RU2471864C1 - Probiotic preparation for viral and bacterial infections toxisporin, method for preparing it, bacillus licheniformis bacterial strain used as ingredient of probiotic preparation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: probiotic preparation for viral and bacterial infections contains mixed biomass of the Bacillus subtilis (Russian National Collection of Industrial Microorganisms B-3679) and Bacillus licheniformis (Russian National Collection of Industrial Microorganisms B-4161) bacterial strains in a spore form with titre not less than 109 spore/g, and glauconite. The optimal content of the Bacillus subtilis bacterial spores mixed with the Bacillus licheniformis bacterial spores makes 50%. The preparation is prepared by culture of the bacteria specified above in a glucose-molasses medium at pH - 6.8-7.0, to produce mixed spores in the amount of 0.1-1 wt % of the preparation weight to be mixed with glauconite to the CFU content not less than 109 spore/g. The preparation is produced with the use of the Bacillus licheniformis (Russian National Collection of Industrial Microorganisms B-4161) bacterial strains.

EFFECT: extended range of probiotics possessing higher antibacterial and antiviral activity and higher therapeutic effectiveness in treating and preventing viral and bacterial infections.

7 cl, 15 tbl, 8 ex

Description

The invention relates to the field of biotechnology, and in particular to probiotic preparations used in medicine and veterinary medicine for the prevention and treatment of gastrointestinal diseases of humans and animals caused by pathogenic and conditionally pathogenic microorganisms, as well as dysbiosis caused by etiologically diverse factors (infections, antibiotics, and chemotherapy, ionizing radiation, acute and chronic intoxications, etc.) and the technology for their preparation.

Currently, a large number of probiotic preparations are known for the prevention and treatment of gastrointestinal diseases in humans and animals (EP 0097484, 1984, CL A23C 1/05; EP 0043962, 1980, CL A23C 19/086; US 4289888, 1979, cl A23C 9/123). Such a preparation, as a rule, contains bacterial cells, residues of culture fluid (QOL), fats, proteins, lactose, gum and other additives. The technology for their preparation includes, as a rule, the preparation of a solution or suspension of the biomass of microorganisms, the introduction of special additives into it, freeze-drying and obtaining the finished product. The process conditions and composition are determined by the characteristics of the microorganisms used and the nature of the task.

So, the drug is known and the method of its production (RU 2080795, 06/10/1997). This preparation contains live Lactobacillus acidophilus cells, gelatin, sucrose, skim milk, water, and the remains of the culture fluid. The method of obtaining it involves cultivating the bacteria Lactobacillus acidophilus in a liquid nutrient medium, introducing a protective medium containing gelatin, sucrose, skim milk, freezing at a temperature of -30 - (-) 35 ° C for 18-12 hours, freeze-drying for 45- 52 hours.

The disadvantage of the drug is its relatively low efficiency, limited scope, as well as the impossibility of long-term storage.

Currently, one of the promising directions for providing a healing effect on the gastrointestinal tract is the use of drugs containing several strains of microorganisms that have a synergistic effect.

Known drug (RU 2160992, A23C 9/12; 12/27/2000) containing viable cells of 2 symbiotic strains of Lactobacillus acidophilus, the remains of the culture fluid (QL) and excipients - gelatin, sucrose, skim milk. The method for its preparation involves culturing bacteria in a liquid nutrient medium, introducing a protective medium containing gelatin, sucrose and skim milk, freezing at a temperature of minus 25-30 ° C for 2-3 hours and freeze-drying for 45-48 hours.

The disadvantage of the drug is its lack of effectiveness with respect to increasing the body's resistance to adverse environmental conditions, insufficient thermal stability, a relatively narrow spectrum of effects on the body.

The closest in technical essence to the claimed group of inventions is a preparation for the prevention and treatment of gastrointestinal diseases of a person containing live cells of strains of Bacillus subtilis VKPM B-7092 and Bacillus licheniformis VKPM B-7038 in spore form with a titer of at least 3 × 10 ¹⁰ spores / g, as well as filler (RU No. 2142287, A61K 35/74, 1999).

The drug is characterized by high antagonistic activity to a wide range of pathogenic and conditionally pathogenic microorganisms, however, it contains live microorganisms that are sensitive to antibiotics, therefore, the drug is not recommended for use in treatment with antibiotics, it does not have antiviral effects, as well as antioxidant and ion exchange activity.

The challenge facing the authors was the creation of a new probiotic with increased efficiency on the basis of symbiotic cultures, which has a more pronounced amylolytic, proteolytic and antagonistic activity against pathogens, and can be stored in a wide range of consumer temperatures.

The task is to create a drug based on the bacterial strains Bacillus subtilis and Bacillus licheniformis, which have increased thermal stability, and are immobilized on a natural ion exchanger and adsorbent - glauconite. It was proposed to use microorganisms that can suppress the growth of both fungi (trichophytes, microsporiums, epidermophytons, candidomycoses, etc.), and pathogenic bacteria - strains of staphylococcus, streptococcus, salmonella, etc.

The technical result consists in expanding the range of probiotics with high antibacterial and antiviral activity, and increasing therapeutic efficacy in the treatment and prevention of viral and bacterial infections.

Studies have shown that immobilization of bacterial spores on glauconite provides them with mechanical protection, a long shelf life, prevents bacterial spores from sticking together, helps to accelerate the germination of bacterial spores and prolongs the action, which increases the effectiveness of the drug and allows its activity to be maintained at the required level for at least 24 hours.

At the same time, glauconite additionally provides the binding and elimination of low molecular weight toxins (methane, hydrogen sulfide, ammonia, etc.), heavy metals and radionuclides, participates in selective ion exchange (removes or reduces the negative effect of aluminum ions on the body, interacts synergistically with magnesium and fluorine, is additional trace mineral source).

The technical result was achieved by creating a group of inventions, including:

bacterial strain Bacillus licheniformis, deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), GNII Genetika under the number VKPM B-4161;

a preparation comprising a mixture of biomass of bacterial strains of Bacillus subtilis and Bacillus licheniformis in spore form and excipient, characterized in that as biomass of bacterial strains it contains the biomass of bacterial strains of Bacillus subtilis 3/28 VKPM B-3679 and Bacillus licheniformis VKPM B-4161 in spore form with titrorm not less than 10 ⁹ spores / g, and as a filler - glauconite in the following ratio of ingredients, wt.%:

a mixture of spores of bacteria Bacillus subtilis and Bacillus licheniformis - 0.1-1.0;

glauconite - the rest;

a method for producing the preparation, comprising separate cultivation of the bacterium Bacillus sub-tilis 3/28 VKPM B-3679 and Bacillus licheniformis L-34 VKPM B-4161 on glucose-molasses medium at pH 6.8-7.0, mixing the obtained biomass strains in spore form in a ratio of 1: 1 between themselves and then in an amount of 0.1-1% wt. from the mass of the drug with glauconite to a CFU content of at least 10 ⁹ spores / g.

