Strains of bacillus sudtilis and bacillus amyloliguefaciens bacteria promoting recovery of microbiocoenoses in soil and animals gastrointestinal tracts, possessing bactericidal, fungicidal and virucidal activity and preparation on basis of same strains

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, veterinary science and plant protection. The bacterial strain Bacillus subtilis IC-1435-1-1 is deposited in the Russian National Collection of Industrial Microorganisms, registration number B-10641. The bacterial strain Bacillus amyloliquefaciens IC-1436-1-23 is deposited in the Russian National Collection of Industrial Microorganisms, registration number B-10642. The bacterial strain Bacillus amyloliquefaciens IC-1437-1-23 is deposited in the Russian National Collection of Industrial Microorganisms, registration number B-10643. The strains are produced by selection and promote the recovery of microbiocenoses in the soil and animals gastrointestinal tract, possess bactericidal, fungicidal and virucidal activity. A preparation on the basis of the strains contains an excipient or water with a bacterial biomass in the spore form Bacillus subtilis, the Russian National Collection of Industrial Microorganisms B-10641, or Bacillus amyloliquefaciens, the Russian National Collection of Industrial Microorganisms B-10642, or Bacillus amyloliquefaciens, the Russian National Collection of Industrial Microorganisms B-10643, or a mixture thereof in ratio 1:1:1 with titre of each bacterial strain not less than 1·104 CFU/g or 1·104 CFU/ml.

EFFECT: preparation possess bactericidal and fungicidal activity.

7 cl, 9 dwg, 12 tbl, 10 ex

 

The invention relates to strains of microorganisms, stimulating the restoration of microflora of the soil and the gastrointestinal tract (GIT) of humans and animals, has antibacterial, fungicidal and virucidal activity, producing interferon α-2 human leukocyte and preparative form based on them and can be used in biotechnology, veterinary medicine, medicine and plant protection for:

receiving drugs against bacterial, fungal and viral infections of animals and plants; use as a microbiological fertilizers, designed to restore soil microbiota; the manufacture of therapeutic and preventive drugs for animals and humans, is able to restore and maintain the microbiocenosis of the gastrointestinal tract; adjust and maintain normal immune status.

Among bacterial fungicides, having the state registration, and registered in the State catalogue of pesticides and agrochemicals permitted for use on the territory of the Russian Federation, there are currently no fungicides, having such a wide range of actions as in the present invention.

The product Integral (liquid-based strain 24D Bacillus subtilis) is registered as an agent intended for use against Bo is Esna grain crops (wheat, the spring barley), vegetables (potatoes, tomatoes and cucumbers) and 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, published 27.04.1999).

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

Among microbiological fertilizers similar to the proposed drug is Lactoferrin, which is based on soil microorganisms Bacillus mucilaginosus, designed to enrich the soil plant-digestible forms of phosphorus and recovery of soil fertility, increase the germination rate and yield in many crops in the 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 h the following fungi which, when introduced into the soil as developing 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. In contrast to the above inventive bacterial preparation on the basis of the proposed strain of Bacillus subtilis VKPM In 10641 has proven itself in the laboratory and field experiments as an effective means to combat such vysokopoligonalnymi diseases of raspberries and black currants, as purple blotch, Fusarium, Alternaria, grey rot, and has no analogues on the territory of the Russian Federation.

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 is used to obtain the drug against fungal diseases of cotton (USSR Author's certificate No. 1717156, CL A01N 63/00, publ. 1992)

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

Known bacterial strain Bacillus subtilis ARRIAM N131 used to obtain the drug against pathogens rot yab is OK and grapes during storage (USSR Author's certificate No. 1706504, CL A01N 63/00, 1992). However, this strain also has a limited action on individual pathogens of fungal diseases of crops.

However, the above strains have not been studied on antagonistic activity and the ability to restore soil microbiocenosis and the digestive tract of animals to evolutionary normal, and do not possess antiviral activity and are not producers of interferon.

Known bacterial strain Bacillus subtilis VKPM B-7036 for obtaining a 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: Phytophthora infestans, Microsporium solani, Fusarium solani (the causative agent of dry rot).

The closest analogue (prototype) is a bacterial strain Bacillus subtilis IC-16, obtained by transformation of strain B. subtilis VKPM B-7048 (IC-9) recombinant plasmid DNA rvmv 105, containing the gene for alpha-2 interferon (RF patent No. 2142287, MKI C12N 1/20, publ. 10.12.1999,). The resulting strain IC-16 deposited in Russian national Collection of Industrial Microorganisms (VKPM), GNII Genetika number VKPM B-7092. Plasmid rvmv 105 is able to replicate in the bacilli and contains the gene for alpha-2 - interferon person, promotoras expression of this gene, the website landing ribosomes (SD) and signal peptide alpha-amylase Century amyloliquefaciens for the secretion of interferon in the culture medium, as well as providing resistance to kanamycin.

The closest analogue (prototype) formulation is the drug Fitop obtained on the basis of the above strain of Bacillus subtilis VKPM B-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 B-7036 with title 7×1010spores/g of 0.1-1.0%; starch 0,2-3,0%; sugar - the rest is up to 100%. The residual 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 does not have a sufficiently broad spectrum of antagonistic activity, and was not analyzed for the ability to restore the microflora of the digestive tract of animals and soil to evolutionary normal.

The technical result of the claimed technical solution is to provide recovery to the evolution of the normal microflora of the soil, the digestive tract of animals and expansion of the spectrum antibacterial, fungicidal and virucidal activity with the use of the drug, obtained on the basis of the inventive strains.

This technical result is achieved by obtaining method aimed village is tion of bacterial strains:

Bacillus subtilis IC-1435-1-1 for the restoration of microflora of the soil and the gastrointestinal tract of animals with bactericidal, fungicidal and virucidal activity and deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10641 (certificate of Deposit attached);

Bacillus amyloliquefaciens IC-1436-1-23 for the restoration of microflora of the soil and the gastrointestinal tract of animals, which have bactericidal and fungicidal activity and deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10642 (certificate of Deposit attached);

Bacillus amyloliquefaciens IC-1437-1-23 for the restoration of microflora of the soil and the gastrointestinal tract of animals, which have bactericidal and fungicidal activity and deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10643 (certificate of Deposit included).

This technical result is achieved by the fact that the drug is characterized by a content of the filler or water with a biomass of bacteria in spore form of Bacillus subtilis IC-1435-1-1 (VKPM B-10641), or Bacillus amyloliquefaciens IC-1436-1-23 (VKPM B-10642), or Bacillus amyloliquefaciens IC-1437-1-23 (PMBC V), or mixtures thereof in the ratio of 1:1:1 with a title for each strain of bacteria is not less than 1·104CFU/g or 1·104CFU/ml

As a powdered sorbent preparation contains the bran powder, or zeolite, or a polysaccharide, or a monosaccharide, or a disaccharide, or a mixture of polysaccharide with a monosaccharide or disaccharide in a ratio of from 1:100 to 1:10.

As the polysaccharide product contains starch, monosaccharides - glucose, as the disaccharide is sucrose.

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

If the drug is starch (polysaccharide) helps to speed up the germination of spores of bacteria, which increases the efficiency of its action.

Powdered sorbent on the basis of 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.

Characteristics of the inventive strains. Strains of bacteria Bacillus amyloliquefaciens has been selected to suppress bacterial and fungal growth.

