EA028962B1 - Metabiotic composition to ensure colonisation resistance of human intestinal microbiocenosis - Google Patents

Abstract

The invention relates to medicine and pertains to a means for ensuring colonisation resistance of human intestinal microbiocenosis in dysbiotic conditions associated with antibiotic impact. Beneficial action on endogenous microflora in the biotope is observed, antagonistic effects against bacterial and viral pathogens are ensured, and non-specific body resistance is supported. The claimed symbiotic composition is performed as a solid dosage form in capsules and includes a metabiotic component (wt.%) including sterilised dried culture media comprising metabolites of Bacillus subtilis VKPM No. B-2335 (1.9), Enterococcus faecium L-3 (4.5), Lactobacillus delbrueckii TS1-06 (4.5) and Lactobacillus fermentum TS3-06 (4.5), oat flakes as a prebiotic component (20.0), zeolite as a carrier adsorbent (64.3) and calcium stearate or aerosil as a processing aid (0.3).

Description

The invention relates to the field of medicine and relates to means for ensuring colonization resistance of the human intestinal microbiocenosis with dysbiotic disorders associated with exposure to antibiotics, due to the beneficial effect on the components of the indigenous biotope microflora, providing antagonistic effect against pathogens of bacterial and viral nature and non-specific resistance of the organism. The inventive synbiotic composition is made in a solid dosage form in the form of capsules and includes (wt.%) A metabiotic component comprising sterilized dried culture liquids containing metabolites VasShik KiShk VKPM No. B-2335 (1.9), Eudessososik Haesst L-3 (4, 5) Spaciosus 5 4e1biesky T81-06 (4,5) and BasaciosStick Goetheshit T83-06 (4,5), oat flakes as prebiotic component (20,0), zeolite as adsorbent carrier (64,3) and stearate calcium or aerosil as a technological additive (0.3).

028962

The invention relates to the field of medicine and relates to means for ensuring colonization resistance of the human intestinal microbiocenosis for dysbiotic disorders associated with exposure to antibiotics, due to the beneficial effect on the components of the indigenous biotope microflora, providing antagonistic effect against pathogens of bacterial and viral nature and nonspecific resistance of the organism.

The actual problem of modern medical science is connected with the study of possible violations of the human microecology, and above all, violations of a very important system of the body - the intestinal microbiocenosis. Microecological changes in the intestinal microflora are recorded in almost 90% of the population [1] and many researchers explain the observed increase in the frequency and severity of acute infectious diseases, torpid current and chronic inflammatory processes by developing or already occurring microbiota disorders accompanying the main disease. Representing the extracorporeal organ, the microflora of the human body performs or regulates the diverse functions of maintaining normal homeostasis [2]. Changes in the composition of normal microflora lead to the development of processes affecting the health of people [3-5].

The value of intestinal microflora in the vital activity of the human body is related to the fact that it has a direct impact on its condition, performing a number of vital functions: providing colonization resistance, forming an immune response, digesting food, regulating the motor function of the intestine, detoxification.

One of the most important functions of normal microflora is to ensure colonization resistance - a set of mechanisms that ensure the ability of the microbiota and macroorganism, cooperating cooperatively, protect the ecosystem from the effects of pathogenic microflora, imparting stability to the normal microflora, preventing the colonization of the body by foreign microorganisms and ensuring its anti-infective protection [5-7] .

The absence of colonization resistance due to various reasons leads to a significant weakening of the body’s anti-infective protection, an increase in the number and spectrum of pathogenic and conditionally pathogenic microbes, their translocation from the primary localization sites, and, consequently, to an increase in the body's susceptibility to the occurrence of infectious diseases of a bacterial and viral nature [5, 8, 9], the development of endogenous infections, superinfections of various localizations [10-12]. The absence of colonization resistance is one of the starting factors for the occurrence of various diseases.

Colonization resistance of the intestinal microbiocenosis is determined by many factors and, above all, depends on the quantity and quality of normal microflora, the antagonistic activity of its constituent microbes, their habitat conditions, including competition for nutritional factors and others. The main mechanisms for ensuring colonization resistance include the activation of the immune system [4].

Therefore, the colonization resistance of the intestinal microbiocenosis is mainly associated with the state of the biotope microbiocenosis and the mechanisms of local non-specific immunological resistance of the organism [1, 9]. In turn, the violation of the mechanisms of immunological homeostasis is one of the important reasons for the development of dysbiotic disorders of the intestinal microbiocenosis. Protective reactions against viral and bacterial pathogens can be enhanced by activating the immune system [13], when the stimulating signal is nonspecific, directed not selectively against the pathogen that caused the infection, but enhances overall immunological resistance.

A variety of adverse effects on the human body cause changes in the immune response and can affect the qualitative and quantitative characteristics of the normal intestinal microflora. When dysbiotic disorders of the intestinal microbiocenosis, clinical conditions arise associated with the absence of colonization resistance, digestive disorders, impaired detoxifying function of the intestinal microflora and changes in the immune response.

The development of intestinal dysbacteriosis is promoted by a variety of exogenous and endogenous factors [4, 5, 15]. At present, stress and overwork, physical and psycho-emotional stress, smoking, unbalanced nutrition, eating disorders, overeating or having a snack on the run, environmental problems, many chronic diseases and a number of other pathological conditions are quite common. In such cases, due to the fact that the microbiocenosis is an evolutionarily established and well-integrated system that ensures the smooth operation of all organs and systems and is based to a certain extent on the principle of self-regulation, the resulting changes in symbiotic microflora in most cases are restored spontaneously.

Everything is completely different after the irrational use of antibiotics. In clinical practice, a similar situation, in which, above all, dysbiotic disorders of the intestinal microbiocenosis occur, is now common. Treatment with antibiotics is often a standard solution for a wide variety of diseases, including such common as sore throat, bacterial infections of the upper and lower respiratory tract.

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(tonsillitis, maxillary sinusitis (sinusitis), pharyngitis, bronchitis, pneumonia and others), urogenital infections, acute intestinal infections and many other pathological conditions. The use of antibiotics is based on well-known basic schemes for emergency prevention and treatment of infectious diseases of a bacterial nature [16–19].

It should be noted that in order to achieve an adequate antibacterial effect, such drugs, as a rule, are taken frequently, long-term and in large dosages. Antibiotics, on the one hand, can effectively inhibit the excessive growth of pathogenic and conditionally pathogenic microorganisms, but on the other hand, with commonly used schemes for their dosing, they often cause the development of a number of side effects from various organs and systems of the body. First of all, they have a negative effect on the state of the human intestinal microbiocenosis due to the lack of selectivity of their action on microorganisms. Under the influence of antibiotics, as a rule, in 2/3 of the patients, relatively long-lasting (within 2-3 months) disorders of the intestinal microbiocenosis and, above all, the balance of the qualitative and quantitative composition of the microflora occur, resulting in symptoms of dysbiosis.

It is necessary to take into account the fact that antibiotics are toxic, have a non-specific bactericidal effect and can provoke the appearance of pathogenic microorganisms that are resistant to the action of traditionally used antibacterial agents, as a result of which antibiotic therapy requires the subsequent restoration of the intestinal microbiocenosis (imbalance in the system "macroorganism and normal microflora") .

Thus, the practical importance of adequate correction of intestinal microbiocenosis disorders in patients receiving antibiotics is beyond doubt. When correcting dysbiotic disorders of the intestinal microflora, a complex of measures is taken taking into account the clinical manifestations of the underlying disease and the severity of dysbiotic disorders of the intestinal microbiocenosis, as well as laboratory research data on the biotope microflora and the state of reactivity of the organism.

To ensure effective correction of violations of the human intestinal microbiocenosis, measures such as

selective decontamination of pathogenic and conditionally pathogenic microflora; normalization of the intestinal microflora; correction of the body's immunological reactivity.

Correction of dysbiotic disorders is carried out in accordance with the Industry Standard [20] and is carried out in stages (in 2 stages) and in accordance with the degree of their manifestation. In cases of dysbacteriosis 2 and 3, the degree of severity of disturbances that have arisen, correction begins with the appointment at the first stage of means that have selective antibacterial activity and provide selective decontamination of pathogenic and conditionally pathogenic microflora. These are various bacteriophages staphylococcal, klebsiellezny, pyobacteriophage, intestitis bacteriophage, pseudomonas, and others. With low sensitivity to bacteriophages, antibacterial preparations (furazolidone, chlorophyllipt, metronidazole, nifuroxazide, intrix, and others) and antifungal agents (ketoconazole, fluconazole, natamycin, and others) are used. At the second stage, it is mandatory to conduct a course of substitution therapy to normalize their own indigenous intestinal microflora. A significant role is played by the differentiated use of various immunobiological preparations.

The spectrum of immunobiological preparations currently recommended for the correction of dysbiotic disorders of the intestinal microbiocenosis is rather wide. Of these, probiotics, which include specially selected strains of living microorganisms or specific substances of microbial, plant or animal origin, which exhibit antagonistic activity against pathogenic and conditionally pathogenic microflora, stimulate reparative processes in the intestinal mucosa and increase the immunological reactivity of the organism are most widely used due to the production of acids, antibiotic substances, the release of various enzymes, vitamins and others. Microorganisms that are part of probiotics are able to synthesize substances with antimicrobial properties, compete with pathogenic microorganisms for nutrients and microbial adhesion sites, modify toxins or toxin receptors. Certain types of microorganisms non-pathogenic for humans and substances of microbial origin, with the natural route of administration, have beneficial effects on the physiological functions, biochemical and behavioral reactions of the organism through the optimization and stabilization of its microecological status [21]. These include, above all, bifidobacteria and lactose-containing agents for regulating the balance of the intestinal microflora.

Bifidobacteria and lactobacilli constitute the basis of the intestinal microflora and play a crucial role in ensuring the normal functioning of the biotope. Bifidobacteria, being saccharolytic microbes, produce a large amount of acidic products. The resulting lactic and acetic acids help to enhance the absorption of calcium, iron and vitamin ионов ions. Production of lysozyme, bacteriocins, alcohols by bifidobacteria, and high antagonistic activity against various pathogens prevent their penetration into the upper gastrointestinal tract.

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pathogens prevent their penetration into the upper gastrointestinal tract (GIT). Bifidobacteria have a high ability to synthesize amino acids, proteins, many B vitamins, which are subsequently absorbed in the intestine. Therefore, with persistent, severe dysfunction of bifidobacteria, a complex of protein-vitamin-mineral deficiency can develop. With a decrease in the level of bifidobacteria, the colonization resistance of the intestinal microbiocenosis disappears, translocation of pathogens to the upper intestine and their excessive growth occur.

The main lactobacilli that colonize the gastrointestinal tract are BaFyCasch asyorIy1 and 8, BaSFYasChic Sake !, BasFsChik p1ap1agisch, LasFsChik Gegteshit. Suppression of putrefactive and pyogenic microbes and the antagonistic activity of lactobacilli are associated with the production of lactic acid, alcohol and lysozyme, products with high antibacterial activity, interferons, interleukin 1 and some others. The disappearance of lactobacilli leads to a shift in the reaction of the medium to the alkaline side and a sharp deterioration in the functional state of the gastrointestinal tract.