Optimum results are achieved when using spores of microorganisms in an equal ratio, but it is possible to use them with a slightly larger or smaller fraction of one of the strains in the mixture, for example, in an amount of 40-60% of the total weight of the mixture.

A high titer of bacterial spores during cultivation (10 ¹² spores / g or more) is ensured by using glucose-molasses medium, optimal for selected microorganisms, having the following composition:

(NH ₄ ) ₂ SO ₄ - 10.0 g / dm ³ ; KH ₂ PO ₄ - 4.0 g / dm ³ ; molasses - 50.0 g / dm ³ ;

glucose - 20.0 g / dm ³ ; soy peptone 2.0 g / dm ³ ; yeast extract - 3.0 g / DM ³ and cultivation under the following conditions: temperature 37 ° C; aeration -1.0 air volume / medium volume per minute; mixing speed 250 rpm; pO ₂ - 40-60% of saturation, cultivation time 24-36 hours.

Microorganisms before the mixing stage, as a rule, are freeze-dried, after which the freeze-dried biomass of Bacillis subtilis 3/28 VKPM B-3679 and Bapillis licheniformis L-34, VKPM B-4161 with glauconite is mixed or the biomass is sprayed onto glauconite in dryers or vacuum apparatuses.

The stamps used in the composition have the following characteristics.

The bacterial strain Bacillus subtilis 3/28, deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), GNII Genetika under the number VKPM B-3679. The strain was isolated from roughage and was subjected to multi-stage selection to suppress bacterial and fungal growth.

The strain is not pathogenic - Conclusion of the All-Russian Scientific Center for the Safety of Biologically Active Substances (VSC BAS), OJSC, 2008

Strain VKPM B-3679 produces biosporicin, a broad-spectrum antibiotic that inhibits the growth of fungi, staphylococci, streptococci and Pseudomonas aeruginosa.

With the best options, experiments were carried out to suppress growth: trichophytes, microsporiums, epidermophytons, candidomycoses and other fungi.

With prolonged storage of the strain, the substance is lyophilized. For the reproduction of bacteria, meat-peptone agar (MPA) is used, and cultivation is carried out at a temperature of 37 ° C.

As a component of a probiotic preparation used to suppress pathogenic and conditionally pathogenic microflora, which has anti-inflammatory and immunocorrective effects, it is described in patent RU 2367454, September 20, 2009.

The studied culture is characterized by the absence of hemolytic activity (Table 1).

Table 1 Physiological and biochemical properties of B.subtilis strain VKPM B-3679 Growth under anaerobic conditions - Fermentation Glucose + arabinose + xylose + mannitol + Disposal citrate + propionate - Hydrolysis starch + urea - Nitrate reduction + The formation of gas from NO ₃ ^- under anaerobic conditions Discoloration of methylene blue + Arginine dihydrolase - Lecithinase - Hyaluronidase - Hemolytic activity - The formation of poly ^β- hydroxybutyric acid globules on glucose agar Lysozyme activity +

According to the data, the sectioned strain of Bacillus subtilis is characterized by physiological and biochemical characteristics typical of this species, which can cause a wide range of positive effects when used.

Antibiotic sensitivity. Studies of the antibiotic resistance of B. subtilis strain against antibiotics and antibiotic substances widely used in healthcare practice showed that the strain is resistant to ampicillin, benzylpenicillin, aztreonam, ceftazidime, ceftisoxime and polymyxin E (Table 2).

table 2 Antibiotic sensitivity of B. subtilis strain VKPM B-3679 Study drug The diameter of the zones of growth inhibition of crops, mm B.subtilis 3A Amoxicillin 18 ± 0.1 Ampicillin 10 ± 0.3 Meslocillin 20 ± 0.3 Methicillin 18 ± 0.1 Oxacillin 14 ± 0.3 Benzylpenicillin 8 ± 0.1 Aztreonam 0 Imipenem 36 ± 0.4 Moxalactam 12 ± 0.3 Cephalotin 32 ± 0.5 Cefamandol 37 ± 0,1 Cefoperazone 16 ± 0.1 Cefotaxime 14 ± 0.2 Ceftazidime 8 ± 0.2 Ceftizoxime 0 Ceftriaxone 18 ± 0.3 Amikacin 20 ± 0.2 Gentamicin 24 ± 0.2 Kanamycin 23 ± 0.1 Tobramycin 24 ± 0.3 Vancomycin 12 ± 0.1 Clindamycin 10 ± 0.3 Tetracycline 24 ± 0.3 Chloramphenicol 16 ± 0.2 Polymyxin E 10 ± 0.1 Nitrofurantoin 16 ± 0.2 Trimethoprim 24 ± 0.1 Norfloxacin 24 ± 0.2

The bacterial strain Bacillus licheniformis L-34, deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), GNII Genetics under the number VKPM B-4161. The strain is isolated from milk and obtained by a multi-stage selection method. The strain was selected for inhibition of bacterial and fungal growth. With the best options, experiments were carried out to suppress the growth of pathogenic strains: staphylococcus, streptococcus, salmonella, trichophytes, microsporiums, epidermophytons, candidomycoses, etc.

Cultural, morphological and biochemical properties of the strain Vas.licheniformis VKPMV-4161. Strain VKPM B-4161 refers to aerobes; gram-positive bacillus capable of forming spores. The sizes of vegetative cells (0.5-0.8) microns and (1.5-3) microns, sometimes reach up to 6 microns. The spores are elliptical, mainly located centrally, no more than one in the sporangia cell, without a distinct bloated sporangia.

Vegetative cells of B.licheniformis VKPM B-4161 grow well on rich nutrient media (meat-peptone broth - MPB and meat-peptone agar - MPA) at a temperature of (37-40) ° С. There is no growth in the presence of 0.02% azide, but there is growth on Saburo agar, with 7% NaCl and with the addition of 0.001% lysozyme.

Weak growth is observed in anaerobic agar.

On meat and peptone broth, pH (7.30.1) after 48 hours of incubation at a temperature of (371) ° C forms a turbidity of the medium, and on the surface - a dry film; on Hottinger’s meat and peptone agar, pH (7.30.1) —weakly convex dry opaque whitish colonies, polymorphic, fine-grained with a diameter of (4-6) mm, growing into the nutrient medium.