The strain of Bacillus subtilis VKPM B-10641 obtained by selection of recombinant bacterial strain Bacillus subtilis IC-16. The specified strain, for example, who was little more than breeding for the suppression of bacterial and fungal growth and on the basis of the number of the produced interferon α-2 human leukocyte. The original recombinant strain-prototype Bacillus subtilis IC-16 (from which the selection is received of the claimed strain of Bacillus subtilis IC-1435-1-1) constructed by transformation of strain .subtilis VKPM B-7048 (IC-9) recombinant plasmid DNA rvmv 105, containing the gene for alpha-2 interferon (RF patent No. 2142287, MKI C12N 1/20, publ. 10.12.1999,) and deposited in Russian national Collection of Industrial Microorganisms (VKPM), GNII Genetika number VKPM B-7092. Plasmid rvmv 105 is able to replicate in the bacilli and contains the gene for alpha-2 - interferon person, a promoter for the expression of this gene, the site of the landing of ribosomes (SD) and signal peptide alpha-amylase .amyloliquefaciens for the secretion of interferon into the culture medium. 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 raspberry. The determination of the activity of interferon was performed on cell cultures using virus encephalomyocarditis mice.

The strain Bacillus amyloliquefaciens IC-1436-1-23 obtained by breeding strains of bacteria Bacillus subtilis IC-9. The indicated strains were subjected to selection for suppression of bacterial and fungal growth. The source is the strain-prototype Bacillus subtilis IC-9 deposited in Russian national Collection of Industrial Microorganisms (VKPM), GNII Genetika number VKPM B-7048. In connection with the change of methods of identification of microorganisms in the Russian Federation and other countries the work was performed by re-species identification of the claimed onselectionchange strain IC-1436-1-23 in the all-Russian collection of industrial microorganisms (VKPM) method detection by 16s pPHK, which was identified as Bacillus amyloliquefaciens by assigning the claimed strain collection of the Institute of Genetics of the number VKPM B-10642.

The strain Bacillus amyloliquefaciens IC-1437-1-23 obtained by selection of the bacterial strain Bacillus licheniformis IC-1. The indicated strains were subjected to selection for suppression of bacterial and fungal growth. The original strain-prototype Bacillus licheniformis IC-1 deposited in Russian national Collection of Industrial Microorganisms (VKPM), GNII Genetika number VKPM B-7038. In connection with the change of methods of identification of microorganisms in the Russian Federation and other countries the work was performed by re-species identification of the claimed onselectionchange strain IC-1437-1-23 in the all-Russian collection of industrial microorganisms (VKPM) method detection by 16s pPHK, which was identified as Bacillus amyloliquefaciens by assigning the claimed strain collection of the Institute of Genetics of the number VKPM B-10643.

Cultural, morphological and biochemical properties of the bacterial strains Bacillus subtilis VKPM B-10641, Bacillus amyloliquefacies VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643.

The strains belong to the gram-positive facultative aerobic rod-shaped bacteria. With growth mesopatamia agar bacterial strain Bacillus subtilis VKPM B-10641 form rough colonies of white color, growing into the surface of the medium. Cells are rod-shaped, oval endospores, are Central or paracentral, don't inflate the cell when sporoobrazovanie. The length of the cell (2-3) µm, width - (0,7-0,8) mm. Capsule form. A gram-stained positively. Strains of bacteria Bacillus amyloliquefaciens VKPM B-10642 and Bacillus amyloliquefaciens VKPM B-10643 form rough colonies are cream in color. Cells are rod-shaped, oval endospores, are Central or paracentral, don't inflate the cell when sporoobrazovanie. The length of the cells of 2.0-3.8 μm, a width of 0.5-1.0 μm. Capsule form. Strains are oligophyllum, multiply at 25-45°C, optimum growth occurs at a temperature of 33 to 37°C. After 48 hours growth at 37°C in mesopatamia agar culture has opaque colonies homogeneous powdery consistency, with smooth edge. pH: minimum of 5.7; maximum of 8.0; the optimum of 7.0 to 7.2. Strains proliferate at 37°C in mesopatamia agar and mesopatamia broth.

Biochemical properties of the strains. Do not grow under anaerobic conditions, do not form acetylaminophenol of glucose in reactiives - Proskauer, oxidized with the formation of acid, glucose, mannose, fructose, ribose, lyxose, cellobiose, trehalose, maltose, turanose. Hydrolyzing starch, urea and esculin. Have lecithinase, the catalase activity. Tolerant to sodium chloride concentrations in the nutrient medium in the range of 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, bacterial strain Bacillus subtilis VKPM B-10641 produces interferon α-2 human leukocyte.

For long-term storage of strains of bacteria spore mass is freeze-dried. For mass propagation of bacteria use mastopathy broth. The cultivation is carried out at a temperature of 37°C.

Determining the number of activity units of interferon α-2 human leukocyte produced by the claimed strain of Bacillus subtilis VKPM B-10641.

Five outbred white mice weighing 18-20 g using an automatic pipette is administered orally 0.1 cm3water suspensions of cells of strain at a concentration of 50 mg/cm3during 5 days.

After 5 days from the beginning of the introduction of strain in 5 mice were selected for the study sample (separately from each vividly the nogo). Animals were subjected to euthanasia by the method of cervical dislocation. Aseptically dissected abdominal cavity and extracted by 0.5 cm distal and proximal intestine. The extracted fragments of intestine was placed in pre-weighed sterile microprobing with poured in it 0.5 cm3eagle medium MEM. Microprobing with fragments of intestine were weighed. All samples prior to analysis were stored at minus 80°C. Samples of the intestine homogenized in a microtube using a Teflon homogenizer. Was osvetleni by centrifugation at 10,000 rpm for 15 min. the Supernatant was additionally osvetleni and sterilized by filtration.

In the wells was added to 0.05 cm3solution of neutral red (1.1 g/cm3in the medium Needle MEM. Were incubated for 90 minutes the Contents of the wells were removed and the wells were washed three times with saline. The tablets were dried in a laminar air stream. To each well was added 0.1 cm3ethanol acidified with hydrochloric acid to pH 4.0). Incubated for 30 min and measured the optical density at a wavelength of 490 nm.

Determination of the specific activity of interferon in the biological substrates of experimental animals was determined using 2-3 days in monolayer cell cultures of L-929 and L-68, sensitive to interferon α-2 lenoci the ary man. Culture cells were grown on beds, using growth medium Needle MEM with a double set of amino acids with 10% serum fruits cows liquid with the addition of benzatinbenzilpenicillin sterile 100 units/cm3and streptomycin sulfate 100 u/cm3. Cells were incubated at a temperature 36,0±1,0°C. After the formation of a full monolayer of cells was removed from mattresses with a mixture of Versene and trypsin in a 1:1 ratio. The monolayer cells were filled with the specified mixture was kept at room temperature for 2-5 min until the swelling and the beginning of the detachment of cells from the surface of the mattress, after which the mixture of Versene with trypsin was decanted, and the cells were dispersively 40-50 cm3supportive environment (nutrient medium Needle with antibiotics without serum of fetuses of cows). In the resulting suspension were counting the number of cells in the cell Goriaev and brought the cell concentration before (150000-300000) cells/ml by adding growth medium. Thus prepared suspension cells were dispensed in 96-well culture tablets 200 mm3into the hole. Incubated tablets with cell culture at a temperature 36,0±1.0°C in an atmosphere with 5,0±0,5% CO2within 48-72 hours before the formation of a complete monolayer.

To determine the activity (titer) indicator of virus was prepared by ten-fold dilution of the culture of the virus obtained from the collection of the cult of the, in a supportive environment. Later in cultural tablets were made in 100 mm3cooked breeding with each dilution of at least four holes. Inoculated and control cell cultures were incubated for 24 hours at a temperature 36,0±1.0°C in an atmosphere with 5,0±0,5% CO2. The titer of the virus was taking the reciprocal of the dilution of the drug at which the cell culture 50% of the holes were completely amazed at the cytopathic effect of the virus.

Activity (titer) of virus was determined by the method of Spearman - Cerberus by the formula:

,

where Dmaxdecimal logarithm of cultivation, above which have been 100% cell death (+);

d is the decimal logarithm of the step dilution (1,0);

n is the number of holes on each dose (4);

p is the number of holes that gave death (+) in Dmaxand subsequent dilutions.

To determine the activity of interferon in the filtrate homogenates fragments proximal, distal intestine of experimental animals and its contents prepared them twice cultivation and double cultivation of a reference standard interferon in a supportive environment (4 cultivation above and below the estimated titer).

