EA043409B1 - VETERINARY PROBIOTIC PRODUCT - Google Patents

Description

Field of technology

The invention relates to biotechnology (feed production, agriculture, veterinary medicine), in particular to probiotic-type veterinary preparations, and can be used for the treatment and prevention of diseases in animals and poultry, including bacterial and fungal infections.

Prior Art

Probiotics are preparations of living microorganisms that, when introduced into the human or animal body, have a positive effect on the physiological, biochemical and immune reactions of the host by optimizing the composition of its intestinal microflora. Probiotics are increasingly used in veterinary medicine as feed additives.

Probiotics consisting of bacteria producing lactic and propionic acids (Lactobacillus, Bifidobacterium, Propionibacterium, Enterococcus), non-pathogenic bacteria of the genus Escherichia, spore-forming bacteria (Bacillus, Closthdium), yeast (Saccharomyces), thermophilic streptococci (Streptococcus) have become widespread as veterinary biological products. .

Of particular interest are probiotics based on spore-forming bacteria of the genus Bacillus. It is known that bacteria of the genus Bacillus suppress the growth and development of pathogenic and opportunistic microflora of the intestines of animals and birds. Microorganisms of the genus Bacillus have a number of valuable properties: they produce many different enzymes, amino acids, proteins, vitamins, antibiotics, and nucleotides. Representatives of this genus are characterized by high adaptability to environmental conditions and the ability to form spores. In this regard, various strains of bacteria of the genus Bacillus are widely used as probiotic preparations, both in the form of monopreparations containing only one strain of the genus Bacillus, and in the form of combined preparations combining several strains of bacteria of the genus Bacillus or bacteria of different genera in various combinations.

Thus, a preparation is known from the prior art that includes a mixture of biomass of bacteria of the genus Bacillus of the following strains: Bacillus subtilis VKPM V-7048, Bacillus licheniformis VKPM V-7038 and the recombinant strain Bacillus subtilis VKPM V-7092 in equal proportions in spore form with a titer of at least 3x101 ° spores/g (RU 2142287, 12/10/1999). The drug has a wide spectrum of antiviral and antibacterial activity, however, the presence of genetically modified components in its composition imposes restrictions on its use.

A known drug for the prevention and treatment of purulent-inflammatory diseases and dyspepsia in animals (RU 2172175, 08/20/2001). The biological product includes the biomass of Bacillus subtilis 12 B-DEP and a filler in the following ratio of components, wt.%: biomass of B. subtilis 12 B-DEP - living microbial cells - (1-2)x10 ⁹ in 1 ml of solvent 92-95 wt.% , filler 5-8 wt.%. The biological product suppresses the development of veterinary strains of pathogenic microorganisms of the following genera: Streptococcus, Eshehchia coli, Pseudomonas, Sthaphilococcus, Proteus, Shigella, Hexaera, has broad resistance to antibiotics and is effective in treatment.

A probiotic medicinal product for farm animals and poultry is known, which contains a mixture of suspensions of bacterial strains and a protective medium (RU 2326692, 06/20/2008). As a protective medium, the product contains a sucrose-gelatin mixture with the following ratio of components, wt.%: sucrose - 15; gelatin - 5; 9% sodium chloride solution - 80. As a mixture of strain suspensions, the product contains suspensions of the following strains, wt.%: Azomonas agilisc B-2586 - 8; Azotobacter chroococcum B-3162 - 7; Azotobacter vinelandii B-2587 - 7; Bacillus subtilis B-5225 - 7; Bacillus subtilis B-4828 - 9; Bifidum bacterium globosum B-2584 - 10; Eschehchia coli B-4412 - 8; Enterococcus faecium B-2898 - 7; Lactobacillus acidophilus B-3488 - 7; Lactobacillus acidophilus B-2585 - 7; Propionibacterium freudenreichii B-6561 - 7; Pseudomonas sp. B-3908 - 7; Pseudomonas sp. B-2589 - 7; Saccharomyces cerevisiae Y-365 - 2. The product allows you to normalize the intestinal microbiocenosis of healthy animals and poultry, reduce the incidence of purulent-inflammatory and dyspeptic diseases, reduce mortality and increase body weight gain in young animals.

The drug Vetosporin-active is a free-flowing powder of activated carbon with sorbed cells of living bacteria of the probiotic strains Bacillus subtilis 11B and Bacillus subtilis 12B, black and odorless. Used for the prevention and treatment of poisoning, diarrhea, allergies, indispensable for poor-quality feed, necessary after taking antibiotics and anthelmintic drugs, for stress, hypothermia and overheating.

Enzymsporin is an innovative feed probiotic additive for optimizing digestion processes, increasing productivity and safety of pigs, cattle and poultry. Designed to reduce the level of intestinal colonization by opportunistic pathogenic microorganisms. Enzymsporin contains three strains of spore-forming bacteria of the genus Bacillus in equal proportions, namely: Bacillus subtilis BKM B-2998D (VKPM B-314), Bacillus licheniformis BKM B-2999D (VKPM B-8054), Bacillus subtilis BKM B-3057D (VKPM B -12079). Fillers - whey powder, maltodextrin, corn flour.