Biochemical properties. Vegetative cells of B.licheniformis VKPM B-4161 hydrolyze casein and starch, are catalase-positive. The Voges-Proskauer reaction is positive.

The strain is not pathogenic - Conclusion JSC VSC BAV 2008

Strain VKPM B-4161 produces proteolytic enzymes that inhibit the growth of fungi, staphylococcus, streptococcus and Pseudomonas aeruginosa.

The therapeutic effect of the drug is the effective elimination of opportunistic microflora and the restoration of normoflora.

The drug, based on the Bacillus bacteria spore association, has received the code name TOXISPORIN (the applicant has filed a trademark application).

TOXISPORIN can be used both in dry and in liquid forms, by introducing it into feed or food or on its own. For treatment, the drug is administered orally at the rate of 1 kg of the drug per 1 ton of feed or from 3 to 10 g per head until the body recovers.

The drug is effective for all types of mammals in the treatment and prevention of colibacterioses, dysfunctions and dysbacterioses of various nature, plague, parainfluenza, influenza, dyspepsia, salmonella, infectious rhinotracheitis in cattle, viral diarrhea, rotavirus enteritis, dysentery, staphylocitic ulcer disease as well as for the prevention of immunodeficiency conditions.

The drug has both antibacterial and antiviral activity due to the high antagonistic activity of the microbial association of spores of bacteria of the genus Bacillus and the action of glauconite, it also stimulates cellular and humoral factors of immunity and is able to increase nonspecific resistance of the body.

The bacterial association immobilized on glauconite is highly resistant to the digestive juices and enzymes of the gastrointestinal tract of mammals and the ability to rapidly colonize the stomach and intestines. When taken orally with water or food, bacteria multiply in the gastrointestinal tract for 3-6 days, then they are completely eliminated from the body, without causing negative consequences even with an overdose.

Propagating, bacteria with their proteases lyse all proteins unusual for the mammalian organism, denatured proteins and nucleoproteins. At the same time, bacterial toxins, elements of tumor neoplasms and other defective cells are destroyed. Healthy cells remain intact due to the presence of anti-enzyme inhibitors in them.

The proposed method is sufficiently technological, does not require the use of expensive enzymes at the stage of cultivation, does not require purification and concentration of the drug; removal or redistribution of moisture combined with obtaining the finished form of the drug.

The essence and advantages of the claimed group of inventions are illustrated by the following examples.

Example 1. Strains B.subtilis VKPM B-3679 and B.licheniformis VKPM B-4161 were cultured on Gromyko medium (meat-peptone agar + wort agar in a ratio of 1: 1) at 37 ° C for 48 hours. Bacteria were washed off the agar surface with a 7% NaCl solution and warmed up at 121 ° C for 15 min. The resulting lysate was diluted with physiological saline to obtain variants with different cell concentrations (according to the optical turbidity standard). In another embodiment, the bacteria were washed off the agar surface with a 7% NaCl solution and diluted with physiological saline to obtain variants with different cell concentrations (according to the optical turbidity standard).

Option 1

Biomass lysate of B. subtilis and B. licheniformis strains - 1 × 10 ⁸ CFU / ml

Option 2

Biomass lysate of B.subtilis and B.licheniformis strains - 1 × 10 ⁹ CFU / ml

Option 3

Biomass lysate of B. subtilis and B. licheniformis strains - 1 × 10 ¹⁰ CFU / ml

Option 4

Biomass lysate of B. subtilis and B. licheniformis strains - 1 × 10 ¹¹ CFU / ml

Option 5

Biomass of B.subtilis and B.licheniformis strains - 1 × 10 ⁸ CFU / ml

Option 6

Biomass of B.subtilis and B.licheniformis strains - 1 × 10 ⁹ CFU / ml

Option 7

Biomass of B.subtilis and B.licheniformis strains - 1 × 10 ¹⁰ CFU / ml

Option 8

Biomass of B.subtilis and B.licheniformis strains - 1 × 10 ¹¹ CFU / ml

Studied the antagonistic activity of the obtained variants of the drug in relation to test cultures (table 3).

Table 3 Antagonistic activity of various probiotic variants Drug options Zone of growth inhibition of test cultures, mm Staphylococcus aures Salmonella typhimurium Shigella zonnei Candida albicans one 22 9 9 17 2 25 eleven 9 twenty 3 28 12 10 22 four 26 10 10 19 5 6 10 9 21 6 6 12 10 23 7 29th 13 12 25 8 27 eleven 12 24

As can be seen from the data shown in table 3, the optimal number of microbial cells in 1 ml of the drug is - 1 × 10 ⁹ -1 × 10 ¹⁰ CFU / ml of both living cells and their lysates. A further increase in the number of bacterial cells does not significantly change the specific activity of the drug against test cultures of microorganisms.

Example 2. The study of the antagonistic activity of strains of B. subtilis VKPM B-3679 and B.licheniformis VKPM B-4161

Antagonistic activity against test cultures is checked by the method of delayed antagonism. Shigella sonnei, Salmonella typhimurium are used as test cultures. S.stenly, S.reading, S.derby, Staphylococous aureus, Pseudomonas aeruginose, Proteus vulgaris, Klebsiella pneumoniae, Candida albicans, C.tropicalis, etc. The test microbes used must satisfy the following requirements: be in S-form, have typical morphological and enzymatic properties.

Used strains of test microbes gave a positive kerato-conjunctival test in guinea pigs.

Staphylococcus test strains formed a 3-5 mm hemolysis zone around their colonies after 18-20 hours of growth on blood agar, coagulated the rabbit blood plasma for 2 hours, caused necrosis for 48-72 hours with an intradermal injection of 200 million rabbits microbial cells of daily culture in 0.2 ml of saline.

The study of the antagonistic activity of B. subtilis VKPM B-3679 and B. licheniformis VKPM B-4161 strains was carried out in a dense Gause N2 medium.

Recipe for dense medium Gauze N2 per 1 liter:

1. Hottinger broth (700 mg% total nitrogen) - 30 ml

2. Peptone - 5 g

3. Sodium chloride - 5 g

4. Glucose - 10 g

5. Microbiological agar - 15 g

6. Distilled water - up to a total volume of 1 liter.

After mixing the components, the medium was heated until the agar was completely dissolved and filtered through a cotton-gauze filter into 0.3-0.4 l flasks. The flasks with the medium were sterilized in an autoclave at a pressure of 0.1 MPa for 15 minutes. After cooling to a temperature of (45 ± 2) ° C, the medium was poured into sterile Petri dishes. Petri dishes with the medium were placed in a thermostat and kept at a temperature of (371) ° C for (24 ± 2) hours to verify sterility.

