbacteriosis cattle, treatment of infectious diseases bacterial and protozoal etiology, prevention of viral diseases and immunodeficiency (primary and secondary) States, increasing the safety of young cattle in the early periods of development, improve productivity of dairy cattle, more gain of young beef cattle, improve the quality and grading of milk and meat, renewing the fertility of breeding stock.

1. The bacterial strain Bacillus licheniformis IC-831-1-2 with
bactericidal and fungicidal activity and deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10561.

2. The bacterial strain Bacillus licheniformis IC-832-1-2 with bacteria is IGNOU and fungicidal activity and deposited in Russian national 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 Russian national 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 Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10564.

5. The drug with bactericidal and fungicidal activity, characterized by a content of the filler with the biomass of bacteria in spore form of Bacillus licheniformis In PMBC-10561, or VKPM B-10562, or VKPM B-10563, or VKPM B-10564, or their mixture with the title of each bacterial strain is not less than 1·103CFU/g or 1·103CFU/ml

6. The preparation according to claim 5, characterized in that the filler it contains water.

7. The preparation according to claim 5, characterized in that the filler it contains powdered sorbent.

8. The preparation according to claim 7, characterized in that as a powdered sorbent contains a zeolite, or a polysaccharide, or a monosaccharide, or a disaccharide, or a mixture of polysaccharide with a monosaccharide or polysaccharide in a ratio of from 1:100 to 1:10.

9. The preparation of claim 8, characterized in that it contains polysaccharide starch, as monosaccharide - glucose is, as the disaccharide is sucrose.



 

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2 tbl, 2 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology and a Cupriavidus eutrophus VKPM B-10646 bacteria strain which produces polyhydroxy alkanoates (PHA) and a method of producing said strain. The strain is isolated from Ralstonia eutropha VKPM B-8562 during long multi-step selection based on effectiveness of synthesis of multicomponent PHA. PHA is obtained by culturing the strain in conditions of aeration and mixing on a liquid salt medium with limited nitrogen. The medium contains a growing substrate with an additional carbon source. The growing substrate used is glucose or fructose or 3-butyric acid or a gaseous mixture - hydrogen, oxygen and carbon dioxide, or synthetic gas mixed with oxygen. The additional carbon source used is a solution of potassium 3-valarate or solution of potassium 3-valerate and potassium 3-hexanoate, or solution of potassium 3-valerate, potassium 3-hexanoate and acrylate, or solution of potassium 3-valerate, or solution of potassium 3-hexanoate and acrylate, or solution of 3-butyric acid and 4-butyrolactone, or solution of 3-butyric acid, 4- butyrolactone and potassium 3-valerate, or solution of 3-butyric acid, 4- butyrolactone and potassium 3-hexanoate, or solution of 3-butyric acid, 4- butyrolactone, potassium 3-valerate and potassium 3-hexanoate.

EFFECT: invention enables to obtain polyhydroxy alkanoate producer with high output.

2 cl, 1 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: synthetic nutrient medium for growing microorganisms contains citric acid, asparagine, potassium phosphate twice substituted, zinc sulfate, magnesium sulfate, iron sulfate, sodium chloride, sodium phosphate twice-substituted, glycine, succinic acid, distilled water and if necessary agar with specified component ratio.

EFFECT: invention makes it possible to increase nutritional properties of synthetic nutrient media.

2 cl, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method involves preliminary selection of concentration of a disinfectant solution at which bacteria exhibit partial sensitivity to said disinfectant. The bacteria are exposed to the disinfectant solution at the given concentration. Grown colonies of bacteria are collected after exposure to said disinfectant and then sown on a solid culture medium, followed by culturing at 37°C. Bacteria colonies isolated from those grown at the previous step are selected in amount of 1-299 colony-forming units (CFU/ml). The collected isolated colonies are resown on a culture medium and grown at conditions needed for growth of bacteria of that type, followed by dividing the grown colonies into two parts. One part of the grown bacteria colonies is exposed to the disinfectant at the selected concentration, and the other part is exposed to the disinfectant at bactericidal concentration. If upon exposure to the disinfectant at bactericidal concentration, there is no growth of bacteria or their growth ranges from 1 to 299 (CFU/ml), then the cycle of selecting bacteria grown at the previous step, their resowing on the culture medium, growing colonies of said bacteria at conditions needed for growth of said type of bacteria and exposing the grown colonies to the disinfectant at the selected and bactericidal concentrations is repeated once more until after exposing the column to the disinfectant solution at bactericidal concentration, growth of bacteria on the culture medium is equal to 300 (CFU/ml), which indicates bacterial resistance to the disinfectant.