From the wells of cultural tablets remove growth medium and added 100 ál of the prepared dilution of the experimental and control the designs, with each dilution of at least 4 wells with cell culture. To control the dose indicator of the virus left 16 wells with cell culture, and for monitoring the state of the monolayer of cells - 4 holes. In these 20 holes were made in 100 μl of maintenance medium used to prepare the dilution of samples. Inoculated and control cell cultures were incubated for 24 hours at a temperature 36,0±1.0°C in an atmosphere with 5,0±0,5% CO2. Then to each well with the experimental samples and the control of the interferon made calculated in advance, the dose indicator of virus - virus encephalomyocarditis mice, corresponding to 100 TCD50in the amount of 0.1 cm3on each hole.

Accounting and analysis of results of measurements conducted on a vertical multichannel spectrophotometer-fluorimetry FL-600 using software KS-4 Bio-Tek Ink., USA. The concentration of human alpha-2 interferon obtained by the method of suppressing the JRC, was not less than 30 IU/g of sample, which is much higher than in the original strain-prototype .subtilis VKPM B-7092.

Example 1. The study of epatajnosti strains of bacteria Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643.

The study was conducted in the laboratory of biotechnological control of APF "Research center".

The pathogenicity of each microo the body naturally was assessed 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 mice weighing 18-20 g in the amount of 40 individuals of both sexes and the rats of Wistar breed weighing 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 mesopatamia broth.

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

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

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

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

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

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

After an autopsy sowing organs (heart, lung, liver, spleen and blood were made mastopathy and blood agars in Petri dishes method prints. P is Seva incubated in a thermostat at a temperature of 37±1°C for 24 hours. The result was taken into account 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 bacteria Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643 are non-pathogenic.

Example 2. Study the antagonistic activity of bacterial strains Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643.

Obtained through breeding strains of bacteria Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643 are characterized by a high antagonistic activity against pathogenic and conditionally pathogenic Mick is organizmov.

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 sonnet, 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 bacterial strains Bacillus subtilis and Bacillus amyloliquefaciens was made mesopatamia agar.

Recipe mycopathologia agar 1 DM3:

- peptone meat enzymatic - 10.0 g;

- yeast extract 5.0 g;

- sodium chloride 5.0 g;

- microbiological agar - 15,0,

- 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 min, After cooling to a temperature of 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 callitrichaceae.

Culture of pathogenic microorganisms mentioned above, were grown on Petri dishes for 18±2 hours at mesopatamia 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,

Next, 0.5 g of the drug was dissolved in 0.5 cm3. The obtained suspension was sown stroke using a microbiological loop diameter Petri dishes with mesopartner 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 (mesopartner agar) without the studied Association antagonists. Obtained experience in data presented in tables 1, 2 and 3.

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 Bacillus subtilis and Bacillus amyloliquefaciens for obtaining microbial biomass.

To obtain biomass dispute bacterial strains Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643 cultivated in a liquid nutrient medium of the following composition:

- peptone meat enzymatic - 10.0 g;

- yeast extract 5.0 g;

- sodium chloride 5.0 g;

- distilled water to a total volume of 1 DM3.

pH 7,2±0,2.

1 DM3fermentation of a nutrient medium used 10 cm3seed with a titer of 108CFU/cm3. The process of growing biomass was carried out in a fermenter for 48±2 hours. At the specified method of cultivation can be obtained a title dispute (5-10)×1010CFU/cm3.

Example 4. Getting ready forms of the drug on the basis of the bacterial strains of Bacillus subtilis and Bacillus amyloliquefaciens.

First cook the mixture is collected and tested for quality raw biomass with filler. As filler can be used water is (for treatment plants using spray) with a title dispute is not less than 1·10 4CFU/ml or powdered adsorbent, such as zeolite. As the sorbent also use the powder from bran or 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 1·104-106CFU/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 bacteria spores of Bacillus amyloliquefaciens In PMBC-10642 with a title dispute is not less than 1·104CFU/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 bacteria spores of Bacillus amyloliquefaciens In PMBC-10642 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 subtilis VKPM B-10641 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 subtilis VKPM B-10641 with powder of bran with a title dispute is not less than 1·106CFU/g

5.5. Part 5. The mixture of biomass bacteria spores of Bacillus amyloliquefaciens In PMBC-10643 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 bacteria spores of Bacillus amyloliquefaciens In PMBC-10643 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 biomass of bacteria spores of Bacillus amyloliquefaciens In PMBC-10643, Bacillus amyloliquefaciens VKPM B-10642, Bacillus subtilis VKPM B-10641 in the ratio of 1:1:1 with a title dispute each type of bacteria is not less than 1·104CFU/g with a mixture of starch and powder-based glucose in the ratio 1:20.

5.8. Part 8. The mixture of biomass spores of the bacteria Bacillus subtilis VKPM B-10641 with starch with a titer of at least 1·106CFU/g

5.9. Part 9. The mixture of biomass spores of the bacteria Bacillus subtilis VKPM B-10641 with sucrose with a titer of at least 1·106CFU/g

5.10. 10. The mixture of biomass spores of the bacteria Bacillus subtilis VKPM B-10641 with glucose titre not less than 1·106CFU/g

5.11. Composition 11. An aqueous solution of biomass bacteria spores of Bacillus amyloliquefaciens In PMBC-10643 with a title dispute is not less than 1·104CFU/ml of This solution was stored in a dark place for no more than 1-2 days.

5.12. 12. An aqueous solution of biomass spores of the bacteria Bacillus subtilis VKPM B-10641 with a title dispute is not less than 1·104CFU/ml of This solution was stored in a dark place for no more than 1-2 days.

5.13. Composition 13. An aqueous solution of biome is with spores of the bacteria Bacillus amyloliquefaciens VKPM B-10643, Bacillus amyloliquefaciens VKPM B-10642, Bacillus subtilis VKPM B-10641 in the ratio of 1:1:1 with a title dispute each type of bacteria is not less than 1·104CFU/g This solution was stored in a dark place for no more than 1-2 days.

5.14. Composition 14. An aqueous solution of biomass spores of the bacteria Bacillus subtilis VKPM B-10641 with a title dispute is not less than 1·106CFU/ml

5.15. 15. An aqueous solution of biomass bacteria spores of Bacillus amyloliquefaciens In PMBC-10642 with a title dispute is not less than 1·106CFU/g

5.16. The composition 16. An aqueous solution of biomass bacteria spores of Bacillus amyloliquefaciens In PMBC-10643 with a title dispute is not less than 1·106CFU/g

Example 6. Data storage the dry form of the drug on the basis of strains of Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643.

To establish the shelf-life and storage conditions of the drug was carried out relevant studies.

Within 96 months of storage the dry form of the drug at the temperature of 30±0.5°C titer 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 medicine, veterinary medicine and agriculture.

Example 7. Data on the inhibitory activity of bacterial strains Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643 against phytopathogens.

For testing strains of Bacillus subtilis and Bacllus amyloliquefaciens in terms of their ability to inhibit pathogenic fungi used method agar blocks. The suspension of the drug with a titer of 1×108CFU/cm3made in mesopotania agar, cooled to a temperature of 36±1°C. For the cultivation of pathogenic fungi used a potato-glucose agar.

Inoculated strains (each separately) medium were poured in 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 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 introducing strains). On the basis of the obtained data was determined inhibitory activity, which was calculated by the formula:

IA=[(Dto-Dabout)/Dto]×100,

where Dtothe diameter of colonies in the control variant;

Daboutthe diameter of the colony in a test version.

Table 4 shows the values of inhibitory activity against fungal phytopathogens tested bacterial strains obtained in the experiment.

Thus, the inventive strains of bacteria Bacillus subtilis and Bacillus amyloliquefaciens showing antagonistic activity against indicated in table 4 fungal phytopathogens.

Example 8. Data of field trials to study the effects of drugs on the basis of strains of Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-1642, Bacillus amyloliquefaciens VKPM B-10643 on the incidence of raspberry purple blotch and defeat currant neighbour.