Cultures of Shigella sonnei, Salmonella typhimurium, Staphylococcus aureus, Candida albicans and other above test cultures were grown on Petri dishes for (18 ± 2) hours on meat peptone agar (MPA). 1 ml of physiological saline was poured into sterile tubes and suspensions of test strains were prepared with a concentration of (3.0 ± 0.2) 10 ⁹ cells / ml.

For testing, four samples of 2 g were taken from each series. Next, 0.5 g of the preparation based on the B. subtilis strain was diluted in 1 ml of physiological saline. The resulting suspension was streaked using a microbiological loop along the diameter of a Petri dish with a dense Gause medium N2. Crops were incubated in an incubator at 37 ° C for 72 hours. Then, the test microbe seed was seeded to the grown culture by the method of perpendicular strokes.

The results were taken into account after 18 hours of incubation at 37 ° C according to the size of the zones of lack of growth of test cultures. The growth control of test cultures was controlled by their parallel seeding on plates with the same dense Gauze N2 medium without the studied culture association.

Strain B.subtilis VKPM B-3679 is characterized by the following indicators of zones of growth inhibition of the test culture (mm):

Shigella sonnei - 17-22

Salmonella typhimarium - 21-25

S.stenly - 13-17

S.reading - 14-18

S.derby - 15-18

Staphylococcus aureus - 22-27

Pseudomonas fluoresocens - 8-10

Pseudomonas vulgaris - 10-12

Pseudomonas aeruginose - 7-9

Proteus vulgaris - 12-14

Klebsiella pheumoniae - 13-15

Candida albicans - 25-32

C. tropicalis - 22-26

Strain B.licheniformis 4161 is characterized by the following indicators of growth inhibition zones of the test culture (mm):

Shigella sonnet - 22-27

Salmonella typhimurim - 26-30

S.stenly - 18-22

S.reading - 19-23

S.derby - 20-23

Stapgylococcus aureus - 27-33

Pseudomonas aeruginose - 12-15

Proteus vulgaris - 17-19

Klebsiella pneumoniae - 18-20

Candida albicans - 30-34

C. tropicalis - 27-31

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

It should be noted that the strains do not exhibit antagonistic activity against representatives of normal microflora - bifidobacteria, lactobacilli and Escherichia coli and relative to each other.

The spectrum of the biological activity of the B.subtilis strain VKPM B-3679 was additionally studied by diffusion of the produced biologically active substances (BAS) into the agar along the growth retardation zones of test cultures.

Test cultures at a concentration of 10 ⁹ CFU / ml were seeded onto the surface of an agar medium (100 μl of suspension / cup). Drills (6 mm in diameter) in agar were used to make wells into which 100 μl of B. subtilis culture lysate was added. Cups were cultured at 37 ° C for 24-48 hours. The activity of the drug was determined by zones of inhibition of growth of strains.

Table 4 The spectrum of biological activity of B.subtilis strain VKPM B-3679 Test culture Zone of growth inhibition of test cultures, mm S.aureus 209 13 ± 1.8 S.aureus 3H 14 ± 2.0 E.fecalis 115H 17 ± 1.9 E.coli 22H 9 ± 1.1 Enterobacter ssp. 3h 10 ± 1.4 Candida albicans 5H 16 ± 2.0 Helicobacter pylori 2 14 ± 1.3 H.pylori 1 11 ± 0.7 H.pylori 5 15 ± 1.4 H. pylori 38 12 ± 1.2 H. pylori 11 13 ± 0.9 H. pylori 54 16 ± 1.7

The data in table 4 indicate that the probiotic strain B.subtilis VKPM B-3679 produces biologically active substances with a wide spectrum of specific action.

Example 3. With industrial technology, the cultivation of strains of B. subtilis and B. licheniformis is carried out separately in a fermenter with glucose-molasses medium containing: (NH ₄ ) ₂ SO ₄ - 10.0 g / DM ³ ; KH ₂ PO ₄ - 4.0 g / dm ³ ; molasses - 50.0 g / dm ³ ; glucose - 20.0 g / dm ³ ; soy peptone - 2.0 g / dm ³ ; yeast extract - 3.0 g / dm ³ , pH 6.8-7.0, at a temperature of 37 ° C, aeration rate of 1.0 air volume / volume medium per minute; mixing speeds of 250 rpm; pO ₂ - 40-60% of saturation for 24-36 and 30-36 hours, respectively.

The process is considered complete if the cell concentration is 3-3.5 and 1 billion / ml, respectively, and the ratio of spores and vegetative cells is 1: 1.

At the end of the incubation of the strains B.subtilis and B.licheniformis and mixing in a ratio of 1: 1, the mixture is concentrated on bactofuge with periodic unloading of the paste. The separation of the culture fluid is carried out at a rotor speed of 6000 rpm The feed rate of the culture fluid is 80-100 l / h. Unloading is done every hour. The yield of paste by volume is 10-15% of the volume of culture fluid. The solids content in the paste is 18-20%. At the end of the separation, the paste is cooled to 20 ° C, poured into ampoules (bottles) or into stainless steel cassettes, after which it is frozen and dehydrated in a vacuum dryer or spray dried without the addition of protectors.

Freeze-dried spore concentrate Bacillis subtilis 3/28 strain VKPM B-3679 and Bacillis licheniformis L-34 strain VKPM B-4161 is mixed with glauconite in the mixer to a CFU content of at least 1-3 × 10 ⁹ in 1 gram.

Example 4

Getting the drug by dehydration.

At the end of the incubation of the strains of B. subtilis and B. licheniformis and mixing them in a ratio of 1: 1, according to the technology of example 4, 10% of the weight parts of the mass of the final form of the drug are selected. A portion of biomass in liquid form is mixed with the estimated amount of glauconite until a residual moisture content of 4-6% is reached, after which the preparation is packaged. If necessary, the drug is dried in a vacuum drying unit (on shelves). The final cell content is not less than 1 × 10 ⁹ CFU / g.

Example 5. Obtaining the drug in vacuum drying units.