Although there are many probiotic products available on the market, the widespread use of antibiotics as growth promoters in livestock and poultry production

- 1 043409 led to the problem of the formation of resistant microflora. Therefore, an important area of research is the search for alternative growth stimulants and the creation of new, more effective probiotic preparations for industrial livestock and poultry farming.

Disclosure of the Invention

The objective of the invention is to create a probiotic composition capable of quickly and effectively restoring intestinal microflora when damaged, as well as increasing its resistance to negative microbiological influences.

This task is achieved by developing a new probiotic composition for use in veterinary medicine based on cultures of microorganisms of the genus Bacillus, including a mixture of biomass of live and spore forms of bacteria of the Bacillus subtilis 1719 strains in a titer of 1-1.5x108 CFU/ml and Bacillus subtilis 3H in a titer of 1-1 .5x108 CFU/ml.

In some embodiments, the probiotic composition further includes a pharmacologically acceptable carrier, diluent or excipient.

In some particular embodiments of the invention, the probiotic composition is in the form of an oral suspension.

In some particular embodiments of the invention, the probiotic composition is in the form of a lyophilisate.

In some particular embodiments of the invention, the bacteria Bacillus subtilis 1719 and Bacillus subtilis 3H are immobilized on a carbon enterosorbent.

This objective is also achieved by using the presented probiotic composition for the prevention and/or treatment of diseases of the gastrointestinal tract.

In addition, the objective is achieved by implementing a method for the prevention and/or treatment of diseases of the gastrointestinal tract in livestock, poultry or fur farming, including the use of a probiotic preparation according to the invention.

In some embodiments of the invention, the probiotic composition is administered orally before feeding or along with food or drinking water at a rate of 0.5-2 ml per 10 kg of animal or bird weight.

In some particular embodiments of the invention, the probiotic composition is administered at the rate of 1 ml per 10 kg of animal or poultry weight.

In some particular embodiments of the invention, the probiotic composition is added to the feed by sprinkling it 20-30 minutes before feeding.

This objective is also achieved by developing feed additives containing the proposed probiotic composition for inclusion in feed, concentrates, premixes or any other dry or liquid feed.

In some embodiments, the feed additive further includes plant, mineral, processing and/or balancing additives.

In some particular embodiments, herbal additives are selected from the group consisting of essential oils, plant extracts, meal, cake; mineral additives are selected from the group consisting of calcium carbonate, feed chalk, monocalcium phosphate, bone meal, sulfur; technological additives are selected from the group consisting of potassium sorbate, sodium benzoate, sodium pyrosulfite, hexamine; balancing additives are selected from the group consisting of protein, protein-vitamin, protein-vitamin-mineral additives, urea food concentrate.

The amount of probiotic composition in the feed additive is calculated to ensure the introduction of 0.5-2 ml of probiotic composition per 10 kg of animal or poultry weight.

The effective synergistic interaction of Bacillus subtilis 1719 and Bacillus subtilis 3H strains ensures the achievement of the following technical results:

A new probiotic composition has been developed based on cultures of microorganisms of the genus Bacillus, capable of quickly and effectively restoring intestinal microflora in case of damage, as well as increasing its resistance to negative microbiological influences, having the following important properties:

has an inhibitory effect against pathogenic and conditionally pathogenic microorganisms, both bacterial and fungal in nature, without suppressing the intestinal microflora, increases the barrier functions of the body, is resistant to most antibiotics, increases the bioavailability of feed, activates intestinal function, does not contain genetically modified microorganisms, microorganisms included in the drug are not pathogenic, not toxic to warm-blooded animals;

The proposed probiotic composition can be used both individually and in a group method with mixed feed, concentrates, mineral supplements, milk substitutes, drinks, etc.

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Detailed Disclosure of the Invention

Definitions and terms.

The following terms and definitions apply throughout this document unless otherwise expressly stated. References to techniques used in the description of this invention refer to well known techniques, including modifications of these techniques and replacement thereof with equivalent techniques known to those skilled in the art.

As used herein, the terms includes and including are interpreted to mean includes, among other things. These terms are not intended to be construed as consisting only of.

The term probiotic composition in this description means a composition obtained from suspensions of Bacillus subtilis 1719 and Bacillus subtilis 3H cells, the concentration of each of which is adjusted to 1-1.5x108 CFU/ml.

By (therapeutically) effective amount (therapeutic dose) is meant the amount of a probiotic composition (probiotic preparation) administered or delivered to an animal (bird) at which it is most likely to exhibit the intended therapeutic effect. The exact amount required may vary from subject to subject depending on the species of animal (bird), age, body weight, general condition, severity of the disease, method of administration of the probiotic composition, combination treatment with other drugs, etc. Administration of the probiotic composition (probiotic preparation) according to the invention to a subject in need of treatment and/or prevention of a disease or condition is carried out in a dose sufficient to achieve a therapeutic effect. When carrying out treatment and/or prevention, administration can be carried out either once or several times a day, more often in the form of a course of administration over a period of time sufficient to achieve a therapeutic effect (from one to several weeks to months), with courses of administration of the probiotic composition can be carried out repeatedly (over the course of a year, several years, etc.).