When dysbiotic states occur, the qualitative and quantitative composition of anaerobic bifidobacteria and lactobacilli undergoes violations in the first place. Therefore, drugs based on them are widely used in the practice of doctors of various specialties. Of these, in a number of clinical situations, probiotic complexes Linex and Bifiform [22, 23], the active principle of which are live bacteria, are the drugs of choice. For example, Linex, produced by the pharmaceutical company Lek (Slovenia), includes a balanced complex of lactic acid bacteria: lactobacilli lacFacDeCor ajorChik, bifidobacterium Vycros yugst 1pGapyk and enterococci Epessosik Haeszt. In contrast to Linex, Bifiform produced by the Danish pharmaceutical company Ferrosan International A / S, in addition to the Bioshobacterium Bifidobacterium (Egst 1pdit (strain ATTS 15707) and Enterococci EPossos Haesst (strain P68) from the microbiological laboratory "Christian Hans. a special nutrient medium for their nutrition, growth and reproduction is included.In addition, Bifiform does not contain lactose, as a result it is indicated for patients with its intolerance. The bacteria included in the preparations are intended to regulate the physiological of the intestinal microflora by producing lactic acid, to a lesser extent acetic and propionic acids. The resulting acidic environment is unfavorable for the development of pathogenic and conditionally pathogenic microorganisms. Useful bacteria are lyophilized and packaged in special protective capsules, which ensures their reliable protection against destruction under the influence of gastric juice and allows you to bring in a little modified form directly to the intestine and release it there.

However, in addition to the favorable effects for the drugs Linex and Bifiform, there are a number of significant drawbacks that limit the possibilities of their widespread use in clinical practice. First of all, one cannot assume that the bacteria in their composition are equivalent to their own indigenous microflora and are able to colonize and multiply in the parietal layer of the intestine. One of the main reasons for this is their insufficient, or rather unpredictable biocompatibility with resident bacteria of the human intestine. The individual biocompatibility of microorganisms introduced into the composition of these preparations and their survival are not guaranteed. Accordingly, the probability of the fulfillment by said bacteria, introduced into the macroorganism from the outside, of its main function (substitution), and especially in a short period of time, is practically excluded.

It should also be borne in mind that the mechanism of action of microorganisms contained in these preparations is transient in nature, they do not colonize in the intestine, and therefore their beneficial effect continues almost only during the course treatment. Therefore, as a rule, they are prescribed for a fairly long time (at least 3-4 weeks) and they recommend 1-2 capsules at least 3 times a day. And since the cost of these commercial preparations is high enough, it also significantly limits the availability of Linex and Bifiform for the general population.

Linex and Bifiform are recommended for the prevention and treatment of dysbacteriosis of various etiologies. At the same time, they are used mainly in the correction schemes for intestinal dysbacteriosis only at stage 2 for the purpose of replacement therapy. The compositions of these drugs include bacteria strains with a fairly high level of antibiotic resistance and often recommend their use simultaneously with antibiotics. However, the experience of using these funds in clinical practice with a wide range of antibiotics with recommended schemes of their appointment showed that most of the bacteria with such a combined appointment die, and the activity of those trapped in the intestine in a viable state is extremely low.

However, it is now known that one of the promising ways to overcome the negative effects of antibiotics on the composition of the intestinal microbiota is not their independent use, but in combination with probiotics. It was found that in the case of combined administration with antibiotics, priority should be given to such probiotics that contain non-living microorganisms (Bifidobacterin, Lactobacterin, Sporobacterin, Laminolact, Linex, Bifiform, etc.), often alien to the natural indigenous microflora of the human gastrointestinal tract, which, moreover, can be destroyed by antibiotics, having a negative impact on the immune system and the macroorganism in general, and metabolites of probiotic strains of microorganisms - 3 028962

metabiotics [4, 24, 25].

A modern representative of such metabiotic agents is a composition that is closest to the claimed metabiotic composition in composition, mechanism of action on the body, and the beneficial effect achieved [26].

The composition of the prototype is a balanced complex with pronounced antimicrobial, antiviral and immunomodulatory activity, which includes metabiotic and prebiotic agents, adsorbent carrier and auxiliary technological additive in the following ratio of ingredients, wt.%:

sterilized culture fluid containing metabolites of the bacterium strain Vaschiz zyyshk VKPM No. В-2335 - 1.6-2.3;

zeolite - 43.0-46.0;

a complex of polysaccharides (β-glucan and mannan) - 51.0-54.0;

calcium stearate or aerosil - 0.5-1.5.

In the composition of the prototype composition, the metabiotic agent is represented by a sterilized culture fluid (SCF) containing metabolites obtained during the cultivation and sterilization of bacteria of the species VasSiZiNiNz. An integral part of the composition of the prototype, providing its therapeutic and prophylactic properties, are biologically active substances (BAS), synthesized by Vaschizyna VKPM No. B-2335 during deep cultivation, including various natural antibacterial substances (bacteriocins, lysozyme, catalase) that are selectively inhibit the growth and reproduction of pathogenic and conditionally pathogenic microorganisms in the intestine, without affecting the symbiotic microflora. Microbial producers synthesize nitrogenous bases (purine and pyrimidine derivatives), which represent a separate group of immunomodulatory substances and are contained in the SCJ in significant quantities. This circumstance is important for the composition of the prototype, since nitrogenous bases and their derivatives restore the functional activity of a single macrophage system of the body and significantly increase the effectiveness of nonspecific and specific protection.

The main purpose of the zeolite, which is part of the composition of the prototype and acts as an adsorbent and carrier of bacillus Vaschisynsus metabolites, is to immobilize the metabolites synthesized by bacilli during cultivation and concentrated in the SCJ, and deliver them as a carrier throughout the intestine. The gradual release of biologically active substances immobilized on zeolite allows maintaining a high level of activity of the composition of the prototype.

The composition of the composition of the prototype introduced a highly active immunomodulatory agent, which is a complex of polysaccharide products of enzymatic hydrolysis of polymers of the inner and outer layers of the cell wall of brewer's yeast, namely, β-glucan and mannan. The effect of the data easily digestible in the body of biologically active substances is primarily that β-glucan activates the main cells of the immune system - macrophages.

To improve the processability of the preparation of the composition of the prototype in solid dosage form for oral administration in its composition introduced an auxiliary additive - calcium stearate or aerosil.

An important feature of the composition of the prototype is the ability to effectively increase the nonspecific and specific immune resistance of the body, which is implemented through a complex effect on the immune system of all its constituent components that exhibit immunomodulatory activity. The main purpose: the composition of the prototype is to maintain optimal microecological parameters in the intestine by introducing into the body a BAS that inhibits the development of pathogens. The absence in the composition of the composition of the prototype of living microorganisms can be used in conjunction with antibiotics to improve the microecological conditions, which helps to restore the qualitative and quantitative characteristics of symbiotic microorganisms.

However, along with these favorable effects for the composition of the prototype is characterized by a number of disadvantages:

limited biological activity, which is expressed in insufficient levels of specific antagonistic activity against a number of pathogenic and conditionally pathogenic microorganisms, which is associated with the use of only one type of microorganism in its composition (WasSchizus);

providing the possibility of effective correction of violations of only one component of the indigenous intestinal microflora - lactobacilli;

the lack of information about the possibility of a beneficial effect on such an important component of the intestinal normoflora as bifidobacteria, and stimulating the growth and reproduction of these beneficial microorganisms.

The noted drawbacks significantly limit the widespread use of the prototype composition in clinical practice in order to correct colonization resistance of the intestine.

nickname reduced due to dysbiotic disorders of the biotope microbiocenosis when prescribing antibiotics for the treatment of pathological conditions.

The aim of the invention was to improve the efficiency of the correction of dysbiotic disorders of the intestinal microbiocenosis, caused by the use of antibiotics and leading to a decrease in the colonization resistance of the biotope, through the use of a multi-component metabiotic composition based on a balanced complex of biologically active substances that provide a beneficial effect on the main components of the intestinal microflora, specific antagonistic activity in against a wide range of pathogens and conditionally pathogenic microorganisms, non-specific immunological resistance of the organism by stimulating the functional activity of immunocompetent cells, containing no hazardous substances, non-toxic, non-sensitizing, cumulative, toxic effects on reproductive function, mutagenic effect and long-term negative effects, stable when stored.

Achieving this goal is possible by creating multicomponent metabioticheskoy composition qualitative and quantitative composition of which will normalize the intestinal microbiocenosis both by selectively stimulating the growth and reproduction of lactobacilli and bifidobacteria indigenous component normoflora biotope and provide specific antagonist activity against a broad spectrum of pathogenic and conditionally pathogenic microorganisms and by stimulating the system of nonspecific immunolo geological resistance and increase nonspecific resistance of the organism.

When forming the qualitative and quantitative composition of the claimed metabiotic composition intended to ensure colonization resistance of the human intestinal microbiocenosis, it was assumed that its main components should

provide an effective beneficial effect on the main components of the indigenous intestinal microflora - lacto-and bifidobacteria, contributing to their growth and reproduction;

to ensure colonization resistance of the biotope microbiocenosis, providing an effective effect on the intestinal microflora due to the manifestation of specific antagonistic activity against a wide range of pathogenic and conditionally pathogenic microorganisms of bacterial nature, with which antibiotic therapy is usually associated, ensuring their selective decontamination;

increase the antiviral defense of the organism due to the activation of the production of cytokine such as interferon in the cells of the macrophage system;

influence the factors of nonspecific resistance of the organism and stimulate the functional activity of immunocompetent cells to eliminate dysfunctional changes in the immune system, developing under the influence of pathogens, and also due to the prescription of antibiotics.

To this end, the claimed metabiotic composition includes: a metabiotic component, which is a rational combination of metabolites produced by probiotic strains of four microorganisms, a prebiotic component with an immunomodulating activity, an adsorbent carrier and an auxiliary technological additive.

In contrast to the composition of the prototype in providing a complex effect on the state of the intestinal intestinal microbiocenosis, both due to the beneficial effect on the indigenous component of the biotope microflora, and to ensure a specific antagonistic effect in relation to a wide range of pathogens, not one type of microorganism (Vas Shikkyykyk) participates, but an additional three - Eisgossossik Gassinp. LioBuSchik bs1ñtstski and LacLesChik Gsgtsp1shp, Metabiotic component is a rational combination of the metabolites of the probiotic bacterial strains VashShyk xpvp private jpm, bp 2335;

At the same time, the bacterium strain VasSchik Quintora PKPM No. B-2335 was introduced in the form of sterilized dried culture liquid, since it is in it that a unique set of biologically active substances is synthesized, synthesized by bacilli in the process of their cultivation. Among the BAS, natural antibacterial substances (bacteriocins, lysozyme and others) are of particular value, which selectively inhibit the growth and reproduction of pathogenic and conditionally pathogenic microorganisms in the intestine, without affecting the symbiotic microflora, and also make a significant contribution to the ability of the claimed metabiotic composition influence the factors of nonspecific resistance of the organism and stimulate the functional activity of immunocompetent cells.

The ability of VasSHICKYSHShK to increase the phagocytic activity of macrophages and thereby exhibit an immunomodulatory effect is associated with the synthesis of nitrogenous bases and their derivatives (adenine, guanine, thymine, uracil, cytosine), which represent a separate group of immunomodulatory substances and are contained in SKZ in the value components, which are in the SKZ in the substance values, and are contained in the SCJ in the value elements, which are in the SKZ in the substance values, and are contained in the SCJ in the values, and they are contained in the SCW in the values, which are in the SCW in the substance, and are contained in the SKZh in the values, which are in the SKZh in the substance, and are contained in the SCW in the values, which are in the SCW. (tab. 1). For the claimed metabiotic composition, this is of great importance, since the indicated BAS contribute to the restoration of the functional activity of the single macrophage system of the body, which inhibits 5 028962

due to exposure to pathogens and antibiotics, and a significant increase in the effectiveness of non-specific protection.