At the end of the incubation of the B.subtilis and B.lichenifbrmis strains and mixing 1: 1, according to the technology of Example 4, 10% by weight of the final form of the preparation was selected. A portion of biomass in liquid form is sprayed onto the estimated amount of glauconite gushing in air heated to 90 ° C in a drying unit until a residual moisture content of 4-6% is reached, after which the preparation is unloaded and packaged. The final cell content is not less than 1 × 10 ⁹ CFU / g.

Example 6. The study of the stability of the drug. The effect of the production technology on the stability of Toxisporin was studied using the example of a study of 9 experimental series of a preparation obtained in three different ways. The results are shown in table 5.

Table 5. The stability of Toxisporin obtained by various drying methods Drying method Indicators of Toxisporin during storage, CFU / g 6 months 12 months 18 months 24 months Sublimation 3 × 10 ⁹ 3 × 10 ⁹ 3 × 10 ⁹ 2 × 10 ⁹ Capillary chemical dehydration 4 × 10 ⁹ 3 × 10 ⁹ 2 × 10 ⁹ 2 × 10 ⁹ Vacuum Drying Oven 2 × 10 ⁹ 2 × 10 ⁹ 2 × 10 ⁹ 10 ⁹

As the results show, after storage of the drug at room temperature for 24 months. the content of live microbial cells in no batch was lower than 10 ⁹ CFU / g, which indicates the equivalence of these methods of production.

Example 7. The study of the ability of the drug Toxisporin to adsorb rotavirus from a liquid.

The adsorption activity of the probiotic Toxisporin against rotaviruses was studied by analyzing the change in the amount of probiotic in the virus-containing fluid. We used the culture fluid of cattle rotaviruses - Tiverval and human strains HRV-134 on green monkey kidney cell cultures with 0.25% trypsin solution (20 PFU). For studies, the following doses of the probiotic Toxisporin in a virus-containing liquid (mg / ml) were selected: 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40 , 0, 45.0, 50.0.

10.0 ml of virus-containing liquid was added to vials with various weights of Toxisporin. The vials were vigorously shaken for 10 minutes, centrifuged at 3000 rpm for 10 minutes. The supernatant was selected and examined for the presence of an infectious-active virus by titration in a cell culture using a CPD of ₅₀ / ml viral antigens in RNGA.

The obtained values of the titer of the viruses were converted into% adsorbed virus according to the formula: A = 100 · (1-T / To)%,

where T is the titer of the virus in the supernatant after adsorption,

That is the original titer of the virus in the culture medium.

As a result of the study, the ability of the probiotic Toxisporin to adsorb rotavirus from the culture fluid was established.

It was found that the introduction of the probiotic Toxisporin in an amount of 1.0 mg / ml the amount of adsorbed virus for strain HVR-134 was 45.0 ± 1.0%, for strain Tiverval - 65.0 ± 0.5%. An increase in the dose of Toxisporin to 10.0 mg / ml increased the percentage of adsorbed virus for all studied strains.

Sorbed: 73.0 ± 1.0% of HRV-134 strain and 80.0 ± 0.6% of Tiverval strain. An increase in the dose of Toxisporin to 15.0 mg / ml led to a further increase in the percentage of adsorbed rotavirus: 95.5 ± 0.3% for the Tiverval strains and 89.4 ± 0.4% for the HRV-134 strain. A further increase in the dose of Toxisporin in the virus-containing fluid did not cause changes in the achieved values.

Studies have shown the ability of the probiotic Toxisporin to adsorb rotaviruses from a liquid medium, which gives reason to use it for the prevention and treatment of rotavirus diarrhea in animals and humans.

Example 8. The therapeutic efficacy of the drug Toxisporin in gastrointestinal diseases of piglets.

Evaluation of the therapeutic effect of the drug was carried out on suckling pigs of 2-3 weeks of age (26 goals, of which 8 were control) and weaned piglets at the age of 2-3 months (40 goals, of which 10 control). For this purpose, animals with signs of diarrhea were selected: an oppressed state, a low need for food, the presence of liquid feces with an unpleasant odor, and increased body temperature. Sick animals of the experimental groups received the drug in different doses 2 times a day for 6 days. Before use, the device was dissolved in warm boiled water and given to sucking pigs from a rubber pipette, and weaned piglets were mixed with food. Piglets-suckers of the control group were treated with tilon (1.0 ml inside for 4-5 days), and weaned piglets with a mixture of farmazin and trichopolum (4-5 days). Observations were carried out within 60 days from the start of the experiment. The scheme for the formation of experimental and control groups of piglets depending on the applied doses of the drug and the results of clinical observation are presented in table 6.

Table 6 Therapeutic efficacy of Toxisporin in gastrointestinal diseases of piglets Groups of animals Heads Treatment course Dose and Multiplicity Recover, goal. Fallen goal Efficiency% Sucking pigs 1 gr.-3.0 g per goal. 10 6 days 0.5 × 2 10 - one hundred 2 gr. - 2.5 g per goal. 8 6 days 0.25 × 2 8 - one hundred 3 gr. - control 8 4-5 days - 5 one - Weaned piglets 1 g - 10.0 g per goal. twenty 6 days 1.0 × 2 twenty - one hundred 2 gr. - 5.0 g per goal. 10 6 days 0.5 × 2 9 - 90.0 3 gr. - control 10 4-5 days - 6 - -

From the data presented in the table shows that the high therapeutic efficacy of Toxisporin (100%) is observed with a dose of 3 g per head and 2.5 g per head to pig suckers once a day for 6 consecutive days. In weaned piglets, 100% effectiveness of the drug was observed in the group where animals were treated with Toxiosporin at the rate of 10.0 g per head once a day. The duration of the disease in piglets of the experimental groups was 5-7 days, in piglets of the control groups - 7-10 days. One pig of the control group fell on the 4th day of illness.

Thus, an effective therapeutic dose of Toxisporin for suckling pigs is 3.0 g per head once a day for 6 days in a row (100% effectiveness), and for weaned pigs - 10.0 g per head once a day for 6 days in a row (100% efficiency). Feces from piglets of all groups were taken for bacteriological studies before and after the experiment and quantitative and qualitative bacterial composition of the intestinal microflora was determined in 1 g. Identified bacteria were determined biochemical, hemolytic, adhesive properties and pathogenicity in white mice (table 7).