EFFECT: invention enables to simulate bacterial resistance to a disinfectant, evaluate build-up of resistance and bactericidal potential of hospital strains to disinfectants during their practical use.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method provides test inoculation of a nutrient medium containing pancreatic fish flour hydolyzate, fermentative meat peptone, NaCl, Tween-80, CaCl2, sodium thiosulphate (Na2S2O3 × 5H2O), ferrous ammonium sulphate ((NH4)2SO4 × FeSO4 × 6H2O), sorbite, bromthymol blue, irgasan (DP-300), rifampicin, NaOH, agar and distilled water in the preset proportions. Pancreatic fish flour hydolyzate, peptone, NaCl, agar are dissolved with heating, sterilised at 121°C for 20 min and thereafter added in a hot medium of the other components specified above. The inoculations are incubated on the nutrient medium in aerobic conditions at temperature 37°C and/or 28°C for 24-48 h and assessed by the presence of black-centre green or dark grey colonies surrounded by a cloudy precipitate zone in the nutrient medium.

EFFECT: invention allows simplifying and providing higher specificity of Shewanella bacteria recovery and identification.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, and concerns a lactic bacteria Lactobacillus reuteri DSM 17938 strain stimulating IL-10 production and hence CD4+CD25+TR-cell proliferation used for making a probiotic product. The probiotic product contains Lactobacillus reuteri DSM 17938 strain and additionally medium-chain triglyceride oil.

EFFECT: use of the probiotic product promoted a favorable effect on intestinal colic in a newborn baby.

3 cl, 3 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: inoculate contains in a mixture or in a combination, main L-cystein of formula HSCH2CH(NH2)CO2H and at least one B.animalis lactis strain. Said cysteine and at least one B.animalis lactis strain are contained or have the form of at least one frozen granule and/or at least one lyophilizate. The pH value of the solution produced by thawing of at least one granule and/or dissolution, of at least one lyophilizate in the relation making 1 to 2 g of the lyophilizate per 8-10 ml of H2O, makes at least 4. L-cysteine contains in the inoculate in an amount within 1 g per 1·1014 CFU to 1 g per 3.5·1010 CFU, of at least one B.animalis lactis strain or all used B.animalis lactis strains whereas the same are numerous. Using the offered inoculate provides stimulation of B.animalis lactis growth and/or metabolism on a lactic substratum to produce a fermented diary product.

EFFECT: high probiotic value of the product.

21 cl, 9 dwg, 3 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is offered in application of at least one strain of probiotic bacteria specified in Lactobacillus plantarum 299, DSM 6595, Lactobacillus plantarum 299v, DSM 9843, Lactobacillus plantarum HEAL 9, DSM 15312, Lactobacillus plantarum HEAL 19, DSM 15313, Lactobacillus plantarum HEAL 99, DSM 15316, Lactobacillus paracasei 8700:2, DSM 13434 and Lactobacillus paracasei 02A, DSM13432 for preparing a composition for treating and/or preventing a viral infection caused by 'cold' virus, and a related method for treating and/or preventing. A number of days for which 'cold' symptoms are experienced has been also reduced in a group of patients having been taking a probiotic.

EFFECT: what is shown is intensified immune protection against antiviral infection that promotes decreasing 'cold' episodes in comparison with the controls.

16 cl, 13 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a method of sublimation dehydration of high-disperse biologically active materials found in a microdrop state characterised by the fact that the microdrop powder is frozen at temperature -35°C to -45°C for 1.5 to 8 h, and then dehydrated.

EFFECT: invention provides reduced duration of the dehydration process of biologically active materials.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents a method for producing fine-grained biologically active materials containing active substances, characterised by the fact that the liquid with biologically active substances is dispersed to the microdrop state in a layer of dry fine-grained inert hydrophobic aerosil in the proportion 10:1.5 to 10:6, to form thereby liquid microdrops surrounded by hydrophobic aerosil particles and powdered which, if necessary, are dried by a technologically acceptable method.

EFFECT: invention provides increasing dispersion of biologically active materials.