The term feed additive means an ingredient, premix, additive that can be used to obtain feed compositions (for example, compound feed).

The term animal as used herein refers to mammals, primarily but not limited to farm animals (including, but not limited to, cattle, swine, horses, beasts of burden, fur-bearing animals, domestic animals, captive wild animals).

Unless otherwise defined, technical and scientific terms in this application have their standard meanings commonly accepted in the scientific and technical literature.

The developed probiotic composition based on cultures of microorganisms of the genus Bacillus includes bacterial strains Bacillus subtilis 1719 and Bacillus subtilis 3H, selected based on the study of morphological, physiological-biochemical, genetic and other biological properties and unexpectedly exhibiting a synergistic effect.

Strain B. subtilis 1719, deposited in GISC named after. L.A. Tarasevich under No. 277, has probiotic properties, manifested in the elimination of opportunistic and pathogenic microorganisms (shows antagonism against opportunistic and pathogenic microorganisms of various taxonomic groups), with the restoration of the quantitative and qualitative composition of normal microflora in experimental dysbiosis, has an immunomodulatory effect on the macroorganism , has low adhesive activity, resistant to gentamicin, polymyxin and erythromycin. Strain B. subtilis 3H, deposited in the State Medical University under No. 248, has antagonistic activity, inhibiting the growth of pathogenic and opportunistic bacteria: staphylococci, Proteus, Klebsiella, Escherichia coli, streptococci, Pseudomonas aeruginosa and yeast fungi, and is resistant to rimphapicin. Thanks to the production of enzymes, it promotes the breakdown of proteins, fats, carbohydrates and fiber, improving the digestion and absorption of food. Strains B. subtilis 1719 and B. subtilis 3H are absolutely safe - they have low toxicity, toxigenicity and virulence.

In some embodiments of the invention, the probiotic composition also contains a protective environment to ensure the safety of bacteria during technological processing, obtaining the final formulation and subsequent storage. As a protective medium, the composition may contain, for example, sucrose-gelatin medium, milk powder, gelatin, lactose, sucrose, etc.

The probiotic composition of the invention may further comprise a solvent. For example, distilled water, saline solution, etc. can be used as a solvent.

The probiotic composition of the invention may additionally also contain an excipient commonly used in the manufacture of various formulations. When formed into tablet forms, it may contain, for example, dextrans, polyglucin, starch, polyvinylpyrrolidone, sucrose, lactose, calcium stearate, glucose, sodium bicarbonate, aluminum hydroxide, methylcellulose, talc, etc.

In some embodiments, the probiotic composition may be in the form

- 3 043409 lyophilisate. Such freeze-dried probiotic preparations may also further contain an excipient and/or dispersant. For example, lactose, sucrose, skimmed milk powder are used as a filler; for example, Tween-80 is used as a dispersant. A dispersant is introduced into the composition in order to uniformly distribute the composition (feed additive) in the main diet. A dispersant is also necessary to regulate the sedimentation characteristics of the aqueous solution of the probiotic composition. The presence of a dispersant contributes to the homogeneity of the aqueous solution of the composition, which is very important when using a probiotic feed additive by feeding.

The probiotic preparation may also be presented in the form of a suspension containing the probiotic composition in the form of a suspension in a 7% sodium chloride solution. Such a preparation may also contain excipients such as, for example, starch, Tween, dispersing agents, acesulfame and/or saccharin.

The probiotic preparation can be encapsulated or immobilized on various types of carriers or sorbents, for example, aerosil, cellulose, activated carbon, carboxymethylcellulose, hydroxyethylcellulose, chitosan, etc. Sorbed forms of probiotics accelerate detoxification and reparative processes in the body. In private embodiments of the invention, a carbon enterosorbent is used as a sorbent, for example, activated carbon of the BAU-A, BAU or OU-VK brands.

Pharmaceutically acceptable carrier, solvent and/or excipient means that the carrier, solvent and excipient used are not toxic or harmful to a subject when used in the probiotic compositions of this invention, including oral administration by the methods presented herein.

Method for preparing a probiotic composition according to the invention.

The technology for obtaining a veterinary product based on the strains of Bacillus subtilis 1719 and Bacillus subtilis 3H is carried out in several stages:

1) obtaining biomass of bacteria Bacillus subtilis 3H;

2) obtaining biomass of bacteria Bacillus subtilis 1719;

3) receipt of the finished product.

1. Obtaining biomass of bacteria Bacillus subtilis 3H.

For maximum accumulation of biomass, the most effective substrate for cultivating the strain is modified medium No. 9 of the following composition: iron sulfate - 0.01 g/l; magnesium sulfate - 0.1 g/l; manganese chloride - 0.01 g/l; calcium chloride - 0.08 g/l; tryptone - 5 g/l; potassium hydrogen orthophosphate - 0.3 g/l; ammonium citrate - 2.0 g/l; yeast extract - 3.0 g/l; lactose - 10.0 g/l; microbiological agar - 3.0 g/l; pH 7.3±0.2.