Table 7 Bacteriological studies of feces of piglets before and after giving Toxisporin in therapeutic doses Groups of animals Research dynamics Before giving the drug After treatment Piglets - Suckers 1 group 3.0 g per goal. Bifido-lactobact. - 10 ⁵ , E. coli - 10 ⁶ ; P.mirabilis - 10 ⁵ , Salmonella - 10 ³ , C.freundi - 10 ³ . Bifido-lactobact. - 10 ¹⁰ , E. coli - 10 ⁷ , C.freundi - 10 ³ . 2 group 2.5 g per goal. Bifido-lactobactobacter-10 ⁵ ; E. coli-10 ⁶ -10 ⁷ ; P.mirabilis - 10 ^5; K.pneumonia - 10 ⁶ . Bifido-lactobactobact - 10 ⁹ ; E. coli - 10 ⁸ . Control group Bifido-lactobact. - 10 ⁵ , E. coli - 10 ⁶ -10 ⁷ , P.mirabilis - 10 ⁵ , Salmonella - 10 ³ , K. pneumonia - 10 ⁵ Bifido-lactobact. - 10 ⁸ , P.mirabilis - 10 ⁵ , Salmonella - 10 ³ , K. pneumonia - 10 ⁵ Piglets - weaners 1 group of 10.0 g per goal. Bifidolactobact. 10 ⁷ , E. coli 10 ⁷ -10 ⁸ , P. mirabilis 10 ⁵ , Salmonella 10 ³ , K. pneumonia 10 ⁶ , Staphilococcus 10 ³ . Bifido-lactobact. - 10 ¹⁰ , E. coli - 10 ⁷ . 2 group 5.0 g per goal. Bifidolactobact. 10 ⁶ , E. coli 10 ⁷ , P. mirabilis 10 ⁵ , Salmonella 10 ³ , C. freundi 10 ³ , Staphilococcus 10 ³ . Bifido-lactobact. - 10 ⁸ , E. coli - 10 ⁷ , P.mirabilis - 10 ⁵ , C.freundi - 10 ³ , Staphi-lococcus - 10 ³ . Control group Bifidolactobact. - 10 ⁷ , E. coli - 10 ⁷ -10 ⁸ , P.mirabilis - 10 ⁵ , P.vulgaris - 10 ⁵ , Salmonella - 10 ³ . Bifido-lactobact. - 10 ⁸ , E. coli - 10 ⁷ -10 ⁸ , P.mirabilis - 10 ⁵ , P.vulgaris - 10 ⁵ , Salmonella - 10 ³ .

From the table it follows that the content of bifidobacteria and lactobacilli in 1 g of the intestinal contents of sucking piglets before giving the drug was at a fairly low level and amounted to 10 ⁵ mcl E. coli and P. mirabilis were isolated from all sucking piglets of both the experimental and control groups. From the sucking pigs of the 1st experimental group, in addition, Salmonella and C.freundi were isolated; K.pneumonia was also isolated from sucking pigs of the 2nd experimental and control groups.

The same microflora was isolated from weaned piglets as from suckling pigs, as well as Staphylococcus. Isolated Staphylococcus were coagulase-positive, which is one of the signs of the pathogenicity of these microorganisms.

The conditionally pathogenic microflora isolated from piglets to the setting of the experiment possessed virulent properties. Microorganisms such as P. mirabilis, E. coli, and Salmonella formed hemolysis zones on blood agar. These properties were especially pronounced in enteropathogenic E. coli (mainly β-hemolysis predominated).

Adhesive properties were determined in RA with anti-adhesive sera K88, K99, F41, P987. Enteropathogenic E. coli isolated from piglets of both the experimental and control groups had various adhesive antigens - 80% of the E. coli isolates contained the K88 antigen, 12% of the K99 antigen and 8% of the E. coli isolates contained the F41 antigen. In addition, 8 P. mirabilis isolates gave positive RA with K99 adhesive serum. In this case, the control was the same microorganisms grown at room temperature (in this case, adhesive antigens are not formed).

The pathogenicity of the isolated microorganisms was determined on white mice, which were injected intraperitoneally with daily cultures of microorganisms at a concentration of 500 million cubic meters. Mice fell on 2-3 days. At autopsy of the dead mice, the original bacteria were isolated.

The results of the experiments are presented in table 8.

Table 8 Virulent properties of bacteria isolated from piglets Research cultures Before giving the drug After treatment hemol. adhesion. toxig. hemol. adhesion. toxig. E.coli 22 22 22 fifteen fifteen fifteen P.mirabilis fifteen eighteen fifteen 12 5 10 P.vulgaris 2 - 2 - - - C.freundii eleven 2 eleven 8 - 8 Salmonella 9 3 9 3 - one K.pneumonia 6 - 6 3 - 3 Staphylococcus 15 cultures coagulative 7 coagulative positive cultures

From the table it follows that after treatment with Toxisporin, there was not only a decrease in the frequency of isolation of opportunistic and pathogenic bacteria, but their virulent properties also weakened. This is especially noticeable in the group of weaned piglets, where the number of coagulase-positive strains of staphylococci halved, indicating a high antagonistic effect of the drug in relation to these bacteria.

In the control group of weaned piglets, no significant changes in the composition of the intestinal microflora were observed by the end of treatment. The intestinal microflora was normalized slowly, and the cell concentration was 10 ⁸ μl. in 1 g of intestinal contents. The same microflora was distinguished as before the setting of the experiment.

The results of bacteriological studies suggest that the cause of digestive upset in piglets was the association of opportunistic microorganisms with virulent properties. In the study of feces after treatment of piglets with various doses of the drug Toxisporin, the following was noted:

- in the group of suckers, where the piglets received the drug in a dose of 3 g, there was an increase in the concentration of bifidobacteria and lactobacilli up to 10 ⁹ -10 ¹⁰ mcl in 1 g of feces, and in the group of sucking piglets, which were given the drug at a dose of 10 g, the concentration of bifidobacteria and lactobacilli increased to - 10 ⁸ -10 ⁹ m cells. Piglets of the control group showed a low content of bifidobacteria and lactobacilli (10 ³ -10 ⁵ m cells);

- salmonella was not found in the piglets of the experimental groups after treatment, while in the control groups these microorganisms were found on all days of the study;

- in the group of weaned piglets, by the end of treatment, there was an increase in the content of bifidobacteria and lactobacilli up to 10 ¹⁰ mcl in 1 g of feces, especially in the group where 10.0 g of the drug Toxisporin was used - per 1 head;

- on the 6th day from the moment of setting up the experiment, weakening of the virulent properties of bacteria isolated from the intestines of the piglets of the experimental groups was established.

Hematological studies were also carried out, which consisted in the analysis of the blood leukogram of the sick piglets in the control and experimental groups (Table 9).