Particularly significant is the fact that the metabolites of VasSHIQPM VKPM No. B-2335 represent the lactogenic factor and exhibit a lactogenic effect, which is expressed in stimulating the growth and reproduction of the lactobacilli of the indigenous intestinal microflora. This causes favorable effects due to the normalization of the intestinal microflora and contributes to the colonization resistance of the biotope.

Metabolites of the probiotic strain Enterococcus Eyagosossik Haesht L-3 are included in the claimed metabiotic composition in the form of sterilized dried culture fluid. As part of the inventive metabiotic composition, the metabolites of the probiotic Enterococcal strain Ept1osossik Haesht L-3 (enterocins A and B) [27] represent a bifidogenic factor and have a beneficial effect on the bifidobacteria of the indigenous intestinal microflora, stimulating their growth and reproduction.

The probiotic strains of the bacteria Bosocyscic bilischi T81-06 and Boscicaccicles Cgtrosch8383 under cultivation actively synthesize metabolites (bacteriocins) with the protein component lactocin B [28, 29]. The composition of the inventive metabiotic composition of the metabolites introduced in the form of sterilized dried cultured liquids and make a significant contribution to the provision of specific antagonistic activity of the claimed metabiotic composition to a wide range of pathogens.

In the composition of the claimed metabiotic composition, the prebiotic component is represented by oat flakes and fulfills its main purpose, which is to provide the nutritional needs of representatives of the indigenous intestinal microflora, as well as the cells of the microorganism, and exhibits immunomodulating activity.

Oatmeal is able to optimally ensure the restoration of intestinal microflora, since the proportion of dietary fiber in them is 11%, which, moreover, contains 35% of β-glucanes that are very valuable for the human body. When correcting intestinal dysbiotic conditions, the role of agents with a high content of dietary fiber and allowing to optimize the conditions of vegetation of symbionts is undeniable. Dietary fibers (a set of various water-soluble polysaccharides) are not digested (not split into monosaccharides) by endogenous secretions of the human gastrointestinal tract and reach the colon in unchanged form, where they are metabolized by anaerobic symbiotic microflora to short-chain fatty acids. In turn, short-chain fatty acids (acetate, propionate, butyrate, valerate) are the main energy substrate for epithelial cells of the mucous membrane of the large intestine [30], stimulating proliferation, cell differentiation and the formation of mucin mucus. In addition, dietary fibers, fixing symbiotic microorganisms on their vast surface, contribute to a sharp increase in their content per unit of intestinal volume and an increase in the metabolic activity of the intraintestinal environment.

The immunomodulatory effect of β-glucan entering the body as part of the prebiotic component is due to the fact that it is transported through the epithelial barrier of intestinal tract cells to the lymph, which interacts with key cells of the immune system (macrophages) by attaching to specific receptors on their cellular membranes. Influencing macrophages, β-glucan enhances their phagocytic activity, namely mobility and the ability to find and eliminate alien, including pathogenic and conditionally pathogenic microorganisms. In addition, β-glucan also stimulates cells such as neutrophils ΝΚ (natural killer cells) and LAC (lymphokino-activated killer cells), thereby further enhancing the antibacterial activity of the claimed metabiotic composition. Also, β-glucan makes it possible to activate production in the cells of the macrophage system of such a cytokine as interferon, thereby increasing the antiviral activity of the claimed metabiotic composition.

The adsorbent in the composition of the claimed metabiotic composition is intended, first of all, for immobilization of the metabolites produced by VasSHyk in the process of their cultivation and concentrated in the SCF. The feasibility of using a zeolite as a component of the claimed metabiotic composition is determined by the unique chemical structure of its molecules, with the result that the zeolite has a high sorbent activity against bacterial metabolites of VasChik bacteria. Optimal conditions arise for the zeolite to perform the main functions — the adsorbent and carrier of the VasShikkishshk metabolites. Without being absorbed in the gastrointestinal tract and passing through the transit, the zeolite transports the metabolites immobilized on it along the entire length of the intestine. The release of metabolites occurs gradually and allows a sufficiently long period of time: (up to 1 day) to maintain the required level of activity of the claimed metabiotic composition. The particles used in the zeolite have a size of not more than 500 μm and an oval-shaped crystal, which eliminates the formation of microtraumas in the intestine and is completely safe [31-33].

To improve the technological process of obtaining the proposed metabiotic composition as an auxiliary technological additives use SC or Aerosil.

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The effect of the inventive metabiotic composition, made in the encapsulated form, begins in the stomach, where the protective capsule dissolves under the influence of gastric juice. The metabolites of probiotic bacterial strains that are part of the claimed metabiotic composition, the degradation of which during the transit through the gastrointestinal tract is practically excluded, enter the intestine, where their beneficial effects are manifested.

As can be seen, the ingredients are chosen in such a way that the claimed metabiotic composition includes a balanced complex of biologically active substances that contribute to the colonization resistance of the human intestinal microbiocenosis due to an effective beneficial effect on the main components of the indigenous microflora of the biotope, ensuring a sufficient level of antagonistic activity against a wide range of pathogens, as well as non-specific immunobiological resistance of the organism, and their number in one capsule is % imprint:

sterilized dried culture fluid containing metabolites of the probiotic bacterial strain Vaschis sine VKPM No. В-2335 - 1.9;

sterilized dried culture fluid containing metabolites of the probiotic bacterial strain EpGosossosiz Haesst L-3 - 4.5;

sterilized dried culture fluid containing metabolites of the probiotic bacterial strain LacTiOnSeur OePegeski TZ1-06 - - 4,5;

dried dried culture fluid containing metabolites of the probiotic bacterial strain Lascaris: Schiz Hepnepht T333 - 4.5;

zeolite - 64.3;

oatmeal - 20.0;

calcium stearate or aerosil - 0.3.

The possibility of achieving the objectives of the invention is confirmed by the results of the research presented in the following examples.

Example 1. Obtaining the claimed metabiotic composition in the form of encapsulated solid dosage forms for oral administration.

The inventive metabiotic composition is made in the form of an encapsulated solid dosage form for oral administration, which not only greatly simplifies its use, but also provides reliable protection for the BAS contained in it against factors that can cause their degradation.

The claimed metabiotic composition is first obtained in powder form, for which the following sequence of actions is carried out. Microorganisms VashSchiz ziOpz VKPM No. B-2335 are grown by the method of deep cultivation in a biological reactor. After the end of the cultivation process, SFC with microorganisms is centrifuged to separate and then remove living cells of microorganisms, and then sterilized. Before sterilization, the SCJ is mixed with an adsorbent, a zeolite, previously ground to a particle size of not more than 500 microns. The resulting mass is subjected to lyophilization, in which there is an immobilization of biologically active metabolites of the producer on the particles of zeolite. Next, a mixture is added to the mass, including oat flakes and sterilized dried culture liquids containing metabolites of probiotic bacterial strains Baconus Scorpius 0e1bthcd T81-06, Bccienne Sci-0s1ttescip T81-06 and Eshegossiz Haesstb-3, then added CK or Aerosilne syringes, and then added CI or AeroSilver I-S alves. for the purpose of mixing and drying. Then the mass is sifted on a vibrating screen and transferred to the stage of encapsulation. Filling capsules carried out on the machine for filling capsules.

Sterilized dried culture fluids containing metabolites of probiotic bacterial strains of Lacin Thurghienerus Téléxierus T81-06 and Laciouchaszíz Olléryttesky T81-06 are administered in the form of dried product "Avena-L" (Avena LLC, St. Petersburg) [34].

Sterilized dried culture fluid containing metabolites of the probiotic strain of bacteria EHeossossiz Haesst L-3 is administered as a dried product "Avena" (Avena LLC, St. Petersburg) [35].

The composition of the inventive metabiotic composition is injected oatmeal, including dietary fiber (11%), which is 35% represented by β-glucans [36].

Natural zeolite is used as an adsorbent - a natural microporous silicate mineral from the Kholinsky deposit (Buryatia) (KRAFT LLC, St. Petersburg) [37, 38].

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As an auxiliary additive, SC [39] or aerosil [40] are used.

As can be seen, the process of obtaining the claimed metabiotic composition in a cansulated solid dosage form for oral administration is simple, and the ingredients used for this are produced in an industrial environment, affordable and inexpensive.

Example 2. Safety assessment of the proposed metabiotic composition.

The safety of the inventive metabiotic composition was assessed by sanitary-chemical analysis, microbiological studies, as well as determining toxicological characteristics, including acute toxicity after intragastric administration, chronic toxicity and cumulative ability, irritant and sensitizing effects, and possible long-term effects.

Sanitary-chemical analysis.

Sample preparation was carried out according to GOST 26929-94 "Raw materials and food products. Sample preparation. Mineralization for the determination of toxic elements. Interstate standard." Lead and cadmium were determined according to GOST 30178-96 "Raw materials and food products. Atomic absorption method for the determination of toxic elements. Interstate standard". Arsenic was determined according to GOST 26930-86 "Raw materials and food products. Method for determination of arsenic". Mercury was determined according to the "Guidelines for the detection and determination of total mercury content in food products by flameless atomic absorption" (No. 5178-90).

The content of organochlorine pesticides (toxicants) was determined according to the Methodological Guidelines for the Determination of Residual Amounts of Organochlorine Pesticides (No. 1766-77) and the official methods of analysis of AOAC ("Synthesis of Synthetic Hydrocarbons") (1984), 1984, 141B, CBSA, 1984, 141P, CBSA, 1984, 141P, APC, 1984, 141P, CBS, APC, 1984, POS. 537538).

The studies were performed using laboratory VLR-500 weights, a Carlo Erba gas-liquid chromatograph (NKOS 5700 model), and exchange cells of the Italian company Tehprop1A81.

The results of the measurements are presented in Table. 2 and indicate that in the claimed metabiotic composition, the content of toxicants (organochlorine pesticides HCTs and DDT) and hazardous substances (lead, cadmium, mercury, arsenic) does not exceed the permissible levels set by the Technical Regulations of the Customs Union "On Safety TS 021/2011" On Safety food products "(Appendix 3, section 10) [41]. The same pesticides as aldrin and heptachlor are not found in the claimed metabiotic composition.

Microbiological studies.

Microbiological studies were carried out in accordance with the requirements of the guidelines MUK 4.2.577-96 "Methods of microbiological control of products for children, medical nutrition and their components" and MUK 2.3.2.721-98 "Determination of safety and efficacy of biologically active food additives", GOST R 52814 -2007 "Food products. A method for detecting bacteria of the genus 8-150a11a", GOST R 52816-2007 "Food products. A method for detecting and determining the number of bacteria of the group of intestinal sticks (coliform bacteria)", GOST 10444.12-88 "Food products. The method is determined I yeasts and molds. " GOST 30726-2001 "Food products. Methods for detecting and determining the number of bacteria of the species E8c Nepsia soi".