The cultivation of bacteria is carried out by periodic cultivation for 72 hours under conditions of constant aeration at a temperature of 37°C, pH 7.3±0.2.

The output product is a cell suspension containing live bacteria and their spores in a culture medium.

2. Obtaining biomass of bacteria Bacillus subtilis 1719.

For maximum accumulation of biomass, the most effective substrate for cultivating the strain is VK-2 medium with the addition of glucose or sucrose. The composition of the VK-2 medium per liter of distilled water is: manganese chloride - 0.01 g/l; calcium chloride - 0.05 g/l; sodium chloride - 5.0 g/l; sodium phosphate disubstituted 12-water - 2.0 g/l; potassium phosphate disubstituted 3-water - 2.0 g/l; glucose - 10.0 g/l; dry enzymatic peptone for bacteriological purposes - 10.0 g/l.

The cultivation of bacteria is carried out by periodic cultivation for 72 hours under conditions of constant aeration at a temperature of 37°C, pH 7.3+0.2.

The output product is a cell suspension containing live bacteria and their spores in a culture medium.

3. Receiving the finished product.

The resulting cell suspensions of Bacillus subtilis 1719 and Bacillus subtilis 3H are adjusted with water to a concentration of 1-1.5x108 CFU/ml of each strain and then mixed to obtain a probiotic composition.

To obtain a probiotic preparation in the form of a suspension for oral administration, the resulting probiotic composition is diluted in a 7% NaCl solution.

To obtain a probiotic preparation in the form of a lyophilisate, the resulting probiotic composition is mixed with a cryoprotector (for example, sucrose-gelatin protective medium), then the stabilized microbial suspension is poured into ampoules of 1-3 cm ³ , frozen at -40°C for 24 hours and lyophilized in for 36 hours until the residual humidity is 3-4%.

To obtain a probiotic preparation in the form of an immobilizate on a carbon enterosorbent, powdered activated carbon, consisting of particles up to 30 microns in size, is mixed with the probiotic composition in a ratio of 0.1 g of carbon per 1 ml of suspension. The resulting mixture is kept at

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4°C for 40-60 minutes with constant stirring until bacterial cells are completely immobilized on the enterosorbent. Next, the product is washed once in an isotonic NaCl solution, transferred to containers in which the resulting biological product is freeze-dried, frozen at -40°C for 24 hours and lyophilized for 36 hours to a residual moisture content of 3-4%.

The proposed probiotic composition can be used in the form of feed additives intended for introduction into feed compositions (for example, feed, concentrates, premixes or any other dry or liquid feed). The composition of such feed additives may also include additional components. For example, herbal, mineral, technological and/or balancing additives, in quantities specified in the recipe for a specific type of animal or bird.

Herbal additives are parts of plants or their derivatives, including herbs, various extracts, essential oils, meal, cake. Such additives usually improve the taste of the feed, having a positive effect on the appetite and fertility of animals and poultry.

Mineral supplements include elements such as calcium, phosphorus, magnesium, iron, zinc, iodine; mineral additives can be selected, for example, from the group consisting of, but not limited to, calcium carbonate, chalk, monocalcium phosphate, bone meal, sulfur.

Technological additives are intended, first of all, to increase feed hygiene and improve its performance, including nutritional performance – to improve the health and productivity of animals and poultry, sensory – to improve feed consumption, zootechnical – to quickly digest nutrients. Technological additives include, for example, organic acids (formic, lactic, silicon, propionic), potassium sorbate, sodium benzoate, methenamine, sodium pyrosulfite and others.

Balancing additives are designed to balance the feed supplied to animals and poultry. Balancing additives include protein, protein-vitamin, protein-vitamin-mineral and other concentrates, such as urea food concentrate.

The developed probiotic compositions can be used for the prevention and/or treatment of eating disorders, prevention and/or treatment of various diseases (including diseases of the gastrointestinal tract), the pathogenesis of which is associated with exposure to infectious agents, including simultaneously with the use of antibiotics, in livestock, poultry and fur farming.

As a result of the studies, it was unexpectedly found that the combination of strains Bacillus subtilis 1719 and Bacillus subtilis 3H is highly effective in suppressing the activity of pathogenic microorganisms in the body of an animal (bird), showing a more pronounced effect than when exposed to these strains taken separately.

The conducted studies showed that the bacterial strains Bacillus subtilis 1719 and Bacillus subtilis 3H included in the probiotic composition do not compete with each other, but enter into a synergistic relationship, having a complex effect on the macroorganism:

exhibit antagonistic activity against pathogenic and opportunistic microorganisms due to the formation of antibiotics, bacteriocins, lysozyme, as well as due to competition for habitats and nutrients;

produce amino acids, vitamins and other biologically active substances consumed by the macroorganism;

have an immunomodulatory effect; carry out the destruction of toxins and allergens.