Table 9 Blood leukogram of suckling piglets Piglets at the age of 14 days (norm on the Kar-way) Clinically healthy Sick piglets After treatment 1 group 2 group Control group White blood cells Basophils - 0 0 0 0 0 Eosinophils 0.6-1.4 0.62 0.9 1,0 1,0 1,5 Neutrophils: YU 0.5-1.1 0.45 0.9 1,0 1,0 1,0 P 4.3-9.7 9.95 10,4 9.6 10.25 10.75 FROM 29.1-36.9 30,4 31.3 33.6 41.0 40,0 Lymphocytes 51.2-56.8 55,4 52.1 54.0 37.5 43.25 Monocytes 3.2-4.88 3.4 6.3 3.6 4.75 3.25

When analyzing the leukogram of sick sucking piglets before treatment, first of all, pronounced monocytosis was noted, indicating a bacterial infection and increased phagocytic activity of these cells. Microscopy of blood smears in monocytes showed bacterial inclusions in the cytoplasm. Many lymphocytes with cytoplasmic protrusions were also noted, which indicates intoxication of the body.

Analyzing the leukogram data of sucking piglets who received various doses of the drug, it can be stated that after stopping the administration of Toxisporin in piglets of the 1st group receiving 3 g of the drug per head once a day, the leukogram indicators corresponded to those of healthy piglets.

Group 1 piglets treated with Toxisporin 2.5 g once a day showed an increase in the number of segmented neutrophils in combination with lymphopenia, which is probably due to an insufficient dosage of the drug and the presence of an inflammatory process in the body. A similar picture of the leukogram in the post-therapeutic period was observed in piglets of the control group, which were treated with chemotherapeutic drugs.

The blood leukogram of sick weaned piglets is presented in table 10.

Table 10 Blood leukogram of weaned piglets 3-4 month old piglets (Karput norm) Clinically healthy Sick piglets After treatment White blood cells 1 group 2 group Control group Basophils 0.2 0 0 0 0 0 Eosinophils 1.1-3.3 2.0 1,0 1,6 0.5 0.7 Neutrophils: 0.4-0.8 0.4 0.53 0.6 0.5 0.6 YU 9.6-13.3 14.2 9.6 9.3 11.0 11.0 P 27.2-32.0 32.8 17.8 23.7 27.5 34,4 FROM 49.0-57.8 47.1 64.0 61.0 58.5 50.3 Lymphocytes 1.9-5.5 3,5 9.7 4.0 2.0 3.0

As follows from table 10, the leukogram of diseased weaned piglets was characterized by eosinopenia, neutropenia, severe monocytosis and elevated lymphocytes. When analyzing the leukogram of sick weaned piglets before treatment, the following was established:

in sick animals, compared with clinically healthy animals, eosinopenia was noted, which characterizes acute intoxication of the body. The decrease in the number of eosinophils is associated with the destruction of these cells, as well as with migration to tissues to eliminate antigen-antibody complexes;

the observed decrease in the number of neutrophils, especially segmented, in sick piglets is explained by the destruction of these cells due to the intensification of phagocytosis as a result of the introduction of microbial elements. Microscopy revealed phagocytized bacterial substances in the neutrophil cytoplasm;

severe lymphocytic and monocytosis also indicated a disease of bacterial etiology. Lymphocytes with cytoplasmic protrusions were found in blood smears, which is an indicator of high intoxication of the body;

in comparison with suckling pigs, the weaning process was more pronounced, which is confirmed by bacteriological and hematological studies.

After treatment, weaned piglets noted the following:

in the group of piglets treated with the drug at a dose of 3.0 g once a day, eosinopenia was noted; the number of segmented neutrophils increased by approximately 60%, without reaching the norm, i.e. there was some neutropenia, indicating prolonged phagocytosis in the course of the action of the drug Toxisporin. The number of lymphocytes decreased slightly, but remained at a higher level compared with clinically healthy animals. The number of monocytes was significantly reduced, which also indicates the protective function of these cells;

in piglets treated with Toxisporin 10.0 g per head once a day, the number of eosinophils was almost the same as in clinically healthy piglets. Neutropenia was noted, which may indicate an overdose of the drug. The number of lymphocytes has changed slightly. Monocyte count decreased to normal;

in animals of the control group there was eosinopenia, which indicates a fairly stable and prolonged intoxication of the body. The number of neutrophils, especially mature ones, increased almost 2 times and was higher than in clinically healthy piglets of this farm.

Thus, the nature of the blood leukogram of the weaned piglets of the experimental and control groups indicates an inflammatory process in the animal organism. Nevertheless, the clinical condition of the animals, the high therapeutic efficacy of the drug Toxisporin, as well as the results of bacteriological studies indicate the preferred therapeutic dose - 3.0 g of the drug per head with a single use for 6 days.

When studying the effect of Toxisporin on the growth rate of weaned piglets of the experimental groups put on fattening, Toxisporin was introduced into the feed for the piglets of the experimental groups at a dose of 3 g per head per day for 1 month. In the experiment, the same livestock of weaned piglets was used, with which the drug was administered for prophylactic purposes. Animals of 2 experimental groups were combined into one group (Table 11).

Table 11 Change in live weight and average daily gain in fattening piglets Indicator Group of animals experienced control Live weight kg 43.3 ± 2.16 43.6 ± 1.22 The average daily gain, g 624 ± 26.9 534 ± 42.8 In% to the control group 116.8 100.0 Total gain, kg 67.4 58.2 The average daily consumption of feed, kg 2.18 2.18 Combined feed consumed per 1 kg of growth, kg 3.27 3.78 In% to the control group 86.5 100.0

It was established that Toxisporin promotes the assimilation of feed by animals and the increase in live weight.

In the pigs of the experimental group treated with Toxisporin, the average daily weight gain increased to 667 g (P <0.02), in animals of the control group, the total for the experiment was 576 g.

Thus, the average daily weight gain in pigs of the experimental group increased by 15.8% compared with the control.

In addition to increasing the growth rate of piglets, the use of Toxisporin in the feed composition improved the efficiency of feed use, which resulted in a decrease in their cost per 1 kg of the live weight gain of the fattening pigs of the experimental group by 16.9-19.4%.

As a result of fecal studies of experimental animals, it was found that the use of the drug Toxisporin for fattening pigs led to an increase in the digestibility of nutrients in animals of the experimental group - fat and BEV by 3.2 abs.% And 4.4 abs.%, Respectively.

Compared to control analogues, animals of the experimental group were digested better by a statistically significant value (P <0.01) or in percentage terms by 13.8 abs.% (Table 12).