11 ex, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a method of combination dehydratation of high-disperse biologically active materials containing active substances in a liquid phase, implemented in stages and differing by the fact that the liquid phase with the active substance in a microdrop state stabilised by a dry high-disperse hydrophobic disconnector in ratio 1:3-1:22, is dehydrated initially at atmospheric pressure and then by mixing with a high-absorbency sorbent with residual moisture less than 1% and dried up if needed.

EFFECT: invention provides higher activity of the active substances.

9 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutical industry and represents a preparation containing a biologically active substance, characterised by the fact that it contains a powder based on dried microdrops of the active substance stabilised by a dry high-disperse inert hydrophobic disconnector representing silicone dioxide with the components of the preparation being in the certain mass ratio per 1 g of the preparation.

EFFECT: invention provides higher dispersity of the preparation and preservation of the active substances in the preparation.

13 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely ophthalmology, and concerns refractive error correction in nystagmus. That is ensured by preoperative transconjunctival injections of the preparation Disport 7-17 Units in muscular sacs of two or four horizontal rectus muscles of both eyes. If observing pendular and pendular jerk nystagmus, Disport is injected in the four horizontal muscles, while jerk nystagmus requires the injections only in the sacs of the two muscles enabling eye turn to a nystagmus relative rest area. In 7-14 days, a standard LASIK or Photorefractive Keratectomy operation follows.

EFFECT: method provides simplified treatment process with higher effectiveness, and decreased number of complications.

3 cl, 3 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: strain of microorganism Bacillus smithii TBMI12 MSCL P737 is resistant to metronidasole. Strain is applied as food or feed additive, and as component of probiotic compositions. Probiotic composition of said strain in form of endospores is applied as probiotic in order to perform antibacterial impact, colonise gastrointestinal tract and stimulate immune system, as antibacterial medication.

EFFECT: invention ensures resistance to diseases and prevention of bacterial infections with application of strain Bacillus smithii TBMI12 MSCL P737.

11 cl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to ophthalmology, and can be applied in treatment of vertical squint with hyperfunction of inferior oblique eye muscle. For this purpose temporally in area of lower-internal angle of orbit near the place of inferior oblique eye muscle attachment transcutaneous injections of medication of botulinic toxin A in dose 20 UN are made. Preliminarily medication is diluted in 1.5 ml of physiological solution of sodium chloride. Then, immediately after this introduction rehabilitative conservative treatment is performed until eye-moving function is restored.

EFFECT: in case of refusal from surgery method creates conditions of maximal patient's plasticity to perception of typical conservative treatment of said type of squint, which leads to restoration of sight in shorter terms, prevention of disease recurrence.

1 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, and concerns a lactic bacteria Lactobacillus reuteri DSM 17938 strain stimulating IL-10 production and hence CD4+CD25+TR-cell proliferation used for making a probiotic product. The probiotic product contains Lactobacillus reuteri DSM 17938 strain and additionally medium-chain triglyceride oil.

EFFECT: use of the probiotic product promoted a favorable effect on intestinal colic in a newborn baby.

3 cl, 3 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to allergology, and can be used for prevention of development of respiratory allergies in a subject. That is ensured by introduction of an effective amount of Lactobacillus rhamnosus GG (LGG) either in a pregnant mother's body, and/or postnatal in a feeding mother's body, or in a subject directly.

EFFECT: introduction of LGG allows preventing an early allergic sensitisation and the following development of respiratory allergies due to higher production of serum antibodies IgA in a subject, and also prevention of allergic inflammation in lungs and respiratory ways.

12 cl, 13 ex, 9 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to microbiological fertilisers for plants, specifically to a microbiological composition based on Rhizobium (symbiotic nitrogen fixer) legume bacteria. The composition contains Rhizobium lupini bacteria and associative nitrogen-fixing bacteria Flavobakterium, with components in weight ratio of 1.0-1.5:1.5-2.0. Due to synergetic interaction of the components, the composition has high physiological activity and has not phytotoxic effect on crop plants. The invention enables to efficiently increase plant biomass in mixed legume-grass seeds by approximately 7.1-7.8% compared to inoculation of seeds with legume bacteria, and by 8.9-9.9% compared to inoculation of seeds with associative bacteria. The degree of inhibition of crop plants or damage falls 1.4-8-fold compared to control crop plants.

EFFECT: microbiological composition increases accumulation of green material by 87-91 centner per hectare or 20-23% compared to control plants - yield in mixed lupine seeds and barley without inoculation of seeds with microbiological preparations.

2 tbl

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