By inhibiting the growth of undesirable microorganisms, the probiotic composition according to the invention creates conditions for the development of normal intestinal microflora, which plays an extremely important and multifunctional role in the life of the host organism: it provides colonization resistance and activates the intestines, participating in digestive, synthetic, immunomodulatory, and detoxification functions. All this makes it possible to achieve an increase in the nonspecific resistance of the macroorganism. Proteolytic enzymes synthesized by the strains of Bacillus subtilis 1719 and Bacillus subtilis 3H (including lipases, lysozyme, amylases and others) promote the breakdown of fats, proteins, fiber, carbohydrates, improve the digestion and absorption of food, which increases the bioavailability of feed and has a positive effect on the increase in body weight of animals and birds.

The most convenient form of use of the probiotic composition according to the invention is its use in the form of feed or drinking additives; it can also be used independently, for example, 20-30 minutes before feeding.

As already indicated, the exact required amount of probiotic composition for administration may vary from subject to subject depending on the species of animal (bird), age, body weight, general condition, severity of the disease, method of administration of the composition, combination treatment with other drugs, etc. . Typically, the amount of probiotic composition is selected at the rate of 0.5-2 ml per 10 kg of animal or bird weight, more preferably at the rate of 1 ml per 10 kg of animal or bird weight.

For example, when used for preventive purposes for cattle (cattle) and

- 5 043409 pigs, the probiotic composition according to the invention is used in a dose of 200 g per ton of feed, when the livestock is affected by mycotoxins - from 400 g to 2.5 kg per ton of feed.

When used for preventive purposes for poultry, the probiotic composition according to the invention is used in a dose of 1-1.5 kg per ton of feed. With increased contamination of feed with mycotoxins up to 5 kg per 1 ton of feed.

The probiotic composition according to the invention, immobilized on a carbon enterosorbent, can, for example, be used as a separate drug for the treatment of poisoning and food intoxication (for example, for feed intoxication, poisoning with alkaloids, heavy metal salts, flatulence, acute dilatation of the stomach) 2-3 times a day day (with water or in tablet form) in the following doses (per animal): cattle - 50-200 g; sheep - 10-50 g; pigs - 5-10 g; chickens - 0.2-1 g. In case of poisoning, for gastric lavage, you can, for example, use a 10-15% suspension of the mixture in water, consisting of a probiotic composition according to the invention, immobilized on a carbon enterosorbent (2 parts), tannin and magnesium oxide (according to 1 part).

When using the probiotic composition according to the invention outside of feeding, it can be used, for example, in the form of a suspension for oral administration at the rate of 0.5-2 ml of suspension per 10 kg of animal (for example, for cattle, sheep, piglets, chickens). The suspension is given to the animal (bird) orally before feeding. Such a probiotic preparation can also be used together with food (irrigate the food with the drug 20-30 minutes before feeding, so that the bacteria have time to open up) or drinking water (the drug is diluted in water, after shaking the bottle with the drug).

In some embodiments of the invention, the use of a probiotic composition is carried out in the form of a lyophilized microbial mass in a dose of 0.5-2 ml per 10 kg of animal weight. The preparation for use is prepared as follows: a small amount of food is poured into a mixer, a probiotic preparation according to the invention, previously diluted in water, is poured into it and mixed for several minutes.

In some other embodiments of the invention, the use of a probiotic composition is carried out in the form of an immobilizer on a carbon enterosorbent in a dose of 0.5-2 mg per 10 kg of animal weight. The preparation for use is prepared as follows: a small amount of food is poured into a mixer, a probiotic preparation according to the invention, previously diluted in water, is poured into it and mixed for several minutes.

The probiotic compositions of the invention can be used either as monotherapy or administered to a subject in need of appropriate therapy or prophylaxis, in combination with one or more other therapeutic agents. For example, along with the effect on the underlying disease and increasing the body’s resistance, the correction of dysbiotic disorders shows: normalization of intestinal motor function, the use of enterosorbents, the prescription of antibacterial drugs, pre- and probiotics, and synbiotics. The probiotic compositions of the invention can be effectively used as part of such complex therapy. In some non-limiting embodiments, for therapeutic purposes, the probiotic composition is intended to be administered twice daily, morning and evening, for 10-14 days. For preventive purposes - once a day for 7-10 days.

The possibility of objective manifestation of a technical result when using the invention is confirmed by reliable data given in examples containing experimental information obtained in the process of conducting research using methods accepted in this field.

It should be understood that the examples given in the application materials are not limiting and are provided only to illustrate the present invention.

Example 1. Study of probiotic properties.

A preliminary assessment of the prospects of the probiotic composition (PC) according to the invention was carried out. The study of the probiotic properties of PC when administered orally was carried out on white mice with an initial body weight of 16-18 g. The study used animals whose weight variation did not exceed 10%. Laboratory mice were divided into groups of 5 animals in each group, respectively.

Objects of research.

Probiotic composition of strains B. subtilis 1719 and B. subtilis 3H in the form of a suspension.

Probiotic composition of strains B. subtilis 1719 and B. subtilis 3H in the form of lyophilisate.

Strains B. subtilis 1719 and B. subtilis 3H immobilized on carbon enterosorbent.

The amount of each strain in the PC during the study was 1x10 ⁸ CFU/ml.