Table 12 Digestibility of nutrients in experimental piglets The indicator, abs.% Group of animals experienced control Organic matter 83.8 ± 1.15 80.6 ± 1.73 Crude protein 79.5 ± 1.27 75.1 ± 2.08 Crude fat 43.2 ± 6.39 40.0 ± 3.55

The use of Toxisporin had a positive effect on the use of nitrogen in the body of piglets of the experimental group (Table 13).

Table 13 Average daily balance and nitrogen use in experimental animals Indicator Group of animals experienced control Taken with food, g 67.5 66.72 Highlighted in feces, g 13.84 ± 0.62 16.67 ± 1.38 Overcooked, g 53.66 ± 0.3 50.05 ± 1.38 Excreted in urine, g 28.50 ± 1.1 27.08 ± 0.51 Deposited in the body, g 25.16 ± 1.15 22.97 ± 1.55 Used, in%: from accepted 37.3 34.44 from overcooked 46.8 45.8

It was established that the “loss” of nitrogen with feces and urine in the piglets of the control group amounted to 43.75 g, and the experimental - 42.34 g, in connection with which 22.97 g of nitrogen was daily deposited in the animals of the control group, compared with the control piglets from the experimental group laid off 2.19 g more nitrogen in the body. Piglets from the experimental group also used nitrogen most efficiently. Of the accepted amount of nitrogen with food, they deposited 37.3% in the body, and 46.8% of the digested nitrogen, against 34.44% and 45.8% in the control.

The effect of the drug Toxisporin on the quality of meat productivity was evaluated after a control slaughter of 10 piglets and carcasses. The results of slaughter and deboning of carcasses are shown in table 14.

Table 14 Productivity Indicators for Fattened Pigs Indicators Meat productivity by animal group experienced control Slaughter live weight, kg 110.7 101.8 Slaughter weight, kg 86.2 78.6 Slaughter yield,% 77.9 77,2 The mass of the cooled half carcass, kg 32,2 30.6 including tissue mass, kg: muscular 18.3 17.3 fatty 10,4 9.8 bone 3,5 3.2 The ratio of tissues to the mass of half carcass,%: muscular 56.8 56.5 fatty 32.8 32,0 bone 10.9 10.5

Analysis of the results of the slaughter of experimental animals showed that piglets of the experimental group showed an increase in slaughter mass by 9.7%, compared with piglets in the control group. The use of Toxisporin did not significantly affect the quality of the carcasses.

Thus, Toxisporin does not significantly affect the slaughter yield and morphological composition of carcasses, however, the mass of carcasses of animals from the experimental group was greater. From them, on average, 1 head per 3.2 kg of meat was obtained more than from control animals.

Table 15 presents the results of a production audit on changes in live weight of piglets, growth and feed costs.

Table 15 Zootechnical indicators of the effectiveness of Toxisporin in pig fattening Indicator Group of animals experienced control Live weight, kg: at the beginning of fattening 39.6 ± 0.23 39.4 ± 0.25 at the end of the fattening 116.0 ± 0.69 108.0 ± 0.78 The average daily gain, g 735 ± 5.97 660 ± 7.57 % to the control group 111.4 100.0 Average daily feed intake 2,30 2,30 Combined feed consumed per 1 kg of growth, kg 3.13 3.48 % to the control group 89.9 100.0

It was established that Toxisporin promotes the assimilation of feed by animals and the increase in live weight. The average daily gain in live weight during the production test in animals of the experimental group compared to the control was 5 g higher (11.4% with a reliable p <0.001), and the cost of feed per 1 kg was 10.1% lower (3.13 kg vs 3.48 kg). Analysis of the results of the slaughter of experimental animals showed that piglets of the experimental group showed an increase in slaughter mass by 9.7% compared with piglets in the control group.

The data showed that Toxisporin is effective for all mammals in the treatment and prevention of colibacterioses, dysfunctions and dysbacterioses of various nature, plague, parainfluenza, influenza, dyspepsia, salmonella, infectious rhinotracheitis in cattle, viral diarrhea, rotavirus enteric infection, dysentery, enteritis, dysentery, enteritis, dysentery, enteritis, dysentery, enteritis, dysentery, enteritis, dysentery, enteritis, dysentery, enteritis, dysentery, enteritis, dysentery, enteritis, dysentery stomatitis, ulcerative dermatitis, as well as for the prevention of immunodeficiency conditions.

A production assessment of the effectiveness of the drug Toxisporin in pig fattening confirmed the results of experiments on weaned piglets on the beneficial effect of the drug on animal homeostasis, growth rate and feed conversion.

The proposed method is sufficiently technological, does not require the use of expensive enzymes at the stage of cultivation, does not require purification and concentration of the drug; removal or redistribution of moisture combined with obtaining the finished form of the drug.

Claims (7)

1. A probiotic preparation against viral and bacterial infections, containing a mixture of the biomass of the bacterial strains Bacillus subtilis and Bacillus licheniformis in spore form and an excipient, characterized in that as the biomass of the bacterial strains it contains the biomass of the bacterial strains Bacillus subtilis VKPM B-3679 and Bacillus licheniformis VKPM B-4161 in spore form with a titer of at least 10 ⁹ spores / g, and glauconite as a filler in the following ratio of ingredients, wt.%:
a mixture of Bacillus subtilis bacteria spores and Bacillus licheniformis 0.1 -1.0 glauconite rest 2. The probiotic preparation according to claim 1, characterized in that the spore content of Bacillus subtilis bacteria in their mixture with Bacillus licheniformis spores is 50%. 3. The method of obtaining the probiotic preparation according to claim 1, which includes separate cultivation of the bacteria Bacillus subtilis VKPM B-3679 and Bacillus licheniformis VKPM B-4161 on glucose-molasses medium at pH 6.8-7.0, mixing the obtained biomass of the strains in spore form in a ratio of 1: 1 between themselves and then in an amount of 0.1-1 wt.% by weight of the drug with glauconite to a CFU content of at least 10 ⁹ spores / g. 4. The method according to claim 3, characterized in that the microorganisms are cultured at a temperature of 37 ° C for 24-36 hours, aeration of 1.0 vol. air / vol. medium per minute, stirring speed 250 rpm, pO ₂ 40-60% of saturation, pH 6.8-7.0. 5. The method according to claim 3, characterized in that the resulting mixture of the biomass of the strains in spore form is subjected to freeze-drying before mixing with glauconite. 6. The method according to claim 3, characterized in that the bacteria are applied to glauconite by spraying. 7. The bacterial strain Bacillus licheniformis VKPM B-4161, used as a component of a probiotic preparation against viral and bacterial infections.