Evaluation of purity and sterility metabolites of probiotic bacteria strains (VasP1i8 8iMSh8 № VKPM B-2335, Ep1etosossi8 Gaessht L3, BasYuasShsch be1tieski T81-06 and T83-06 BasYuasShsch GegtepSht) included in the inventive compositions metabioticheskoy. When assessing the sterility of metabolites, cultivation was carried out at a temperature of 37 ° C on liquid and dense nutrient media. To do this, 1 g of the starting material was diluted in 9 ml of water, phosphate buffer or saline sodium chloride.

In the study of the sterility of the VasPiI8 8SH1818 metabolites, the presence of VasPIi8 and 8SH8 in them was carried out. We carried out seeding of VasP1i8 and Sb18 metabolites into a liquid nutrient medium. To do this, 2 g of powder metabolites were introduced into 20 ml of broth and cultured for 24 hours at 37 ° C. Broth turbidity and precipitate formation (from powder) were noted. When seeding turbid broth on tryptose agar, bacilli were not detected.

They also carried out a check on the purity of the metabolites of VasSi8 8 and 18 (the admissible total infection is less than 10 ² CFU / g). A portion of Vaschn8 8iMSh8 metabolites weighing 10 g was added to 90 ml of sterile physiological sodium chloride solution and mixed thoroughly for 5 min. The supernatant was sterilely taken at 100 μg, seeded on Petri dishes with meat-peptone agar and incubated at 30 ° C for 7 days. or at 37 ° C for 24 hours. In both cases, the growth of extraneous microflora was not observed.

It has been established that the metabolites of VasSi8 and Si18, adsorbed on zeolite, do not contain live bacilli. Foreign microflora also did not grow (less than 10 ² CFU / g).

In the study of the sterility of the metabolites of Ep1etossi8 Haesst b3, they carried out a check on the presence in them of Ep1tossoci8 Haesst b3. For this purpose, the metabolism of the Epithetosi8 Haesst b3 metabolites was carried out in the liquid nutrient medium of Beal lego1H. To do this, 2 g of powder metabolites were introduced into 20 ml of broth and cultured for 24 hours at 37 ° C. When seeding broth on enterococcal agar entere - 8 028962

rococci were not detected.

When checking the purity of the metabolites of the Eighosossic Haesst b3 (permissible total contamination of less than 10 ² CFU / g), the weight of the metabolites of the Eigosossic of Haesst b3 weighing 10 g were added to 90 g of sterile physiological saline solution, thoroughly mixed for 5 minutes. The supernatant was sterilely taken at 100 μg, seeded on Petri dishes with meat-peptone agar and incubated at 30 ° C for 7 days. or within 24 hours at 37 ° C. In both cases, the growth of extraneous microflora was not observed.

It has been established that the metabolites Ep1egossos5 Haesht b3 do not contain live enterococci. The growth of extraneous microflora was also absent (less than 10 ² CFU / g).

In the study of the sterility of the metabolites of BioSchic biennial T81-06, the presence in them of Bacoaccadi5 biennial T81-06 was carried out. For this purpose, we carried out the seeding of the metabolites of Bos1o111 and 5b1Hgieskn T81-06 into Bilo i liquid nutrient medium. Powder of metabolites in the amount of 2 g was introduced into 20 ml of broth and cultured for 24 hours at 37 ° C. When the broth was sown on a selective dense nutrient medium, live lactobacilli Lacobac111 and 5b1Hgieskn T81-06 were not detected.

When checking the purity of the metabolites of LioBicSHIk b1Hegescn T81-06 (permissible total contamination is less than 10 ² CFU / g), the weight of the metabolites of LJBICSHICbEgHiysnn of T81-06 weighing 10 g was added to 90 g of sterile saline sodium chloride, thoroughly mixed for 5 min. The supernatant was sterilely taken at 100 μg, seeded on Petri dishes with meat-peptone agar and incubated at 30 ° C for 7 days. or within 24 hours at 37 ° C. The growth of extraneous microflora was not observed.

It has been established that the metabolites of Basachola 5 β1 hygiene T81-06 do not contain live lactobacilli. The growth of extraneous microflora was also absent (less than 10 ² CFU / g).

In the study of the sterility of the metabolites of LayoBass Schick Gegteyit T83-06, they carried out a check of the presence of Layoaschik Gegteit T83-06 in them. To this end, the metabolism of the Lacrobacter 5 Gegteit T83-06 was sown in the liquid nutrient medium Lactus, 2 g of the metabolite powder was added to 20 ml of broth and cultured for 24 hours at 37 ° C. When sowing the broth on a selective dense nutrient medium, live lactobacilli Lacobac111 and 5 Gegteit T83-06 were not detected.

When checking the purity of the metabolites of Basacut 5 Gegteit T83-06 (permissible total contamination of less than 102 CFU / g), a portion of the metabolites of Basactivus 5 Gegteith T83-06 weighing 10 g was added to 90 g of sterile saline sodium chloride, mixed thoroughly for 5 minutes. The supernatant was sterilely taken at 100 μg, seeded on Petri dishes with meat-peptone agar and incubated at 30 ° C for 7 days. or within 24 hours at 37 ° C. The growth of extraneous microflora was not observed.

It has been established that the metabolites of BioScic Gegteit T83-06 do not contain live lactobacilli. The growth of extraneous microflora was also absent (less than 10 ² CFU / g).

Thus, it was experimentally established that, according to microbiological safety indicators, the claimed metabiotic composition meets the requirements established by the Technical Regulations of the Customs Union TR TS 021/2011 "On food safety" (Annexes 1 and 2, Section 1.9) (Table 3).

Acute toxicity study.

The inventive metabiotic composition was administered to healthy outbred white rats of both sexes weighing 130-150 g and 180-200 g intragastrically in the form of aqueous suspensions through a metal atraumatic probe, slowly immersing it to the stomach. Studies were performed using probit analysis according to Litchfield-Wilcoxon in the modification of Z. Roth [42]. During the experiment, two groups of 10 animals each were formed: experimental (the claimed metabiotic composition was administered twice a day at the maximum tolerated dose of 15 g / kg) and the control (equivalent volumes of distilled water were injected). Animals were divided into groups randomly by randomization. The absence of external signs of disease and the homogeneity of groups according to body weight (± 10%) were considered as criteria for acceptability of randomization.

Observation of experimental animals was carried out for 14 days. with registration of indicators: mortality, time of death of animals, symptoms of poisoning, results of daily observation of general condition and behavior, weighing, results of monitoring feed and water consumption, results of autopsy and macroscopic description of dead and surviving animals at the end of the study (euthanasia was carried out by an overdose of ether), results determine the mass coefficients (MK) of the internal organs.

The introduction of the proposed metabiotic composition in the maximum tolerated doses did not cause the death of experimental animals. No other signs of a negative effect of the claimed metabiotic composition were noted either. In particular, no dyspeptic symptoms were noted. On all days of observation, the animals of the experimental and control groups did not differ in their general condition and behavior.

The results of the study of the dynamics of change in body weight in experimental animals that received the application 9 028962

a metabolic composition, for 14 days. observations made it possible to establish that the dynamics of the body weight of the animals of the experimental and control groups did not practically differ (Table 4).

Analysis of the MK values of the organs of white rats allowed to establish that the acute introduction of the claimed metabiotic composition by them in the maximum tolerated dose did not cause any significant differences compared with the control group of animals treated with distilled water (Table 5).

Thus, the results of toxicometry and observational data on experimental animals for 14 days. after an acute injection, the claimed metabiotic composition can be attributed to non-toxic (relatively harmless by SD Zaagonikov) and low-hazard (hazard class IV according to GOST 12.1.007).

Chronic toxicity and cumulative studies.

Chronic toxicity studies were performed on non-linear white rats-males weighing 180-200 g with the introduction of the proposed metabiotic composition intragastrically through an atraumatic metal probe for 30 days with a daily dose of 1.5 g / kg. Animals of the control group received distilled water in the same volume.

The measurement of basic physiological parameters was carried out after 30 days of chronic daily administration of the claimed metabiotic composition.

No lethal effects were observed during the entire observation period. The general condition and behavior of the experimental animals remained normal and did not differ from the control group during the whole experiment.

The results of the study of integral indicators of total toxicity, are presented in table. 6 and 7 show that none of the analyzed parameters showed statistically significant differences with the control group of animals treated with distilled water (p> 0.05 at the 95% probability level) or pathological deviations beyond the limits of the variation of the physiological norm.

An autopsy of rats killed on the day after the last injection of the inventive metabiotic composition showed that the size, shape and color of their internal organs did not have macroscopic changes compared with the control group. The mucous membrane of the stomach and small intestine was shiny, pale pink, with no signs of irritation or inflammation.

Histological examination of drugs of the lungs, myocardium, liver, kidneys and gastric mucosa of experimental animals treated with the claimed metabiotic composition, dystrophic, inflammatory or necrobiotic changes of organs were not detected.

Consequently, chronic intragastric administration of the claimed metabiotic composition into the organism of experimental animals did not cause dystrophic or destructive changes in parenchymal organs and was not accompanied by irritation of the mucous membranes of the gastrointestinal tract. Thus, according to the integral indicator of cumulation and indicators of total non-lethal toxicity, the claimed metabiotic composition does not have the ability to cumulate and is non-toxic.

A study of possible local irritation.

The local irritant effect of the claimed metabiotic composition was evaluated on the frog esophageal ciliated epithelium model. The cytotoxic effect was studied on Kapa marsh frogs (planed with a body weight of 25 g. After immobilization by destroying the spinal cord and fixing the animals on the cork table, the chest was opened, the esophagus was dissected and dissected in the caudal direction. Two rods were installed above the pharyngeal part of the esophagus. Two bars were installed during the experiment. the time of passage of the cork crumb of a segment of the esophagus with a length of 10 mm, limited by rods, before and after applying distilled water to the mucous membrane of the esophagus mucous membrane or the claimed metabiotic tion of the powder composition in an amount of 50 mg. The duration of exposure was 1 min.

It was established experimentally that the application to the mucous membrane of the esophagus of the claimed metabiotic composition in powder form does not affect the motor function of the ciliated epithelium of the frog esophagus (Table 8). Thus, the claimed metabiotic composition does not have a local irritant effect.

The study of the action on the skin and eyes.

The effect on the skin and the skin-resorptive effect was studied on white female rats weighing 130-150 g. After placing the animals in special houses, their tails for 2/3 were placed for 4 hours in a slurry, which was obtained by mixing the claimed metabiotic composition in powder form with cold water in quantities of, respectively, 90 and 10%. After 1 h and 16 h after the end of a single application, no erythema or swelling of the tail skin was noted (the amount of swelling was estimated by measuring the thickness of the tail in the middle part using a TR-1-10 thickness gauge). It was established that a 20-fold application did not cause the death of experimental animals and did not reveal irritating effects on the skin during the subsequent 5-day observation period.

Morphologically, the skin of the tails at the site of application of the powder had no changes. Epidermis and skin appendages were also intact. The layers of the epidermis are distinct, the basement membrane is preserved. Epithelial cells of the outer and inner root sheaths of the hair follicles and

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connective-woven bag were well expressed.

Macroscopic and microscopic changes in the internal organs of the experimental animals were absent.

The study of morphological, biochemical, hematological and physiological parameters of white rats did not reveal significant differences from the control group of animals whose tails were placed in water.

Therefore, the claimed metabiotic composition does not have an irritant effect on the skin and skin-resorptive effect, i.e. non-toxic when in contact with skin or in contact with it.