Individual supplements were used as comparison objects when analyzing the effects of PC on the microflora of mice: strains B. subtilis 1719 and B. subtilis 3H, which were administered at a dose of 1x10 ⁸ CFU/ml of each strain.

Study design.

The study design involves the use of experimental groups of animals receiving probiotic supplements, as well as a control group that did not receive probiotic supplements.

Laboratory mice were divided into groups of 5 animals in each group, respectively. Introduction

- 6 043409 corresponding doses of feed additives were administered orally for 14 days against the background of experimental dysbiosis. Experimental dysbiosis was caused by administering an antibiotic (doxycycline hydrochloride) to experimental mice in an amount of 0.01% of body weight for 7 days.

Table 1

Comparative assessment of the microflora of experimental mice

Microorganisms Number of microorganisms, CFU/g feces before dysbiosis mice with dysbiosis E. colilac ⁺ 10 ⁵ -10 ⁶ 10 ⁹ Bifidobacterium spp. less than 10 ³ not detected Lactobacillus spp. 10 ¹¹ less than 10 ³

Analysis of luminal microflora was carried out using 3 samples of aseptically taken fecal matter from 5 mice after 7 and 14 days of the experiment. Bacteriological studies were carried out using appropriate standard differential diagnostic media (Kligler agar, blood agar, Clarke's medium, etc.).

Throughout the experiment, the body weight of the experimental animals was monitored every 7 days to confirm the positive effect of PC on body weight gain.

table 2

Comparative effects of PC and individual supplements on the microflora of mice against the background of experimental dysbiosis

Microorganisms Content of microorganisms in samples, CFU/g feces against the background of experimental preparations Control V. subtil is 1719 B. subtilis ZN Suspension Lyophilisate Enterosorbent control time - 7 days E. colilac* 10 ⁹ 10 ⁷ 10 ⁹ 10 ⁶ 10 ⁶ 10 ⁶ Bifidobacterium spp. less than 10 ³ less than 10 ³ Not discovered less than 10 ³ 10 ³ 10 ⁴ Lactobacillus spp. less than 10 ³ 10 ⁵ 10 ³ 10 ¹¹ 10 ¹¹ KG ⁰ control time - 14 days E. coli lac* 10 ⁹ 10 ⁷ 10 ⁸ 10 ⁵ 10 ⁵ 10 ⁴ Bifidobacterium spp. less than 10 ³ less than 10 ³ Not discovered 10 ⁴ 10 ^{5 10th} Lactobacillus spp. less than 10 ³ 10 ⁸ 10 ⁶ 10 ¹¹ Yu ¹¹ KG ⁰

Table 3

Dynamics of body weight of mice during the period of introduction of experimental additives

Feed additive Body weight, g Original 7 days 14 days 21 day 28 days Control 16.8 17.3 17.8 18.2 18.6 Bacillus subtilis 1719 16.5 17.4 18.3 19.1 20.0 Bacillus subtilis ZN 17.1 17.8 18.3 18.9 19.5 PC (suspension) 16.9 17.5 18.7 19.9 21.3 PC (lyophilisate) 16.9 17.6 18.2 19.3 21.2 PC (carbon enterosorbent) 16.7 17.4 18.3 19.5 21.3

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Table 4

Body weight gain in experimental animals

Feed additive Body weight gain over 28 days, % Control 10.8 Bacillus subtilis 1719 21.2 Bacillus subtilis ZN 14.0 Π P (suspension) 26.0 ΠΠ (lyophilisate) 26.6 ΠΠ (carbon enterosorbent) 27.5

Research has shown that throughout the entire observation period, not a single laboratory mouse fell ill or died in the animals of the experimental and control groups. In addition, no deviations in their physiological and functional state were identified. Appetite and water consumption were preserved.

A study of the normal microflora of intact mice showed the presence of Escherichia coli, Bifidobacterium bifidum and Lactobacillus acidophilus in an amount of 105-10 ⁶ CFU/cm ³ , less than 10 ³ and 10 ¹¹ CFU/cm ³ , respectively. After administration of the antibiotic for 7 days, the death of normal microflora and contamination of the intestines with opportunistic microorganisms was observed in mice. The content of E. coli in feces increased to 10 ⁹ CFU/cm ³ , B. bifidum was not detected at all, and the amount of L acidophilus decreased to 10 ² CFU/cm ³ g (Table 1).

The results of the study of the microflora of mice after correction of experimental dysbiosis using PC and comparison drugs are presented in Table. 2. The use of PC in all tested forms led to an increase in the number of bifidobacteria and lactic acid bacteria already on the 7th day after administration, while the individual components proposed for comparison did not show such an effect. Thus, significant bifidogenic and lactogenic activities of PC were revealed. The introduction of the probiotic composition according to the invention under conditions of induced dysbiosis, which promotes an increase in the number of bifidobacteria and lactobacilli, indicates its pronounced probiotic effect.