For testing strains of Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642 on diseases of raspberries and currants in the open ground used industrial planting raspberry varieties Zorenka Altai and currant varieties Sophia. As a chemical standard used 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, according to the scheme experience:

- strains of Bacillus subtilis VKPM B-10641 and Bacillus amyloliquefaciens VKMV-10642;

Topaz 0,1%;

control.

Experiment was repeated four times, square plots of 10 m2the fluid flow - 4 DM3on option. 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 - very strong development of the disease (busy spots more than 50% of the leaf blade surface).

Step is ery damage to the raspberry canes were assessed by a special 4-point scale for purple spot raspberry:

- 0 points - healthy stalk;

- 1 point - 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, %;

and the number of plants relevant score, PCs;

b - point scale;

N is the total number of plants, PCs;

It is the highest point 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, PCs;

b - the number of plants in the experiment, pieces

Biological efficacy of these preparations was determined by the formula:

,

where a is the average prevalence in the control, %;

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

The results of field experiments are presented in tables 5 and 6.

Thus, the processing of raspberry plantations drugs n the basis of strains of Bacillus subtilis VKPM B-10641 and Bacillus amyloliquefaciens VKPM B-10642 in the field experiment were reduced: the development of the disease is almost 3 times; the prevalence is more than 2 times.

Table 5.
The influence of the proposed products on the basis of the bacterial strains Bacillus subtilis VKPM B-10641 and Bacillus amyloliquefaciens VKPM B-10642 to defeat raspberry purple blotch (SHA "Gardens of Siberia, 2009).
OptionPrevalence, %Development %Biological efficiency, %
Control75,025,0-
Bacillus amyloliquefaciens VKPM B-1064227,311,454,4
Bacillus subtilis VKPM B-1064138,6the 9.761,2
Topaz 0,1%38,610,856,8
NDS052,0

Biological efficiency amounted to 54.4-61,2% depending on the term of Provideniya. The yield increase was 0.3 t/ha Treatment plants currant strains of bacteria Bacillus subtilis VKPM B-10641 and Bacillus amyloliquefaciens VKPM B-10642 in a field experience reduced disease development in 2.2 times. Biological efficiency was compared with 41.8 59.7 per cent, depending on the duration of the account.

Table 6.
The influence of the proposed products on the basis of the bacterial strains Bacillus subtilis VKPM B-10641 and Bacillus amyloliquefaciens VKPM B-10642 to defeat currant neighbour (SHA "Gardens of Siberia, 2009)
OptionBiological efficiency, %*
Time records
June 27July 4July 11
Bacillus amyloliquefaciens VKPM B-1064256,548,446,8
Bacillus subtilis VKPM B-1064159,743,541,9
Topaz 0,1%64,556,556,5

Example 9. Research the Finance of the claimed preparation in the form of microbiological fertilizer

Investigated the effects of experimental biological products on the basis of spore-forming bacteria Bacillus subtilis and Bacillus amyloliquefaciens on qualitative indicators harvest of soft spring wheat Bagansky - 95 in Western Siberia.

Research conducted in 2007-2009 in the territory of the Federal Heartset" (Novosibirsk region, sverch - Tula). Type of soil: black soil leached medium, middle, texture - loam. For planting in the experimental area used seeds first reproduction varieties of spring wheat Bagansky - 95.

Experiment was repeated four times, the area of one plot was 42 m2. We studied the efficacy of experimental biological products on the basis of spore-forming bacteria of the genus Bacillus (Bacillus subtilis, Bacillus amyloliquefaciens) in the following ways: 1 - control 1, no pre-sowing seed treatment was not carried out; 2 - control 2, presowing treatment of seeds was carried out by chemical disinfectants (vial-TT); 3 - pre-sowing seed treatment completed drug (composition 13) on the basis of a mixture of bacterial strains Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643 (hereinafter in example 9 - Fitop 8.67) and preparations (compounds 1, 11 and 12) based on monocultures strains (flow mixtures and monocultures 10 ml/t); 4 - was conducted unicornia the feeding vegetative plants at the tillering stage; 5 - completed pre-sowing treatment of seeds with a mixture of strains and foliar vegetative plants at the tillering stage. During the growing season for all variants of the experiment was conducted single herbicide treatment of crops tank mixtures Magnum, VDG (5 g/ha), Dialen-super, V.R. (0.4 l/ha). Topic, ke (0.5 l/ha).

According to the results of three experiments revealed a positive impact of Pitapa 8.67 when it is used for seed treatment and feeding vegetative plants at the tillering stage on yield and quality characteristics of spring wheat. The obtained data are presented in tables 7, 8 and 9.

Table 7.
Thousand seed weight, g Soft spring wheat Bagansky - 95.
Version of experience200720082009Average years of experience
ggggcontrol 1%
Control 1 (no treatment)34,230,8 to 33.832,90,0100,0
Control 2 (seed treatment with chemical disinfectants Vital TT)35,531,134,8to 33.80,9102,7
Treatment of seeds Picopop 8.6735,63135,634,11,2103,5
Treatment of seeds Atopica20,820,522,2of 21.24,1123,8
Treatment of seeds Clallam20,720of 21.220,63,5120,7
Treatment of seeds Picopop 8.67 and Clallam35,731,636,234,51,6104,
Feeding Picopop 8.6734,731,537,634,61,7105,2
Feeding Atopica20,823,7of 21.2of 21.94,8128,1
Feeding Clallam20,72323,622,45,3131,2
Feeding Picopop 8.67 and Clallam31,93131,2of 31.4of-1.5for 95.3
Treatment of seeds Picopop 8.67 and feeding Picopop 8.6734,630,535,733,60,7102,1
Treatment of seeds Atopica and feeding Atopica20,8 2216,719,82,7116,0
Treatment of seeds Clallam and feeding Clallam20,721,316,219,42,3113,5
Treatment of seeds Picopop 8.67 together with Clallam and feeding Picopop 8.67 together with Clallam33,929,630,8of 31.4of-1.595,5

Table 7 provides data on the impact of Pitapa 8.67 on the weight of 1000 grains of wheat. The most significant increase of the weight of a thousand grains observed in the variation of the feeding vegetative plants in Clallam (5.3 g compared to control 1 and 4.4 g compared with control 2). Also showed a significant (NDS) in comparison with control 1 and control 2 the increase of the weight of a thousand grains in variants of experience with the processing of seeds of 1.2 g and 0.3 g and seed treatment followed by foliar feeding during the tillering Picopop 8.67 1.7 g and 0.8, proved Good option, in which Fitop 8.67 and drug Krill used together for the treatment of seeds before the settlement of the PTO. However, foliar top dressing of crops during tillering mixture of these drugs had a negative effect on the thousand seed weight, which decreased compared with both control options. Thousand seed weight in control 2 was higher than the value in the control 1 0.9 G. Other options experience showed lower value of the mass of a thousand grains in comparison with control 2, but higher compared to control 1.

Table 8 presents data on the influence of Pitapa 8.67 on the gluten content in wheat grains. Significant increase in the gluten content in grain was observed when the total use of pre-sowing seed treatment and foliar feeding of plants at the tillering period Picopop 8.67 - 1.6% relative to control 1 and 0.2% relative to control 2. The grain obtained after harvest in control 2, the gluten content was higher than the equivalent value in the control 1 1.4%. The use of drugs Krill and Isofit for seed treatment and foliar application had a negative effect on the gluten content, the percentage of which in these embodiments, the experience was lower than in control 1 and control 2. The increase in gluten observed in variants of experience-sharing for seed treatment and feeding during tillering of Pitapa 8.67 and drug Krill. In other options, which the ants experience marked increased in comparison with control 1 gluten content, however, its increase does not exceed the value of the HCP05and can not be considered significant.