The irritating effect of the claimed metabiotic composition on the mucous membrane of the rabbit eye was also investigated. During the experiment, it was found that after 1 min after a single application of the claimed metabiotic composition in the form of a powder into the conjunctival sac of a rabbit eye in an amount of 50 mg, moderate hyperemia and lacrimation appeared, accompanied by a brief blepharospasm, which can be regarded as a reaction to an indifferent mechanical foreign body. The plethora of blood vessels was maintained for no more than 20 minutes, and after 40–45 minutes all the irritation phenomena that had occurred were completely gone. Therefore, the claimed metabiotic composition has a mild irritant effect on the mucous membrane of the rabbit eye, like any mechanical dust.

Investigation of inhalation exposure to dust.

A single dynamic inhalation effect of the claimed metabiotic composition in the form of powder on white rats-females weighing 130-150 g was produced for 2 hours in standard cells B.A. Kurland volume of 200 liters at a temperature of 20 ° C and air flow rate of 60 l / min. The maximum possible concentration of dust, which in the course of the experiment was determined by the gravimetric method, was achieved with the help of dust sprayers. Air samples were taken every 30 min. During the experiment, the average dust concentration was 53.4 ± 3.1 g / m ³ .

Both during the effects and after them, no experimental deaths were observed in experimental animals, but only signs of dust (mechanical) irritation of the upper respiratory tract (sneezing, grooming) and eyes (lacrimation), as well as anxiety.

After opening the sacrificed animals, the changes were not revealed, except for moderate replenishment of the internal organs. In the airways were determined particles of the powder of the claimed metabiotic composition.

Therefore, the acute inhalation exposure to dust of the claimed metabiotic composition is not dangerous, since it does not cause signs of poisoning and, like any mechanical dust, only slightly irritates the upper respiratory tract and eyes.

The study of sensitizing action.

The study of the sensitizing effects of the claimed metabiotic composition was carried out on guinea pigs. To this end, 0.02 ml of an aqueous dilution of the claimed metabolic composition at a dose of 10 mg / kg was injected once into the skin of the outer surface of the animals with a tuberculin syringe, and after 10 days, the declared metabiotic composition was applied to the pre-cut skin on the lateral surface of the back 2x2 cm powder form at the rate of 20 mg / cm ² . Blood sampling from animals was carried out 3 hours after skin tests were made.

The results of the evaluation of the effect of the proposed metabiotic composition on the indicators of the sensitizing action are presented in Table. 9 and indicate the absence of signs of sensitization in the peripheral blood - eosinophilia or an increase in the level of lysis in the reaction of specific lysis of leukocytes (RSLL) compared with the control. When examining the skin of the back of experimental animals, no signs of irritation were noted. Therefore, the claimed metabiotic composition with epicutane contact does not have a sensitizing effect, that is, does not provoke the development of allergies.

The study of possible long-term effects.

The study of the possible gonadotoxic effect was carried out in accordance with the methodological recommendations on the indicators that are mandatory for the initial evaluation of chemicals [43, 44]. The study of mutagenic activity was carried out according to the frequency of micronucleus formation in polychromatophilic erythrocytes of the bone marrow (from the femur) of white mice in accordance with the methodological recommendations [45, 46].

The results of the study of possible gonadotoxic and mutagenic effects of the claimed metabiotic composition with its introduction for 10 days at a dose of 1 g / kg, carried out 15 days after the end of its introduction, indicate the absence of significant changes in the indicators of gonadotoxicity and mutagenicity (Table 10). Inhibition of spermatogenesis or an increase in the frequency of mutations was also not observed.

Therefore, the claimed metabiotic composition does not have a toxic effect on reproductive function and does not have a mutagenic effect. This indicates its safety in terms of the development of long-term negative consequences in humans.

Example 3. Evaluation of the stability of the proposed metabiotic composition during storage.

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The possibility of preserving the activity and safety of the claimed metabiotic composition in powder form was investigated and the guaranteed shelf life was established in case of storage at different temperatures and without the use of special equipment.

During the experiment, the powder was placed in 100 consumer packages in the form of double plastic bags and stored for 2.5 years at temperatures of 5 ± 2 ° C, 10 ± 3 ° C, 25 ± 5 ° C, 35 ± 6 ° C ( 25 packs for each temperature storage mode). Every 6 months. storage were taken on 5 packs from the appropriate temperature and determined the characteristics of the powder: moisture, amylolytic and proteolytic activity, antagonistic activity and microbiological purity. To assess the possible changes in these characteristics during storage, their values were determined at the beginning of the study.

The data presented in table. 11, indicate that when selected for testing temperature storage conditions (from 5 to 40 ° C), the estimated indicators remain virtually unchanged for 2.5 years. The average values of the indicators were humidity 6.8 ± 0.1%, amylolytic activity 2.0 ± 0.2 units. AA / g, proteolytic activity of 1.0 ± 0.12 units PA / g. The microbiological purity of the claimed metabiotic composition, assessed by the absence of pathogenic microorganisms in it (Rgiyotopak aegitschok, EYBOLAypaseae, §1ParBi1osossik aigeik and VasShik Segeik), was maintained for 2.5 years, i.e. the whole period of research.

Thus, the guaranteed shelf life of the claimed metabiotic composition, made in solid form in the form of a powder, is not less than 2.5 lbs at a temperature not higher than 40 ° C, provided that the packing is preserved. Special temperature storage conditions or the use of special equipment is not required.

Example 4. Investigation of the influence of the VasSHICKIOSHK VKPM metabolites No. B-2335 on the growth and reproduction of probiotic lactobacilli BasBasCyc pIayatit 8K-A3.

In the course of the experiment, йη uigo influenced the metabolites of Vaschik viral PCMM number B-2335 on the growth of lactobacilli basacobaccinus p1ap1atite 8K-A3, which are close to the normal component of the human intestinal microflora of the organism.

Studies were performed using

the method of direct antagonism by applying sterile metabolites of the bacterium VasChickiKiShk VKPM No. B-2335 strain into the wells or onto the surface of solid nutrient medium (MPC-4 agar) immediately after seeding lactobacilli yaObicSchic p1ap1atite 8K-A3 into it, followed by incubation for 24 hours with temperature 37 ° С;

the method of double-layer agar [47], in which a suspension of metabolites of VasCycKiSHC VKPM No. В-2335 was introduced into the lower layer of solid nutrient medium (MPC-4 agar), and lactobacilli were seeded into the upper layer.

Metabolites VasShikKYShk VKPM number B-2335 was administered in concentrations of 1 g or 0.1 g in 10 ml of agar.

Lactobacilli were seeded in various quantities, constituting 7 1§ CFU / ml - 2 1§ CFU / ml.

After the application of sterile metabolites of VasSHIK KYShK VKPM No. B-2335 into the wells or onto the surface of agar seeded with Lactic acid bacterium LactusCyc p1Ayat 8K-A3, experimentally, using the direct antagonism method, it was established that the metabolites VasCyQiCyCVPM No.--30-23-A-23-A-3-A-3-U-2335 the specified strain of lactobacilli, zone of inhibition of growth of lactobacilli are absent.

The use of the bilayer agar method confirmed that the metabolites of VasSchik KiVKPM No. B-2335, when administered at the indicated concentration, do not inhibit the growth of Lactic acid bacterium p1ap1atit 8K-A3. At the same time, it was experimentally established that the metabolites of VasSHICKIOSHK VKPM No. B-2335 stimulate the growth of the probiotic Lactobacilli LABOxCyc p1ap1atit 8K-A3 (Table 12). In the presence of these metabolites, a larger amount of Lactus bacterium P1ap1atit 8K-A3 (four or more times) grew in accepted concentrations.

It was also established that the colonies of lactobacilli when sowing 50 colonies per Petri dish and growing in the presence of VasSHICKINSV VKPM No. B-2335 metabolites were 2 times larger, their diameter increased from 1 to 2 mm compared to control (without adding metabolites of the indicated bacterial strain ).

Thus, the lactogenic effect of the VasSHICKIOSHK VKPM No. B-2335 metabolites has been experimentally established, which is expressed in stimulating the growth and reproduction of probiotic lactobacilli LactusBicPlAyatit 8K-A3. This indicates that the metabolites of VasShik KiYShk VKPM No. B-2335 in the composition of the claimed metabiotic composition represent a lactogenic factor, which is due to their positive effect in restoring the intestinal normal flora.

Example 5. Investigation of the effect of the metabolites of Etossossic HaesstL-3 on the growth and reproduction of probiotic bifidobacteria.

In the course of the experiment, the influence of the metabolism of Exetossik Haesht L-3 on the growth and reproduction of probiotic bifidobacteria Vytobinae Uyit 1 and VygioBasiogt 1opdit OT15, close to the natural component of the intestinal microflora of the intestine, was investigated.

Studies were performed using the method of double-layer agar, as described in example 4.

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Metabolites Epilessian Haesst L-3 was administered in concentrations of 1 g or 0.1 g in 10 ml of nutrient medium for bifidobacteria. Indicator strains of Bifidobacterium Bibiophilus leuch Hydet 1 and Bibiotus Bacteriophilus epitum CT15 were inoculated in various quantities of 7-2 IU CFU / ml.

Experimentally, using the bilayer agar method, it has been established that metabolites of the probiotic strain of bacteria Eoshossosik Haesst L-3 in the indicated concentrations do not have an inhibitory effect on Bifidobacterium B1ToBeasteiit UIDit 1 and B1yTodusIeIt 1Tdit CT15 and apply to the patterns, which are used to put the 15% to 15%. . In the presence of the indicated metabolites, in the accepted concentrations, a greater number of Bifidobacterium Bibiocomplexit Hypdit 1 (three and more times) and Bibiocompatime 1pdit CT15 (four or more times) grew.

Thus, the bifidogenic effect of the claimed synbiotic composition was experimentally established, which included the metabolites Eaygosossik Haesst L-3, which is expressed in stimulating the growth and reproduction of probiotic bifidobacteria B1Docomplex. YyDit 1 and VShDoas1epit 1 pdt ST15.

Example 6. The study of the specific antagonistic activity of the claimed metabiotic composition and the composition of the prototype against conditionally pathogenic microorganisms.

In the course of the experiment, comparative studies were carried out on the specific antagonistic activity of the composition of the prototype, including metabolites of the probiotic strain of bacteria Vas Chick Quyik VKPM No. B-2335, and the claimed metabiotic composition, including the complex of metabolites of probiotic strains of Vas Was bacteria Kiquyyk VKy-2335-2335, 58 -3, LasHoSyScIe TGI0610 and LashoSaGyGeShit T83-06, for conditionally pathogenic microorganisms.

In the particular case, a prototype composition of the following composition was used: SKZh containing metabolites of the VKPM strain No. B-2335 of bacteria VasShikkykyk (2.1 wt.%), Zeolite (44.6 wt.%), Polysaccharides complex (52.4 wt. .%), calcium stearate (0.9 wt.%).

Gram-positive (enterococci, staphylococcus, Listeria) and gram-negative bacteria (Escherichia, Klebsiella, pseudomonads) were used as indicator test cultures (Table 15). Cultures of microorganisms used as an indicator correspond to those that are usually detected in the diagnosis of dysbiotic disorders of the GIT microbiocenosis.

Indicator test cultures of microorganisms were sown in the course of the experiment on a nutrient medium in various quantities, constituting 7-2 1d CFU / ml.