Due to the fact that when using the probiotic composition according to the invention, a result was obtained that was numerically superior to the results (Tables 3 and 4) obtained when testing the B. subtilis 1719 and B. subtilis 3H strains separately, this indicates the presence of a synergistic effect , due to which the highest bifidogenic and lactogenic activity is achieved, which helps to increase immunity, improve metabolism, better absorption of vitamins necessary for the body and, as a result, increase body weight gain in a short time.

Example 2. Determination of the effective dose of the probiotic composition.

The study of the effective dose based on the strains of Bacillus subtilis 1719 and Bacillus subtilis 3H when administered orally was carried out on white mice with an initial body weight of 18-20 g. The study used animals whose weight range did not exceed 10%. Laboratory mice were divided into groups of 5 animals in each group, respectively.

Study design.

Experimental groups of animals were used that received probiotic compositions in various concentrations administered orally, as well as a control group. One type of animal was used in the experiment: white outbred mice. Mice in the experimental groups received a probiotic composition in the form of a suspension containing the specified amount of each strain once a day, 0.01 ml/100 g of animal weight in the following doses: 1 x 10 ⁷ , 1 x 10 ⁸ , 1.5 x 10 ⁸ and 1 x 10 ⁹ CFU/ml. For ease of administration, the required amount of the composition was adjusted to a volume of 0.5 ml with physiological solution. Mice in the control group did not receive any drugs.

The administration of the probiotic composition was carried out after a 24-hour fast orally for 14 days against the background of experimental dysbiosis caused by the administration of doxycycline hydrochloride to experimental mice in an amount of 0.01% of body weight for 7 days.

Analysis of luminal microflora was carried out on three samples of aseptically taken fecal matter from 5 mice after 7 and 14 days of the experiment. Bacteriological studies were carried out using appropriate standard differential diagnostic media (Kligler agar, blood agar, Clarke's medium, etc.).

Research results.

It was found that after administration of the antibiotic for 7 days, antibiotic-associated diarrhea was observed in mice. Analysis of the luminal microflora of fecal matter showed a deterioration in the state of the normal microflora of animals and contamination of the intestines with opportunistic microorganisms. Microflora analysis data are presented in table. 5.

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Table 5

Comparative assessment of the microflora of experimental mice

Microorganisms Number of microorganisms, CFU/g feces before dysbiosis mice with dysbiosis E. coli lac 10 ⁵ -10 ⁶ u ⁹ Bifidobacterium spp. less than 10 ³ not detected Lactobacillus spp. 10 ¹⁰ 10 ³

The results of the study of the microflora of mice after correction of experimental dysbiosis are presented in table. 6. The use of the probiotic composition in all tested doses led to an increase in the number of bifidobacteria and lactic acid bacteria already on the 7th day after administration. The results when administering drugs with a microorganism content of 1x10 ⁷ CFU/ml do not meet the norm; with a microorganism content of 1.5x10 ⁸ and 1x10 ⁹ CFU/ml, the data are comparable to 1x10 ⁸ CFU/ml. When comparing the proposed dosages, we can conclude that the optimal amount for the manifestation of the proper effect of the probiotic composition is necessary and sufficient, the content of microorganisms being 1-1.5x10 ⁸ CFU/ml.

Table 6

Comparative effect of different dosages of probiotic composition on the microflora of mice with experimental dysbiosis

Microorganisms Content of microorganisms in samples Dosage, CFU/ml Control 1*10 ⁷ 1*10 ⁸ 1.5*10® 1*1 about ⁹ control time - 7 days E. coli 1ac+ 10 ⁹ 10 ⁸ 10 ^{6 10th} u ⁵ Bifidobacterium spp. not detected 10 ¹ 10 ³ 10 ³ 10 ³ Lactobacillus spp. 10 ³ 10 ⁵ 10 ^{11 10} 10 ¹¹ control time - 14 days E. coli lac+ 10 ⁹ 10 ⁷ u ⁵ u ⁵ u ⁵ Bifidobacterium spp. not detected 10 ² 10 ⁴ u ⁵ u ⁵ Lactobacillus spp. 10 ² 10 ⁹ Yu ^{11 10} 10 ¹¹

Studies have shown that the optimal amount for the manifestation of the proper effect of a probiotic composition is necessary and sufficient to contain the microorganisms Bacillus subtilis 1719 and Bacillus subtilis 3H in the composition 1-1.5x10 ⁸ CFU/ml.

Example 3. Characteristics of antagonistic activity.

In the course of the studies, the morphological, physiological-biochemical, genetic and other biological properties of various strains of Bacillus subtilis, as well as their combinations in various combinations, were studied.

The most important criterion for the selection of probiotic strains is the study of their antagonistic activity towards pathogenic and opportunistic microorganisms (OPM), which ensures colonization resistance of the gastrointestinal tract. The purpose of the study was to characterize and compare the antagonistic activity of various combinations of Bacillus subtilis strains often used as probiotics and the proposed probiotic composition.

Objects of research.

Strain: Bacillus subtilis 1719, Collection of the State Research Institute for Standardization and Control of Medical Biological Preparations named after. L.A. Tarasevich, No. 277.

Strain: Bacillus subtilis 3H, State collection of pathogenic microorganisms, No. 248.