Table 8.
Gluten content, %. Soft spring wheat Bagansky - 95
Version of experience200720082009Average years of experience
%%%%control 1%
Control 1 (no treatment)3029,821,827,20,0100,0
Control 2 (seed treatment with chemical disinfectants Vital TT)3331,221,728,61,4to 105.3
Treatment of seeds Picopop 8.673230,5 2027,50,3101,1
Treatment of seeds Atopica322520,725,9-1,3for 95.2
Treatment of seeds Clallam3227,720,726,8-0,498,5
Treatment of seeds Picopop 8.67 together with Clallam2927,61925,2-2,092,6
Feeding Picopop 8.673228,922,527,80,6102,2
Feeding Atopica323019,327,1-0,199,6
Feeding Cree is llom 3130,719,727,1-0,199,8
Feeding Picopop 8.67 together with Clallam3328,220,727,30,1to 100.4
Treatment of seeds Picopop 8.67 and feeding Picopop 8.673528,42328,81,6105,9
Treatment of seeds Atopica and feeding Atopica3332,42028,51,3104,7
Treatment of seeds Clallam and feeding Clallam3330,42128,10,9103,4
Treatment of seeds Picopop 8.67 together with Clallam and feeding Picopop 8.67 together with Criollo the 3233,622,429,32,1107,8

Table 9 presents data on the influence of Pitapa 8.67 on wheat yield. The yield was significantly higher in test options: seed treatment with Picopop 8.67 4.3 t/ha and 1.1 kg/ha; treatment of seeds Picopop 8.67 subsequent foliar top dressing Picopop 8.67 in the tillering period of 5.1 t/ha and 1.9 t/ha; treatment of seeds Picopop 8.67 with drug Krill - 6.8 t/ha and 3.6 t/ha compared to control 1 and control 2, respectively. In the variant of the experiment control 2 yields a soft spring wheat exceeded its value in the control 1 3.2 t/ha In the following variants experience yields exceeded those in the control 1, but was lower than the values obtained in the variant of the experiment with seed treatment with chemical disinfectants vial-TT together with the cultivation of seeds Picopop 8.67 and subsequent feeding during the tillering Picopop 8.67 1.9 t/ha higher than in control 2 yields observed in variants of experience with pre-sowing seed treatment drugs Krill and Atopic and then feeding these drugs.

Table 9.
Yield, centner/ha Soft spring wheat Bagansky - 95.
Version of experience200720082009Average years of experience
kg/hakg/hakg/hakg/hacontrol 1%
Control 1 (no treatment)of 17.517,616,117,10,0100,0
Control 2 (seed treatment with chemical disinfectants Vital TT)2017,923,120,33,2118,9
Treatment of seeds Picopop 8.6721,3a 21.521,421,44,3125,1
Treatment of seeds Atopica20,8 22,2of 21.24,1123,8
Treatment of seeds Clallam20,720of 21.220,63,5120,7
Treatment of seeds Picopop 8.67 together with Clallam21,323,926,623,96,8140,0
Feeding Picopop 8.6721,322,22322,25,1of 129.6
Feeding Atopica20,823,7of 21.2of 21.94,8128,1
Feeding Clallam20,72323,622,45,3131,2
Feeding Picopop together with Clallam21,3of 21.22020,83,7the level of 121.8
Treatment of seeds Picopop 8.67 and feeding Picopop 8.6721,320,914,719,01,9110,9
Treatment of seeds Atopica and feeding Atopica20,82216,719,82,7116,0
Treatment of seeds Clallam and feeding Clallam20,721,316,219,42,3113,5
Treatment of seeds Picopop 8.67 together with Clallam and feeding Picopop 8.67 together with Clallam21,320,1a 12.718,00,9105,5

In tables is 10 presents data on the influence of the drug (composition 13) Fitop 8.67 and preparations based on strains of bacteria Bacillus subtilis VKPM B-10641 (12), Bacillus amyloliquefaciens VKPM B-10642 (part 1), Bacillus amyloliquefaciens VKPM B-10643 (part 11) on different groups of soil microorganisms.

Seed treatment formulation (composition 13) Fitop 8.67 based on a mixture of strains and preparations based on monocultures strains of Bacillus subtilis (12) and Bacillus amyloliquefaciens (preparations of compounds 1 and 11) can significantly increase the total microbial and fungal number, and the number of nitrification and ammonification in the soil, and increases the number zymogenous microflora. Similar results were obtained when applying Filipa 8.67 for foliar feeding of wheat plants during the growing season and the joint effect of seed treatment and feeding.

5
Table 10.
The composition of the soil microflora (fractal, on average, over the 3 years of testing).
Version of experienceTBCOGCNitrificationAmmonificationAutochthonous microfloraSimagena microflora, total
123467
Control 1 (no treatment)0,410,21,75,17,4
Control 2 (seed treatment with chemical disinfectants Vital TT)1,00,10,11,80,43,1
Treatment of seeds Picopop 8.675,34,21,52,70,835,1
Treatment of seeds Atopica0,20,30,40,91,42,9
Treatment of seeds Clallam12,53,12,823,01,98,2
Treatment of seeds Picopop 8.67 the n conjunction with Clallam 5,56,71,31,31,217,6
Feeding Picopop 8.675,01,82,410,01.885,5
Feeding Atopica14,52,87,31,23,0143,1
Feeding Clallam2,02,01,00,10,10,2
Feeding Picopop together with Clallam2,11,04,6of 17.01,015,3
Treatment of seeds Picopop 8.67 and feeding Picopop 8.67a 3.91,53,53,0 0,34,8
Treatment of seeds Atopica and feeding Atopica7,51,8the 4.73,20,10,8
Treatment of seeds Clallam and feeding Clallam4,11,76,05,02,034,1
Treatment of seeds Picopop 8.67 together with Clallam and feeding Picopop 8.67 together with Clallam3,31,74,27,00,11,5
Seed treatment with bacterial strain Bacillus subtilis VKMV-10641 (12)4,3111,52,35,945,9
Seed treatment with bacterial strain Bacillus amyloliquefaciens VKPM B-10642 (part 1)1,22,20,6 2,14,090,3
Seed treatment with bacterial strain Bacillus amyloliquefaciens VKPM B-10643 (composition 11)2,01,11,41,0the 4.7116,6

Example 10. Data recovery microflora of the gastrointestinal tract of animals on simulated under laboratory conditions salmonellosis.

The data presented in figure 1-9. Figure 1 - graph of quantitative account of the dynamics isolate Salmonella typhimurium strain 5715 from the feces of rats. Figure 2 is a graph of the IgG titer in the serum of rats in the control group. Figure 3 is a graph of the IgG titer in the serum of rats in group 1, by days. 4 is a diagram showing changes in the titer of IgG in the serum of rats in group 2, by days. 5 is a diagram showing changes in the titer of IgG in the serum of rats in groups. 6 is a graph of quantitative account of the dynamics isolate Salmonella typhimurium strain 5715 from the feces of rats. Fig.7 is a diagram showing changes in the titer of IgG in the serum of rats in group 3, days. Fig - schedule of changes in titer of IgG in the serum of rats in group 4, for days. Fig.9 is a diagram showing changes in the titer of IgG in the serum of rats in groups.

As a test object for infection in laboratory conditions used healthy is x rats of Wistar breed of both sexes weighing 280±10 g from nursery NPF "Research center". 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. As a therapeutic and preventive controls are applied to a cell suspension of Bacillus subtilis strain VKPM B-10641 (aqueous solution, the composition 14) not less than 1×106CFU/ml (hereinafter referred to as the drug Board 1.23). For infection of animals used cell suspension in physiological solution agar culture Salmonella typhimurium strain 5715 (deposited at room 100067 in the Public collections of pathogenic microorganisms state Institute of standardization and control of medical biological preparations. Lautareasca of the Russian Ministry of health) with a concentration of 5×107CFU/ml Infection was carried out by parenteral (intraperitoneal), the infectious dose of 1 ml Dynamic allocation of Salmonella was determined by two methods - seeding the suspension of faeces of rats with differential nutrient medium with subsequent serotyping cultures and solid phase "sandwich"method (using the text-system total IgG - ELISA - BEST"). At the first stage of determining the dynamics of the allocation of Salmonella solid phase "sandwich"method of calibration samples with known concentrations of IgG and analyzed the samples were incubated in the wells stripyoung tablet with them is abilityone monoclonal antibodies (MCAT) for IgG; in the second stage, who contacted the holes IgG was treated with a conjugate of MCAT to light (lambda and Kappa) chains of the immunoglobulins in rats with peroxidase. After washing off the excess conjugate is formed immune complexes immobilized MCAT - IgG - conjugate was detected by enzymatic reaction of peroxidase with hydrogen peroxide in the presence of Chromogen (OPD). After setting the peroxidase reaction stop reagent results took into account photometrically. The concentration of IgG in the samples was determined via a calibration curve.