Solid medium MPC-4 was used as a nutrient medium.

The studies provided for the determination of the growth inhibition of indicator test cultures of conditionally pathogenic microorganisms when exposed to the composition of the prototype or the proposed metabiotic composition and were carried out using

the method of direct antagonism by directly applying the composition of the prototype or the claimed metabiotic composition in the wells or on the surface of a solid nutrient medium immediately after seeding the indicator test cultures, followed by incubation for 24 hours at 37 ° C;

method of double-layer agar, in which the composition of the prototype or the claimed metabiotic composition was introduced into the lower layer of solid nutrient medium, and indicator test cultures of conditionally pathogenic microorganisms were seeded into the upper layer.

When using the method of direct antagonism, it was established that the prototype composition and the claimed metabiotic composition exhibit a specific antagonistic effect on pseudomonads (table 16) and listeria (table 17). Moreover, the antagonistic effect of the claimed metabiotic composition in relation to pseudomonads and Listeria is more pronounced than in the composition of the prototype.

Using the method of double-layer agar, the antagonistic activity of both the prototype composition and the claimed metabiotic composition was evaluated. To do this, 1 g of the composition of the prototype or the proposed metabiotic composition was introduced into the lower layer of agar. In this case, indicator test cultures of conditionally pathogenic microorganisms were introduced into the upper layer of agar.

Also, the composition of the prototype and the claimed metabiotic composition significant antagonistic effects manifest in respect of staphylococci and Escherichia (Tables 18 and 19), the growth of these indicator test cultures is absent when added to the lower layer of agar, 1 g of each of the studied compositions. It was experimentally established that the antagonistic activity of the claimed metabiotic composition exceeds the specified characteristic of the composition of the prototype.

Thus, it has been established experimentally that the claimed metabiotic composition has a sufficient level of specific antagonistic activity against a wide range of conditionally pathogenic microorganisms, which is associated with a high level of activity of bacterial metabolites of Eyagosossik Haesst L-3 (bacteriocins A and B), as well as BosHoGesHikSHyesyy T. -06 and las1bcSHik GegtepSh T83-06 (lactocin B). The most reliably observed effects are demonstrated by the example of test cultures of pseudomonads (frequent pathogens of purulent-inflammatory diseases) and listerias (causative agents of intestinal and urogenital infections).

Example 7. The study of the immunotropic effects of the claimed metabiotic composition and the composition of the prototype.

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Since local immunity plays a significant role in maintaining the colonization resistance of the organism, studies have included an assessment of the ability of the prototype composition and the claimed metabiotic composition to influence the factors of nonspecific resistance of the organism. Phagocytic activity of local macrophages, as well as antibacterial and antiviral substances (lysozyme, interferon) were considered among the local non-specific factors of anti-infective protection.

The studies were performed on white outbred laboratory mice weighing 18-23 g (nursery "Rappolovo" RAMS). A total of 150 animals were used (three groups of 50 individuals each: a control one (an isotonic solution of sodium chloride was obtained) and two experimental animals in which the prototype composition or the claimed metabiotic composition were administered to the animals.

In the particular case, a prototype composition of the following composition was used: SKZh, containing metabolites of the VKPM strain No. B-2335 of bacteria Vaschiz zyShz (2.1 wt.%), Zeolite (44.6 wt.%), Polysaccharide complex (52.4 wt. .%), calcium stearate (0.9 wt.%).

The composition of the prototype and the claimed metabiotic composition was administered orally 1 time per day at a dose of 150 μg / mouse.

Introduced the composition of the prototype and the claimed metabiotic composition for 7 days. Evaluation of the effect of their impact was carried out after 1, 5 and 7 days. To do this, before the introduction of drugs (control), as well as after 1, 3 and 7 days. after the introduction of the animals of each group, blood was collected for the study. At each stage of the study, material was sampled from 10 animals from the group, individually from each mouse after its decapitation.

The dynamics of changes in factors of nonspecific resistance was assessed by determining the functional state of phagocytic system cells (the digestive activity of granular leukocytes), as well as serum concentrations of lysozyme and myeloperoxidase, which have not only a direct bactericidal effect, but also mediators of intercellular interaction during the development of an immune response to any antigenic impact.

The digesting activity of granular peripheral blood leukocytes in experimental animals was assessed by the method of Zelenova E.G. and Nikiforova V.A. (1972). Serum was obtained by the standard method, separating the blood clot from the liquid phase. To assess the functional state of phagocytic cells, the blood was taken into test tubes pretreated with silicone (8guua, Germany) and containing a solution of heparin at the rate of 20 IU per 1 ml of blood.

The phagocytic index (phi), which represents the ratio of the severity of the absorption and digesting functions of phagocytic cells, was determined after 15 minutes and 30 minutes of incubation with a phagocytosis test object — a test microbe (M. 1b5bl5c), grown on a meat-agar agar at a temperature of 37 ° C for 24 h. The index of the digestive activity of granular leukocytes (PI), representing the ratio of the number of phagocytic cells in a smear after 30 minutes of incubation of blood with a phagocytosis test object to the amount of phagocytic cells in a smear after 15 min of incubation of blood with a phagocytosis test object. The values of PI were determined by the formula

PI = B: A, (1)

where A is the number of phagocytic cells per 100 cells, counted in a smear of granular leukocytes, which swallowed the test microbe during 15 minutes of incubation;

B - the number of phagocytic cells per 100 cells, counted in a smear of granular leukocytes, which swallowed the test microbe during 30 minutes of incubation.

The value of PI expressed in arbitrary units (Fig. 1). Experimental data indicate that the use of the inventive metabiotic composition as well as the composition of the prototype contributes to a significant increase in the functional activity of peripheral blood granular leukocytes, since the PI values significantly exceed the level of the control group (differences with the control group are significant at p <0.05). At the same time, the effect of the claimed metabiotic composition is higher than that of the prototype composition (the differences with the “composition-prototype” group are significant at p <0.05).

The concentration of lysozyme in the serum was determined by the method of Bukharin OV et al. (1974). For this purpose, the blood of an experimental animal was collected in a centrifuge tube and serum was obtained. Then 0.1 ml of serum was transferred to a bacteriological tube and 0.9 ml of saline was added. The resulting mixture was stirred and 1.5 ml of the test microbe suspension was added to it, which was previously standardized on a FEK-56M photoelectrocolorimeter until extinction 0.66.

The prepared suspension containing the test microbe and the test serum was mixed and placed in a thermostat for 30 minutes at a temperature of 37 ° C. After the time of incubation, the tubes were removed from the thermostat and the extinction of the material was determined. The obtained extinction values using special tables were converted to values characterizing the concentration of lysozyme in the serum, and expressed in μg / ml.

The level of myeloperoxidase (MPO) in the serum was determined by the method developed on the basis of generally accepted approaches to the identification of this substrate in the blood serum and immune complex 14 028962

tent cells (Ge11 R.C.N. et al. (1968); Rade KS. et al. (1978)). To determine serum MPO concentration in 96-well flat-bottom immunological plates, starting from the second well of row A, 0.1 ml of sera taken from experimental animals were poured. Then they were added with 0.1 ml of reagent, which is a mixture of benzidine and 3% hydrogen peroxide. In the first well of row A of the panel (control), instead of serum, 0.1 ml of saline was poured. After adding the reagent, the panels were shaken, placed in a thermostat and kept at 37 ° C for 60 minutes. After the time of incubation, the panels were removed from the thermostat and viewed on a vertical spectrophotometer EupaYusk (Germany) at a wavelength of 495 nm. The serum enzyme concentration was obtained in extinction units and expressed in arbitrary units.

As shown in FIG. 2-3 data, immunotropic effects of the composition of the prototype and the claimed metabiotic composition are manifested at the level of the cells of the phagocytic system of the blood and the processes associated with the synthesis and secretion into the blood of the enzymes lysozyme and myeloperoxidase. The dynamics of changes in the values of factors of nonspecific resistance in experimental animals of the experimental groups that were injected with the prototype composition or the claimed metabiotic composition are almost the same, as evidenced by the absence of statistically significant differences (p> 0.05). The revealed effects, consisting in a significant increase in the functional activity of factors of nonspecific immunity, were recorded already after 1 day. after the introduction of the investigated compositions and they lasted at least 7 days. from the moment of introduction. That is, the stimulating effect of both the composition of the prototype and the claimed metabiotic composition on the mechanisms of immunological resistance took place after a single injection.

Introduction as a composition of the prototype, and the claimed metabiotic composition for 1, 3 and 7 days. leads to a significant increase in the activity of factors of nonspecific resistance compared with the control. This manifests itself both in an increase in the index of the digestive activity of granular leukocytes, and in an increase in the concentration of lysozyme and myeloperoxidase. Quite a significant increase in the functional activity of these factors is observed after a single injection of the tested compositions already during the first day. On the one hand, this indicates the activation of factors of nonspecific immunity and the possibility of ensuring rapid nonspecific protection of the body against pathogens. On the other hand, this means that the primary mechanisms of action of the studied compositions are associated not only with a direct effect on the gastrointestinal microflora, but also with a direct effect on the components of local immunity, which, inter alia, are associated with the regulation of the balance of endogenous cytokines that enhance protective reactions.

In the case of using the inventive metabiotic composition, which contains, like the composition prototype β-glucan (as part of oatmeal), practically the same efficiency was observed in the established effects in comparison with the composition of the prototype. Thus, the claimed metabiotic composition had a similar effect on the cells of the phagocytic system of the blood and their oxygen-dependent bactericidal mechanism (the concentration of myeloperoxidase in the blood serum increased). At the same time, it is known that an increase in the concentration of serum myeloperoxidase indicates not only the activation of nonspecific immunological resistance, but also an increase in the probability of its activating effect on immunocompetent cells, since their membrane has a corresponding specific receptor.

It was established experimentally that the stimulating effect of the claimed metabiotic composition on the mechanisms of nonspecific resistance is almost the same as that of the prototype composition. The concentration levels of lysozyme and myeloperoxidase in the serum of experimental animals treated with the claimed metabiotic composition, after 1 day. increased, respectively, by 3.9 and 1.9 times in comparison with the control group of animals that were injected with isotonic sodium chloride solution. In comparison with the group of experimental animals treated with the composition of the prototype, the levels of concentrations of lysozyme and myeloperoxidase after 1 day. grew, respectively, 1.05 and 1.09 times. Thus, the claimed metabiotic composition exhibits almost the same immunotropic effects as the composition of the prototype.

Example 8. The study of the interferonogenic properties of the composition of the prototype and the proposed metabiotic composition.

Since non-specific immunological resistance is designed to provide protection not only for bacterial, but also viral infections, the extent to which the composition of the prototype was investigated and the claimed metabiotic composition was able to influence interferonogenesis.

The studies were performed on outbred laboratory mice (nursery "Rappolovo" RAMS) weighing 18-23 g. Experimental groups of animals were formed as described in example 7.

In the particular case, a prototype composition of the following composition was used: SKZh, containing metabolites of the VKPM strain No. B-2335 of bacteria VasShik SUBY8 (2.1 wt.%), Zeolite (44.6 wt.%), Polysaccharide complex (52.4 wt. .%), calcium stearate (0.9 wt.%).