Strain: Bacillus subtilis 534, All-Russian collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms named after. G.K. Scriabin RAS; VKM No. 1666 D.

Strain: Bacillus subtilis 12B, Sporovit, Collection of pure cultures of microorganisms VGNKI.

Veterinary probiotic composition based on Bacillus subtilis 1719 and Bacillus subtilis 3H strains according to the invention.

Test culture strains from the State Collection of Pathogenic Microorganisms of the Federal State Budgetary Institution NTsESMP used to determine antagonistic activity: Staphylococcus aureus Filippov, Shigella Sonnei 170, Proteus mirabilis 24a, Escherichia coli ATCC 25922, Pseudomonas aeruginosa 9022, Staphylococcus aureus 29213, Candida albicans 690.

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Claims (10)

Study design. Studies of 20-hour bacterial culture liquids of strains were carried out using the delayed antagonism method. The studied samples contained 1x10 ⁸ CFU/ml of each strain. The strain culture was sown in a loop diagonally in the center of a Petri dish with agarized meat-peptone medium. The crops were incubated in a thermostat at 37°C for 24 hours. Then test microorganisms (500 million suspensions of daily cultures in physiological solution) were streak-sown onto the grown culture. The results were recorded after 18 hours of incubation at 37°C according to the size of the zones of no growth of test cultures. The growth of the test cultures was controlled by their parallel cultivation on plates with an agar medium without a culture of the test strain. Results. The obtained test results are presented in table. 7. Table 7 Antagonistic activity of combinations of B. subtilis strains Test strain Growth retardation of test cultures, mm (M±t) V. subtilis zn + v. V. subtilis ZN + V. IN. Subtilis 1719 + V. B. Subtilis 1719 +B. Subtilis 12V B. subtilis ZN B. subtilis 1719 B. subtilis 1719 + IN. Subtilis 534 Subtilis 12V subtilis 534 subtilis ZN S. aureus "Filippov" 19±2.6 18+ 2.0 20 ± 1.2 18 ± 1.1 20±0.9 22±1.8 24± 1.6 S. sonnei 170 23± 1.6 20± 1.2 22 ± 1.7 20 + 1.8 22±1.3 24±2.2 26±0.9 R. mirabilis 24a 17± 1.9 17± 1.9 18 ± 1.5 19 ±2.5 18±2.0 19±2.0 22± 2.3 E. soL ATCC 25922 14±1.0 13±2.1 18 ± 1.6 22 ± 1.2 15±2.1 24±0.7 26± 1.5 R. aeruginosa 9022 16 ± 1.8 15 ± 1.7 18 ± 1.5 20 ±1.9 17±3.3 23±2.8 27± 1.8 S. aureus 29213 15± 1.6 18±2.2 19 ± 1.5 19 ±3.0 16±1.5 21±0.8 22± 2.1 C. albicans 690 29±3.1 30±2.0 28+2.2 29±1.7 31±2.8 29±1.1 30±3.8 As can be seen from table. 7, various combinations of probiotic strains of bacteria of the genus Bacillus subtilis exhibit antagonistic activity against a wide class of opportunistic microorganisms. The best indicators of growth inhibition zones were found in the combination of Bacillus subtilis 1719 and Bacillus subtilis 3H, which indicates their more pronounced synergistic effect. Thus, the probiotic composition according to the invention has an inhibitory effect against pathogenic and opportunistic microorganisms, increases the bioavailability of feed without suppressing the intestinal microflora, activates intestinal function, increases the resistance of microflora to negative microbiological influences by activating the function of the immune system and lytic (detoxifying) action of synthesized enzymes. The use of the probiotic composition according to the invention will significantly reduce the cost of treating diseases in animals and poultry and increase their productivity. Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments detailed are provided for purposes of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the spirit of the present invention. CLAIM 1. Probiotic composition, including a mixture of biomass of live and spore forms of bacteria of the strains Bacillus subtilis 1719 in a titer of 1-1.5x10 ⁸ CFU/ml and Bacillus subtilis 3H in a titer of 1-1.5 x10 ⁸ CFU/ml. 2. The probiotic composition according to claim 1, further comprising a pharmacologically acceptable carrier, solvent and/or excipient. 3. Probiotic composition according to claim 1 in the form of a suspension for oral use. 4. Probiotic composition according to claim 1 in the form of a lyophilisate. 5. Probiotic composition according to claim 1, in which the bacteria Bacillus subtilis 1719 and Bacillus subtilis 3H are immobilized on a carbon enterosorbent. 6. A method for the prevention and/or treatment of diseases of the gastrointestinal tract in veterinary medicine, including the use of a probiotic composition according to any of claims 1-5. 7. The method according to claim 6, in which the probiotic composition is administered orally before feeding or along with food or drinking water at the rate of 0.5-2 ml per 10 kg of animal or bird weight. - 10 043409 8. The method according to claim 7, in which the probiotic composition is administered at the rate of 1 ml per 10 kg of animal or poultry weight. 9. Feed additive, including a probiotic composition according to any one of claims 1-5. 10. Feed additive according to claim 9, additionally including plant, mineral, technological and/or balancing additives. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2 -