There were formed the following groups: control; 1 experimental (treatment), in which the Board 1.23 dose of 75 ml/kg were asked orally for 10 days after infection; 2 experimental (treatment), in which the Board 1.23 asked orally in a dose of 100 ml/kg for 10 days after infection; 3 experienced (preventive), in which the Board 1.23 asked orally at a dose of 75 ml/kg for 10 days before infection; 4 experienced (preventive), the Board 1.23 asked orally in a dose of 100 ml/kg for 10 days before infection. The repetition of the experience was applied four times.

Found that in laboratory conditions at artificially simulated rats salmonellosis Board 1.23 has both curative and preventive efficiency.

In three of determining the content of microbial cells of Salmonella in f is kaliah of rats (figure 1), in the two experimental groups during the period of the experiment, the number of CFU/g of Salmonella decreased.

The lower titers of Salmonella compared to the control on the order of the recorded 1 day after the start of treatment (figure 1) when giving the drug at a dose of 100 ml/kg body weight on day 10 after the start of treatment when giving the drug at a dose of 75 ml/kg, which indicates therapeutic efficacy of Wecoma 1.23 in the treatment of salmonellosis.

From the chart data (figure 2) shows that the highest titer IgG at a dilution of 1:128000 in the control group was observed on day 10 of the experiment. Due to the considerable variation of the indicators in the rest of the dilutions due to high concentration of serum in the future, a decision was made about the advisability of considering only the data dilution 1:128000.

1 and 2 experimental groups (Fig 3, 4) the level of IgG in serum was significantly increased by the last day of the experiment.

For comparison, the values change titers of IgG in the serum of animals 1, 2 experimental and control groups was made General chart (figure 5, a dilution of 1:128000).

It is noted that in the first group for the period of the experiment the rate of optical density of IgG increased by 0,212, the second on 0,199, in the control group on 0,041. The difference between the data obtained from the experimental and control groups, statistically significant at the level of the NDS05.

The highest titer of IgG was observed in animals receiving Board 1.23 dose of 75 ml/kg

When determining the prophylactic effectiveness of Wecoma 1.23 in dosages of drug 75 (group 3) and 100 (4) ml/kg animal body weight analysis of changes in the number of microbial units of Salmonella in the feces of infected animals was carried out according to groups twice: after 4 days after infection (day 4 of the experiment) and 10 days after infection (day 24 of the experiment). Determination of changes in the titer of IgG in the serum of rats, we conducted three times: before the application of the drug (day 1 of the experiment); 4 days after infection (day 4 of the experiment) and 10 days after infection (day 14 of the experiment).

In the result set (6)that animals 3 groups at the beginning sickened by Salmonella, however, by the end of the experiment they ceased to isolate Salmonella from faeces that can testify to their clinical recovery.

In group 4, in the faeces of rats Salmonella not detected during the whole period of the experiment, which indicates a good immune protection to the infective agent. In contrast to the control group, where K is the number of CFU/g of Salmonella in the faeces of rats did not decrease during the experiment. Therefore, the Board 1.23 is an effective means for the prevention of Salmonella when used in a dose of 100 ml/kg

When studying the dynamics of changes in titer of IgG in the serum set (7)that in group 3 (dose 75 ml/kg) titer IgG at a dilution of 1:128000 on the 4th day of infection was slightly higher than on day 1 of the experiment (which may explain the disease in the first days of infection), then to 24 days of the experiment, the titer increased significantly, and possibly so in this period we have not found microbial units of Salmonella in the feces.

4 experimental group (dose of 100 ml/kg) titer IgG later, 4 days after infection was relatively higher than on day 1 (Fig), before the application of the strain, and 24 the day of the experiment, the titer increased significantly.

For comparison, the values change titers of IgG in the serum of animals 3, 4 experimental and control groups was made General diagram (Fig.9, dilution 1:128000).

From the graph it is seen that in group 3 during the period of the experiment the rate of optical density of IgG increased 0.114, 4 group - is 0.135, in control - decreased by 0.002. Thus, the results of studies of prophylactic effectiveness of Wecoma 1.23, we can conclude that the most effective preventive dose is 100 ml/kg of body weight.

Thus, the Board 1.23 is effektivnym medication for the prevention of salmonellosis which is used in a dose of 100 ml/kg body weight titre not less than 1×106CFU/ml not only avoids infection (animals remain clinically healthy), but also creates significant tensions of specific immunity, allowing you to maintain a high level of immunoglobulin IgG in the serum.

For drugs on the basis of strains of Bacillus amyloliquefaciens In PMBC-10642 (15) and Bacillus amyloliquefaciens VKPM B-10643 (16) it was determined the effect on the microflora of the gastrointestinal tract of broiler chickens cross the ISA F-15 (table, 12). So, daily use of the drug composition 15 from the strain of Bacillus amyloliquefaciens In PMBC-10642 in the 3rd experimental group during the entire period of growing chickens (35 days) to the greatest extent contributed to the displacement of conditionally pathogenic bacteria. The drug composition 16 on the basis of the strain Bacillus amyloliquefaciens VKPM B-10642 for 5 days with 5-day break in the 2nd experimental group led to a decrease in the number of opportunistic bacteria and a slight reduction in the number of symbiotic lacto - and bifidobacteria. The greatest influence on the species composition of the microflora of the intestinal tract of chickens studied the biological product has had in the 1st experimental group when fed to it within 10 days. In this case, the observed sharp decrease in the numbers of opportunistic bacteria in the period when armlehne strain and a sharp increase in the number of symbiotic lacto - and bifidobacteria 10 days after withdrawal of the drug. The number of lacto - and bifidobacteria, as well as strains of E. coli with normal enzymatic activity increased by 100-1000 times compared to the initial level.

The data presented in table 11, show that the use of the drug on the basis of the strain Bacillus amyloliquefaciens VKPM B-10642 (15) has no negative effects on the species composition of the microflora of the gastrointestinal tract of chickens. Moreover, its introduction into the organism of animals contributes to the increase in both absolute and relative number of symbiotic microorganisms with normal enzymatic activity.

According to the results of studying the influence of the drug composition 16 on the basis of the strain Bacillus amyloliquefaciens VKPM B-10643 (table 12) on the microflora of the gastrointestinal tract of broiler chickens cross the ISA F-15 marked increase in the number of bifidobacteria, lactic acid bacteria and Escherichia coli with normal enzymatic activity in the advanced options 1-3 (application of strain in the culture fluid with a titer of 1×109CFU/ml daily (5 days together with antibiotics); daily with antibiotics; a day (first 5 days in conjunction with antibiotics) at a dose of 3 µl/kg

Indications for use (recommendation). Preparations OS is ove strains of Bacillus subtilis VKPM B-10641, Bacillus amyloliquefaciens VKPM B-10642, Bacillus amyloliquefaciens VKPM B-10643 recommended for use in poultry and livestock production, crop production for:

- prevention and treatment of dysbacteriosis agricultural and wild birds, including chickens, ducks, geese, turkeys, Guinea fowls and others; enhance the natural resistance of the organism of birds; elimination of immunodeficiency caused by infectious (viruses, bacteria, protozoa, intracellular) and noncommunicable (low-quality forage, kuchenne content, the stress resulting from temperature regimes of contents) factors; increase the livability; increase egg production; additional gain of broilers; reduction of feed conversion; reducing the time poultry production;

- prevention and treatment of all types of productive farm animals: dysbacteriosis; infectious diseases of viral, bacterial and protozoal etiology; prevention of viral diseases and immunodeficiencies; increase the safety of youngsters in the early periods of development; productivity; additional gain of young beef cattle; improve the quality and grading of milk and meat; the renewal of the fertility of breeding stock;

processing of seed - seeds, tubers, bulbs, etc. is before sowing; processing of the root system when transplanting plants; processing vegetative mass of agricultural plants and urban landscapes; agricultural irrigation plants and urban landscapes (flowers, shrubs, grass etc); soil cultivation in greenhouses, fields, orchards and other plantations; processing of vegetables, fruits, grains after harvest; processing of hay before putting it into winter storage.