To determine the interferonogenic properties of the composition of the prototype and the claimed metabiotic composition, blood samples were taken from test animals from the retroorbital sinus over time, combining the material from three animals in one sample. Obtained after centrifugation at

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1000 rpm for 10 min the serum was aspirated and stored at minus 20 ° C. The internal organs of the mice were ground with the addition of a physiological solution, the homogenate was centrifuged (4000 rpm) for 20 minutes, and a 10% suspension was prepared from the supernatant in a physiological solution.

The samples thus obtained were stored at a temperature of minus 20 ° C. Titration of interferon (IFN) in the obtained samples was performed by micromethod in cell culture. An L-929 cell culture diluted with growth medium to the required concentration (3-4-10 ⁵ cells / ml) was added to the wells of 96-well plastic panels and incubated in a thermostat for 24 hours. Throughout the titration process, the incubation of cell cultures took place at identical conditions: temperature 37 ° C in an atmosphere with 5% CO ₂ and 98% humidity. After the formation of a cell monolayer at the bottom of the wells, the growth medium was removed from the panel and a supporting medium was added, in which sequential twofold dilutions of the test samples were made (in paired wells). At the end of the daily incubation, the test samples were removed and test encephalomio-carditis virus (EMC) was introduced at a working dilution of 100 CPD50 (cytopathogenic dose at which 50% of the cells die). A day later, the results were recorded using an inverted microscope. The titer of IFN is the reciprocal of the last dilution of serum or sample of the organ homogenate, at which a 50% protection against the virus is observed.

It was established experimentally (Fig. 4) that a single administration of both the composition of the prototype and the claimed metabiotic composition at a dose of 150 μg / mouse each has almost the same fairly pronounced interferonogenic effect, since already after 1 day. after administration of the tested compositions, the level of serum interferon increased, respectively, to 158.4 U / ml and 163.7 U / ml, which is 49.5 times (the prototype composition) and 51.2 times (the claimed metabiotic composition) exceeds the control groups of animals (the differences are significant (p <0.05)). Later, a decrease in the level of serum interferon was observed, however, even at subsequent periods of the study, the estimated index for the studied compositions reliably exceeded the data of the control group of animals (p <0.05).

Studies have shown that the claimed metabiotic composition, as well as the composition of the prototype, in addition to activating the mechanisms of bacterial protection significantly activates interferonogenesis, which represents a key mechanism of antiviral protection. Quite fast production of endogenous interferon allows, at an early stage, to ensure colonization resistance of the human intestinal microbiocenosis and reliable protection of the body against viral pathogens.

Thus, studies have shown that the claimed metabiotic composition, representing a balanced complex of biologically active substances

has a sufficient level of antagonistic effect against a wide range of pathogens. The observed effect is due to the favorable overall effect of the lactobacillus metabolites of LacFBaSchis bs1Biski T81-06 and LacObArSi5GSGPSTTT83-06, as well as the metabolites of the probiotic strain of bacteria Ei1cgosssi8 Gasshb-3. Most reliably, these effects are manifested in relation to pseudomonads (frequent pathogens of purulent-inflammatory diseases) and Listeria (causative agents of intestinal and urogenital infections);

shows lactogenic effect. It has been experimentally shown that the metabolites of Vaschizyus VKPM No. B-2335 in the composition of the inventive metabiotic composition represent the lactogenic factor and stimulate the growth and reproduction of lactobacilli lacFbSchiz p1ai1agit 8K-L3 close to the indigenous component of the human intestinal microflora;

exhibits a bifidogenic effect. It has been experimentally shown that the metabolites of EShsgossis Gassht L-3 cause a bifidogenic effect and stimulate the growth and reproduction of probiotic bifidobacteria VIIIt 1 and Tx115, which are close to the natural component of the indigenous microflora of the human intestine

It has an effective stimulating effect on immunocompetent cells and contributes to an increase in the nonspecific resistance of the organism;

contributes to the fairly rapid production of endogenous interferon, which allows at an early stage to provide colonization resistance and reliable protection of the body against viral pathogens.

In addition, the claimed metabiotic composition does not contain toxic and hazardous components in quantities exceeding permissible levels, belongs to low-hazard substances, does not have a sensitizing effect and the ability to cumulation, undesirable side effects, including toxic effects on reproductive function, mutagenic effect and long-term negative effects, stable when stored for 2.5 years.

The claimed invention meets the criterion of "novelty", since for the first time to ensure colonization resistance of the human intestinal microbiocenosis, it is proposed to use a metabiotic composition made in a solid capsulated dosage form for oral administration and representing a balanced complex of biologically active substances, together providing a sufficient level of antagonistic effect on

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spectrum of pathogens, lactogenic and bifidogenic effects on the indigenous component of the intestinal microflora, contributing to an increase in non-specific resistance of the organism.

The claimed invention meets the criterion of "inventive step", because of the available information did not seem obvious expediency, the inclusion in the composition of the claimed metabiotic composition of the metabolites of probiotic bacterial strains VasShik KiENK VKPM No. В-2335 and Еп1egossossik Haesst L-3 and the possibility of providing them, respectively, lactogenic and bifidogenic effects, as well as the possibility of achieving a sufficient level of antagonistic effect against pathogens of bacterial and viral nature due to the actions of a mixture of lactobacillus metabolites of LacFacDe 0E1Bgieski T81-06 and LacPenGal GhegshepCpp T83-06 and the production of endogenous interferon.

The conformity of the claimed invention to the criterion of "suitability for use" is confirmed by the examples given, which showed a simple technology for obtaining the claimed metabiotic composition in a solid encapsulated dosage form for oral administration, the ingredients of which are produced in an industrial environment are available and inexpensive, and the claimed metabiotic composition has a high storage stability, as well as the results of numerous studies confirming the safety of the proposed metabiotic composition and its efficiency in providing resistance microbiocenosis colonizing human intestinal.

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Table 1

Characteristics of the biological and physico-chemical properties of sterilized culture fluid obtained by deep cultivation of the bacterial strain VasShik KyoPk VKPM No. В-2335

Name of the analyzed indicators Values of the analyzed parameters in the SCJ of the bacterium strain Vaschizus Bay VKPM No. В-2335 Residual concentration of microbial producers (cells), CFU / ml 1 · 10 ⁷ Protein content *, µg · cm ' ³ 541.5 Proteolytic activity (PA), units PA dm " ³ 8.7 Amylolytic activity (AA), units AA-dm " ³ 42.7 pH, units pH 5.6 OP, units OP 0.31 WITH% 0.68 Total concentration of total amino acids, µg cm ’ ³ 568,8 The total concentration of free amino acids, µg cm ' ³ 60,8 Concentration of total, µg cm ³ - hexosamines - glucosamines - diamino-chimelic acid 104.9 95.6 19.6 The concentration of free glucosamine, µg-cm ' ³ 6.5 The concentration of free / bound nitrogenous bases, µg / cm ³ - adenine / Xa ** - guanine / Xg ** - thymine / Xm ** - uracil / Hu ** - cytosine / Xz ** 12.2 / 3.2 0 / 16,3 8.2 / 0 11.4 / 8.6 0 / 12,1 Notes: * - protein content was determined by the method of Lowry; ** - Xa, Xg, Xt, Hu, Xc - unidentified derivatives, respectively, of adenine, guanine, thymine, uracil and cytosine, calculated on the basic substance

table 2

The content in the claimed metabolic composition of toxic elements and

organochlorine pesticides

The studied indicators The content of toxic elements and organochlorine pesticides, mg / kg Standard TR TS 021/2011 (Annex 3, section 10) In the inventive metabolic composition Toxic elements, mg / kg: - lead 5.0 0.04 - cadmium 1.0 0.01 - mercury 1.0 0,003 - arsenic 3.0 0.03 Pesticides, mg / kg: - HCCH (sum of α-, β- and χ-isomers) 0.1 <0.01 <0.01 not detected (<0,001) not found (<0,001) - DDT and its metabolites 0.1 - Aldrin - Heptachlor not allowed (<0.002) not allowed (<0.002)

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Table 3

Microbiological safety indicators of the proposed metabiotic composition

The studied indicators The values of microbiological indicators Standard TR TS 021/2011 (Annexes 1 and 2, Section 1.9) In the inventive metabiotic composition KMAFAM, CFU / g, not more 5X 1 · 10 ² BGKP (coliforms), the mass of the product (g), which are not allowed OD not detected Uncapsules, mass of product (g), which is not allowed 1.0 not detected Pathogenic microorganisms, incl. Salmonella, product weight (g), which is not allowed 10.0 not detected Yeast, CFU / g, not more than 100 one Mold, CFU / g, not more than 100 not detected Spore-forming microorganisms of the species Waschis liyis, product mass (g), which is not allowed 1.0 Live cells of the bacterial strain Waschis sibnz VKPM No. B-2335 were not detected Microorganisms of the species EHegossoss Haesst, mass of product (g), which are not allowed 1.0 Live cells of the strain of bacteria Degosossiz Haessht BZ not detected Microorganisms of the species BasYonSchez 4e1Hsheski T81-06, mass of the product (g), which are not allowed 1.0 The living cells of the bacterial strain Lacuscease cilecreta T51-06 were not found Microorganisms of the species BasUnasSchiz GegtepScht TZZ-06, mass of product (g), which are not allowed 1.0 Live cells of the bacterial strain Lacusceus GegtepS TZ3-06 were not found

Table 4

The dynamics of change in body weight of rats treated with the claimed metabiotic composition

Group of animals View of animals The body weight of the animals (g) after a period of time background 2 days 7 days 14 days Control (treated with distilled water) male rats 193.8 + 3.8 193.2 + 3.1 195.2 + 3.2 200.8 + 4.1 female rats 189.2 + 3.0 189.4 + 3.1 192.3 + 2.2 200.3 + 3.1 Experienced (receiving the claimed metabiotic composition) male rats 189.1 + 3.9 189.3 + 3.7 196.3 + 4.0 199.3 + 2.7 female rats 188.9 + 3.7 191.1 + 1.9 194.3 + 2.2 201.7 + 2.4

Table 5

Mass factors (MK) of organs in white rats with acute administration of the proposed metabiotic composition

Analyzed organ The mass coefficients of organs (mg / 100 g mass of the gel) of animal groups Control (treated with distilled water) Experienced (received the claimed metabiotic composition) male rats female rats male rats female rats A heart 4.0 + 0.2 4.3 + 0.2 4.2 + 0.03 4.2 + 0.08 Lungs with trachea 7.2 + 0.2 7.5 + 0.7 7.6 + 0.1 7.3 + 0.2 Thymus 1.31 + 0.11 1.34 + 0.03 1.27 + 0.12 1.31 + 0.09 Liver 36.3 + 1.3 36.3 + 1.4 32.7 + 1.6 35.6 + 1.1 Spleen 4.3 + 0.2 4.22 + 0.15 4.5 + 0.2 4.4 + 0.2 Kidney (left) 4.3 + 0.1 4.3 + 0.2 5.4 + 0.3 4.7 + 0.08 Adrenal glands 0.12 + 0.02 0.11 + 0.01 0.10 + 0.03 0.12 + 0.01 Brain 8.9 + 0.2 8.7 + 0.2 9.2 + 0.1 8.8 + 0.2 Testicles or ovaries 13.5 + 0.2 0.27 + 0.01 11.7 + 0.2 0.25 + 0.02

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Table 6

Changes in the behavioral responses of non-linear white rats according to the open field method after chronic administration of the claimed metabiotic composition

(registration within 5 minutes), M ± t

Group of animals Indicators of behavioral reactions by the method of "open field" Horizontal motor activity Vertical motive activity Mink reflex Bolyus Integral activity Control (receiving distilled water) 32 ± 3 14 ± 3 11 ± 2 4 ± 1 63 ± 4 Experienced (receiving claimed metabiotic composition) 35 ± 4 15 ± 2 12 ± 3 5 ± 1 69 ± 7

Table 7

The effect of chronic administration of the claimed metabiotic composition on the indicators of total non-lethal toxicity of non-white non-linear rats, M ± t

Total non-lethal toxicity index Value of total non-lethal toxicity indicator Control After chronic administration of the inventive metabiotic composition Body weight, g 201 ± 9 207 ± 1 Mass indices of organs, g / 100 g body weight: - lungs 6.0 ± 0.2 5.4 ± 0.3 - liver 27.4 ± 0.5 26.7 ± 0.4 - kidneys 7.8 ± 1.0 7.3 ± 0.5 - spleen 4.4 ± 0.2 4.2 ± 0.1 - a heart 2.6 ± 0.1 2.5 ± 0.2 Urine indicators: - urine protein, mg% 3.5 ± 0.3 3.3 ± 0.4 - specific weight of urine, g / mm ³ 0.13 ± 0.32 0.12 ± 0.23 Blood biochemical parameters: - blood sugar, mg% 89 ± 3 84 ± 2 - whey protein, g% 8.05 ± 0.11 7.10 ± 0.13 - lipid phosphorus, mmol / l 32.86 ± 0.35 31.10 ± 0.33 - AlAT, mkat / l 0.80 ± 0.4 0.82 ± 0.5 - AsAT, ukat / l 0.73 ± 0.12 0.75 ± 0.11 - alkaline phosphatase, mkat / l 0.74 ± 0.05 0.77 ± 0.10 - thymol test, units cloud 0.81 ± 0.12 0.83 ± 0.5 Morphological blood parameters: - the content of leukocytes, thousand / mm ³ 9.11 ± 0.12 8.84 ± 0.23 - the content of red blood cells, million / mm ³ 6.30 ± 0.5 7.18 ± 0.06 - hemoglobin content, g% 14.1 ± 0.1 14.4 ± 0.2 Physiological indicators: - SPP, V 13.5 ± 0.3 14.1 ± 0.1 - duration geksenalovogo sleep, min 24.9 ± 1.9 23.8 ± 1.1

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Table 8

The influence of the proposed metabiotic composition on the motor function of the ciliated epithelium of the esophagus of the frog

Group of animals amount experiences The transit time of the cork crumb of the esophagus segment with a length of 1 С 'mm, s Original Through the time after application 1-3 min 7-10 min Control (distilled water was applied to the mucous membrane of the esophagus) ten 22.4 ± 1.35 27.2 ± 2.1 24.2 ± 1.5 Experienced (the declared metabiotic composition was applied on the mucous membrane of the esophagus) ten 20.9 ± 1.3 20.1 ± 1.5 23.1 ± 1.2

Table 9

Indicators of sensitization of peripheral blood of guinea pigs after intradermal administration of the inventive metabiotic composition, M ± t

Indicator sensitizing actions The value of the indicator sensitizing action Control After the introduction of the proposed metabiotic composition Leukocytes, thousand / mm ³ 10.2 ± 0.3 9.9 ± 0.2 Eosinophils,% 7 ± 1 6 ± 1 Lymphocytes,% 33 ± 2 34 ± 1 Monocytes,% 5 ± 1 5 ± 1 Neutrophils,% 51 ± 1 53 ± 4 RSLL,% lysis 10 ± 1 9 ± 2

Table 10

Indicators of gonadotoxicity and mutagenicity of the claimed metabiotic composition, M ± t

Indicator of gonadotoxic and mutagenic effects Value of the indicator gonadotoxic and mutagenic effects Control After the introduction of the proposed metabiotic composition Gonad weighting of male rats, g / 100 g body weight 6.15 ± 0.27 6.87 ± 0.31 The number of sperm, 10 ⁶ / ml 152 ± 17 163 ± 29 Duration of sperm movement, min 99 ± 08 102 ± 4 Osmotic resistance, 1M ΝαΟΙ 1.43 ± 0.5 2.39 ± 0.08 The number of analyzed bone marrow cells of mice 4137 ± 98 4142 ± 102 The frequency of cells with micronuclei,% 0.37 ± 0.01 0.39 ± 0.02

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T table 11

The study of changes in the characteristics of the proposed metabiotic composition during storage for 2.5 years

Name of the indicator Value of the indicator in the inventive metabiotic composition Before storage After storage Appearance Powder in the form of granules Powder in the form of granules Colour Light brown Light brown pecked Smell Specific, peculiar to this product Specific, peculiar to this product Humidity,% 6.5 6.8 ± 0.1 Amylolytic activity, units AA / g 2.3 2.0 ± 0.2 Prothetotic activity, units PA / g 1.1 1.0 ± 0.12 Microbiological purity: KMAFAM, CFU / g BGKP (coliforms), CFU in 0.1 g EzsNepsYa con, CFU in 1.0 g Pathogenic microorganisms, including: - Salmonella, CFU in 10 g - yeast, CFU / g - mold, CFU / g 1 · th ¹ not found not found not found 1 not found 1 K) ¹ not found not found not found 1 not found

Table 12

Effect of Vaschiz metabolites § VKPM No. B-2335 on the growth and reproduction of probiotic lactobacilli Lacobicus P. p1ap! Agit 8K-A3

Concentration metabolites VasShiz zyyyz VKPM number B-2335 Tenfold dilutions of LasFbosSchiz p1agLasht 8K.-AZ, seeded with 10 μL ***, CFU / g one 2 3 four five 6 Baseline * lawn thousands thousands hundreds 85 ten Breeding 1: 10 ** lawn thousands thousands hundreds 50 Control lawn thousands thousands 124 12 0 Notes: * - 1 g per 10 ml of nutrient medium; ** - 0.1 g per 10 ml of nutrient medium; *** - dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc.

Table 13

Influence of metabolites of Ep! Egossi§ Haesst E-3 on the growth and reproduction of bifidobacteria VshboSdovit Hybit 1 using the method of two-layer agar

Concentration of metabolites Еп1гососизиз Гаесшт Е-3 Tenfold dilutions of Vshioyaagsht Uyit 1, seeded with 10 μl ***, CFU / g one 2 3 four five 6 7 Baseline * lawn THOUSANDS THOUSANDS THOUSANDS HUNDREDS 85 7 Breeding 1: 10 ** lawn THOUSANDS THOUSANDS HUNDREDS 46 6 0 Control lawn THOUSANDS THOUSANDS 138 14 2 0 Notes: * - 1 g per 10 ml of nutrient medium; ** - 0.1 g per 10 ml of nutrient medium; *** - dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc.

Table 14

Influence of metabolites of Ep! Egossi§ Haesht 1.-3 on the growth and reproduction of bifidobacteria

Concentration of metabolites of Egygossoss Haesst L-3 Tenfold dilutions of VSE-Biokegsht 1op§it OT15, seeded with 10 µl ***, CFU / g one 2 3 four five 6 7 eight 9 Original concentration* lawn lawn lawn lawn THOUSANDS THOUSANDS HUNDREDS 85 ten Breeding 1: 10 ** lawn lawn lawn lawn THOUSANDS HUNDREDS 156 17 2 Control lawn lawn lawn lawn HUNDREDS HUNDREDS 52 four 0 Notes: * - 1 g per 10 ml of nutrient medium; ** - 0.1 g per 10 ml of nutrient medium; *** - breeding: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc.

- 25 028962

Table 15

Characteristics of the used indicator test cultures

Indicator test culture Strains indicator test cultures Source of receipt Gram-positive bacteria 81par 31ARUBOOSOSIZ AEGEIZ 209 Reference strain from the collection of the Department of Microbiology, St. Petersburg State University Psileph Hypopus toposus Reference strain (Germany), provided by the Laboratory of General Pathology, NIIEM SZO RAMS Gram-negative bacteria Essiepsy Use Cond M15 Collection of the Department of Molecular Microbiology of the Scientific-Research Institute of Electrometry of the Russian Academy of Medical Sciences Rezeiboshopaz Rzeybopaz aegispoza 27853 ATCC * Note. * - strain from the American type culture collection

Table 16

Antagonistic activity of the composition of the prototype and the claimed metabiotic composition in relation to pseudomonads

The number of test culture Rezeitopaz aggishpose ATSS 27853, Unit CFU / ml The diameter of the zone of growth inhibition of the test culture Rziiiyotopaz agschrpoza ATSS 27853 (mm) under the influence of prototype compositions claimed metabiotic compositions 7 no zone no zone 3 five eight 2 ten 13

Table 17

Antagonistic activity of the composition of the prototype and the claimed metabiotic composition against Listeria

Number of test culture PTepa toposue £ rpe8 GEO T _g cfu / ml The diameter of the zone of growth inhibition of the test culture had a topos! O _e ez ez (mm) under the influence prototype compositions claimed metabiotic compositions 7 no zone no zone 3 7 9 2 eleven 14

Table 18

Antagonistic activity of the composition of the prototype and the claimed metabiotic composition against staphylococci

The investigated composition (number) Tenfold dilutions of Apulusosiosis 209, seeded with 10 μl * Without breeding one 2 3 four five Claimed metabiotic composition (1 g) thousands hundreds 140 ** sixteen 0 0 Composition prototype (1 ^g ) thousands thousands hundreds 95 7 0 Control lawn lawn thousands hundreds 24 five

Notes: * - dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc.

** - hereinafter - the number of CFU (colony forming units)

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Table 19

Antagonistic activity of the composition of the prototype and the claimed metabiotic composition in relation to Escherichia

The investigated composition (number) Tenfold dilutions of EcSpecia so M15, seeded with 10 μl * Without breeding one 2 3 four five Claimed metabiotic composition (1g) thousands hundreds 84 ** 23 6 0 Composition prototype (1 g) thousands thousands hundreds 67 12 one Control lawn lawn thousands hundreds 35 eight

Notes: * - dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc.

** - hereinafter - the number of CFU

Claims (1)

CLAIM Metabioticheskaya composition for colonization resistance human intestinal microbiocenosis comprising sterilized dried culture broth, comprising metabolites of the probiotic bacterial strain Bacillus znONz VKPM B-2335, zeolite, calcium stearate or aerosil, characterized in that it further comprises a sterilized dried culture fluid containing metabolites Ep1egosossiz Gaesshsh E-3, EASIOBASP1 of his16031 T31-06 and EASO-CONSULIAS of 1363, as well as oatmeal, is made in solid oh dosage form in the form of capsules, and the number of ingredients in one capsule is, wt.%: sterilized dried culture fluid containing metabolites of the probiotic bacterial strain VasShiz zIYSHz VKPM No. B-2335 - 1.9; sterilized dried culture fluid containing metabolites of the probiotic strain of bacteria EH and Protoxicos E-3 - 4.5; sterilized dried culture fluid containing metabolites of the probiotic bacterium strain EasFascis jéesqua T31-06 - 4,5; sterilized dried culture fluid containing metabolites of the probiotic bacterial strain Easlobacterium of T33-06 - 4,5; zeolite - 64.3; oatmeal - 20.0; calcium stearate or aerosil - 0.3. Control □ Prototype Composition θ Metabiotic Composition Claimed