1. The bacterial strain Bacillus subtilis IC-1435-1-1 for the restoration of microflora of the soil and the gastrointestinal tract of animals with bactericidal, fungicidal and virucidal activity and deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10641.

2. The bacterial strain Bacillus amyloliquefaciens IC-1436-1-23 for the restoration of microflora of the soil and the gastrointestinal tract of animals, which have bactericidal and fungicidal activity and deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10642.

3. The bacterial strain Bacillus amyloliquefaciens IC-1437-1-23 for the restoration of microflora of the soil and the gastrointestinal tract of animals with bactericidal fungicidal activity and deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika under registration number VKPM B-10643.

4. The drug with bactericidal and fungicidal activity, characterized by a content of the filler or water with a biomass of bacteria in spore form of Bacillus subtilis VKPM B-10641, or Bacillus amyloliquefaciens number VKPM B-10642, or Bacillus amyloliquefaciens VKPM B-10643, or their mixture in the ratio 1:1:1 with a title for each strain of bacteria is not less than 1·104CFU/g or 1·104CFU/ml

5. The preparation according to claim 4, characterized in that the filler it contains powdered sorbent.

6. The preparation according to claim 5, characterized in that as a powdered sorbent contains a powder of bran, or zeolite, or a polysaccharide, or a monosaccharide, or a disaccharide, or a mixture of monosaccharide polysaccharide, or a mixture of the polysaccharide with the disaccharide in the ratio of from 1:100 to 1:10.

7. The preparation according to claim 6, characterized in that it contains polysaccharide starch, monosaccharides - glucose, as the disaccharide is sucrose.



 

Same patents:

FIELD: agriculture.

SUBSTANCE: method of plant cultivation comprises includes treatment of plant seeds prior to their sowing and of plant in the growing season with a biological product in liquid form. As a biological product, a mixture of strains of bacteria of B. subtilis of RNCIM B-10641, B. amyloliquefaciens RNCIM B-10642 and B. amyloliquefaciens RNCIM B-10643 is used, with a titer of not less than 106 CFU/ml in the form of an aqueous suspension.

EFFECT: invention provides increase in productivity of plants and restoration of soil microbiocenosis.

3 cl, 4 dwg, 5 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to oligonucleotide primers and the method of their use for detection of Lactobacillus delbrueckii subspecies bulgaricus in starter cultures. Proposed synthetic oligonucleotide primers have nucleotide sequences: Lbul4F 5'-GGCCAGCCAGATCGCCAGC-3' and Lbul5R 5'-GACCAGGTCGCTGTCCGGC-3'. The proposed method includes performance of PCR. In case a DNA ferment is detected with size of 409 base pairs. a conclusion is made on availability of Lactobacillus delbrueckii subspecies bulgaricus in the investigated biomaterial.

EFFECT: inventions may be used in milk processing industry for detection and identification of strains and cultures Lactobacillus delbrueckii subspecies bulgaricus in starter cultures used in production of cultured milk foods.

2 cl, 1 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: nutrient medium contains HMF - agar, erythrocytic mass from donor's human blood, serum of cattle and yeast extract at the specified ratio.

EFFECT: invention makes it possible to increase sensitivity of medium to extracted microorganisms, to improve growth properties of nutrient medium and to simplify method of its preparation.

FIELD: biotechnologies.

SUBSTANCE: new strain of bacteria Pasteurella trehalosi is proposed, where the specified bacteria are positive in respect to beta-haemolysis, positive in respect to oxidase, positive in respect to catalase, negative in respect to urease, positive in respect to nitrates, negative in respect to indole, MacConkey-positive, positive in respect to glucose, positive in respect to saccharose, positive in respect to mannitol, negative in respect to arabinose, negative in respect to cellobiose, positive in respect to xylose, negative in respect to salicin, negative in respect to ornithine, negative in respect to esculin, negative in respect to alpha-fucosidase, positive in respect to beta-galactosidase. Also the strain of bacteria Mannheimia haemolytica is proposed. These bacteria are deposited under registration numbers ATCC No. PTA-3667, ATCC No. PTA-3668, ATCC No. PTA-3669.

EFFECT: immunisation of chickens with the purpose to prevent disease caused by above bacteria.

7 cl, 22 dwg, 2 tbl, 6 ex

FIELD: biotechnologies.

SUBSTANCE: nutrient medium contains monosubstituted potassium phosphate, magnesium sulphate, sodium phosphate, iron citrate, glycocol, monosubstituted sodium glutamate, glycerine, agar, potato broth, whey of cattle and distilled water at specified ratios.

EFFECT: invention makes it possible to increase yield of biomass of L-forms of mycobacteria and to reduce time of biomass accumulation.

1 tbl

FIELD: food industry.

SUBSTANCE: invention relates to food industry, in particular to production of food additives based on sea weeds. The method for production of a functional food additive (being a source of food fibres, bioactive polysaccharides, iodine-containing substances, a combination of ω-3 ω-6 polyunsaturated fatty acids) envisages sea weeds treatment. The milled sea weeds are maintained in water at a ratio of 1:10 at a temperature of 20-25°C during 20-40 minutes for swelling. Then one introduces probiotic microorganisms i.e. Lactobacillus genius lactic acid bacteria into the produced jelly-like sea weed substrate at t=20-25°C during 12-24 hours till concentration is equal to no less than 107 CFU/g to accrete biomass. The invention allows to produce a functional food additive based on sea weeds and having a complex preventive and probiotic action. The produced additive is used in the recipe of mince meat products in an amount of 5% of the raw material weight.

EFFECT: invention promotes semi-products quality enhancement, structure and appearance improvement and storage life extension.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns oligonucleotide primers for B.mallei genetic typing. The presented primers are complementary to a differentiation fragment BMA0416 of an equinia agent genome, possess activity of upstream and downstream primers in an amplification reaction and have the following structure: 5'-TGGCGGAGTATGGATGCTG-3' - BmVAT6-Ch2s 5'-GAACGAGAACACCTACGACCTGAT-3' - BmVAT6-Ch2as.

EFFECT: presented invention may be used in practical healthcare for detection of the differentiation DNA fragment BMA0416 of the equinia agent.

3 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns oligonucleotide primers for B.mallei genetic typing. The presented primers are complementary to a differentiation fragment BMA0577 of an equinia agent genome, possess activity of upstream and downstream primers in an amplification reaction and have the following structure: 5' - GAG GAT GAA GGT GCC GTG G - 3' - BmVATl-Chls 5' - GAC AAC TAC TTC ATC GGC TAT CTG - Y - BmVATl-Chlas.

EFFECT: presented invention may be used in practical healthcare for detection of the differentiation DNA fragment BMA0577 of the equinia agent.

3 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Described is a plasmid which codes esterase Psychrobacter cryohalolentis K5T and contains Ndel/Xhol - a DNA fragment of plasmid pET32a with length of 5.366 thousand base pairs, which includes a promoter of bacteriophage T7, a translation enhancer of gene 10 of the bacteriophage T7, a transcription terminator of the bacteriophage T7, a bla β-lactamase gene which determines resistance of cells transformed by plasmid pPC023 to ampicillin, a replication initiation section ori; and Ndel/Sall - a DNA fragment with size of 0.869 thousand base pairs, which contains a gene which codes the mature form of esterase Psychrobacter cryohalolentis K5T with an amino acid sequence SEQ ID NO: 2, given in the description, having an C-end hexahestidine linker. Provided is an Escherichia coli bacteria strain which is modified by said plasmid, a producer of a polypeptide having esterase Psychrobacter cryohalolentis K5T activity.

EFFECT: invention increases the level of biosynthesis of esterase to 20% of total cell protein.

2 cl, 2 dwg, 5 ex

FIELD: agriculture.

SUBSTANCE: biological preparation for stimulation of growth and protection of plants from diseases, increase in productivity and soil fertility contains biomass Bacillus amyloliquefaciens RNCIM B-11008 and humates at the following ratio of components in vol. %: biomass Bacillus amyloliquefaciens RNCIM B-11008 - vegetative cells and spores of 1.24-1.30×1010 CFU/ml of culture fluid and the spore content 94% of the total amount of CFU - 99.0, humates - 1.0.

EFFECT: biological preparation enables to protect plants from fungal and bacterial diseases, to improve the phytosanitary condition of soil and improve its fertility, to increase crop yields and improve the quality of agricultural products.

5 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and pharmacology, and represents a method for probiotic correction of postintoxification psychosis in the patients suffering alcohol dependence syndrome, consisting in restoring the hepatic function ensured by increasing the detoxification and metabolic functions with the use of biologically active additives containing the probiotic culture of bifidus bacteria 10 CFU/g 3 times a day and lactobacilli 108 CFU/g 3 times a day for 5 days.

EFFECT: provided reduced activity of the AST enzymes, activated pigment, protein and synthetic hepatic function, restored normal flora of the colon.

2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and biotechnology and can be used in food industry and medicine. The microbial strain of Lactobacillus plantarum Tensia DSM 21380 produces conjugated linoleic acid (CLA), hydrogen peroxide (H2O2), nitrogen monoxide (NO), contains plantaricin encoding genes and has antioxidant activity. This strain and its liophilised form are used to prepare an antimicrobial and hypotensive probiotic, a dairy product and a medicinal agent which lowers blood pressure. The strain and its liophilised form are also used to increase polyamine turnover in the body, to attain a dominant position among intestinal lactic bacteria in the gastrointestinal tract and to prolong the shelf life of a dairy product.

EFFECT: use of the invention enables to lower blood pressure, suppress undesirable microflora and inhibit oxidative processes.

12 cl, 8 dwg, 31 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: strain Enterococcus hirae BC - 37 having high antagonistic activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number VKPMV-10090 and may be used, for instance, in production of such cultured milk foods as kefir, cottage cheese or ryazhenka.

EFFECT: invention makes it possible to produce a strain having high antagonistic activity and high speed of milk turning sour.

3 ex

FIELD: biotechnologies.

SUBSTANCE: strain Lactobacillus gallinarum I-12, having high antagonistic activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number VKPM V - 10134 and may be used in production, for instance, of such cultured milk foods, as kefir, acidophilic milk, acidophilic-yeast milk.

EFFECT: invention makes it possible to produce a strain having high antagonistic activity and high speed of milk turning sour.

3 ex

FIELD: biotechnology.

SUBSTANCE: strain of Enterococcus hirae "И"-27 with high antagonistic activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under registration number of RNCIM B-10091 and can be used in production, for example, of fermented milk products such as kefir, cottage cheese or fermented baked milk.

EFFECT: invention enables to obtain a strain with high antagonistic activity and a high rate of milk fermentation.

3 ex

FIELD: biotechnology.

SUBSTANCE: strain of Enterococcus hirae "И"-29 with high antagonistic activity, deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under registration number of RNCIM B-10088, and can be used in production, for example, of fermented milk products such as kefir, cottage cheese or fermented baked milk.

EFFECT: invention enables to obtain a strain with high antagonistic activity and a high rate of milk fermentation.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutical industry and represents a composition with anti-infectious activity presented in the solid dosage form for oral administration in the form of a powder, representing a balanced complex containing a probiotic agent, an adsorbent and an excipient; the probiotic agent is sterilised cultural fluid containing metabolites of the strain Bacillus subtilis VKPM (Russian National Collection of Industrial Microorganisms) No. B-2335; the adsorbent is zeolite; the excipient is calcium stearate or aerosil; the composition is characterised by the fact that it additionally contains a high-active immunomodulatory agent representing a complex of polysaccharides - products of polymer enzymatic hydrolysis of internal and external layers of brewers' yeast cell coating in the form of β-glucane and mannan with the ingredients of the composition taken in specific proportions, wt %.

EFFECT: invention provides high effectiveness and versatility with respect to dangerous bacterial and viral infections; it is storage safe and stable.

11 ex, 10 tbl, 10 dwg

FIELD: biotechnologies.

SUBSTANCE: probiotic lacto-amylovorin is produced by means of depth growth of the strain Lactobacillus amylovorus RNCIM B-6253 on the liquid nutrient medium, which contains the following: corn extract - 10 ± 0.01 ml, lactose - 20 ± 0.005 g, chemically deposited chalk - 0.5 ± 0.01 g, triple-substituted citric-acid sodium - 7.5 ± 0.05 g, 3-water acetous sodium - 2 ± 0.01 g, iron sulfate - 0.1± 0.001 g, 5-water manganese sulfate - 0.16 ± 0.002 g, hydrolysate of dry nonfat milk - up to l. Subsequent extraction of the target product in liquid condition or in the form of a dry powder is carried out with preliminary addition of a stabiliser to a cultural liquid or to a protective medium used when producing the probiotic in the form of the dry powder. The stabiliser is selected from: sodium metabisulfite, sodium hydrosulfite and ascorbic acid.

EFFECT: invention provides for stable production of a probiotic with high values of a titre of antimicrobial bodies not only in a cultural liquid, but also after its treatment required for production of different pharmaceutical forms of a preparation.

3 cl, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to veterinary science. The preparation is applicable for preventing and treating gastrointestinal diseases. The preparation (wt %): sterilised culture fluid Bacillus subtilis 3/28 Russian National Collection of Industrial Microorganisms B-3679 - 10.0-15.0; sterilised culture fluid Bacillus licheniformis L-34 Russian National Collection of Industrial Microorganisms B-4161 - 10.0-15.0; sterilised culture fluid Halobacterium halobium - 10.0-15.0; glauconite - 50.0-55.0; cell envelopes of brewers' yeast strain Saccharomyces cerevisiae - 5.0-15.0; calcium stearate - 0.5-2.0.

EFFECT: use of the preparation reduces the intensity of dyspeptic disorders, improves intestinal digestion, effectively harmonises intestinal biocoenosis composition, has immunomodulatory action.

13 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: invention relates to food for infants and/or young children. The food contains indigestible oligosaccharides with polymerisation degree equal to 2 - 200, unviable Bifidobacterium breve in an amount equivalent to 103 CFU - 1013 CFU of B. breve per g of the food dry weight, and viable Bifidobacterium breve in an amount of less than 103 CFU of B. breve per g of the food dry weight. The invention relates to the food application for production of a composition for feeding infants and/or young children.

EFFECT: indigestible oligosaccharides combination combined with unlivable Bifidobacterium breve promotes prevention and/or therapy of atopic illness and food allergy.

19 cl, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbological industry, particularly production of biologically active substances and can be used in producing fusicoccins for different purposes, for both agricultural and medical purposes. The culturing medium contains molasses, raw sugar, potassium nitrate (KNO3), potassium phosphate (KH2PO4), magnesium sulphate (MgSO4 x 7H2O) and tap water in a given ratio.

EFFECT: invention increases the range of fusicoccins.

4 ex

Up!