RU2589818C1 - Metabiotic composition to ensure colonisation resistance of human intestinal microbiocenosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for production of meta-biotic composition to ensure colonisation resistance of human intestinal micro-biocenosis. Meta-biotic component is a rational combination of metabolites of probiotic strains of microorganisms and additionally includes sterilised dried culture liquids containing metabolites of Enterococcus faecium L-3, Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06. Prebiotic component is represented by oat flakes. Composition is presented in a solid dosage form in the shape of capsules, and the number of ingredients in one capsule constitutes, MP: sterilised dried culture liquid containing metabolites of a probiotic strain of bacteria Bacillus subtilis VKPM No. B-2335 - 1.9; the sterilised dried culture liquid containing metabolites of probiotic strain of bacteria Enterococcus faecium L-3 - 4.5; the sterilised dried culture liquid containing metabolites of probiotic strain of bacteria Lactobacillus delbrueckii TS1-06 - 4.5; the sterilised dried culture liquid containing metabolites of probiotic bacterial strain Lactobacillus fermentum TS3-06 - 4.5; zeolite - 64.3; oat flakes - 20.0; calcium stearate or aerosil - 0.3.

EFFECT: use of this composition allows to normalise microbiocenosis of the intestine due to selective stimulation of growth and reproduction of Lactobacilli and bifidus bacteria of indigent component of normoflora of a biotope providing specific antagonistic activity with respect to a wide range of pathogenic and opportunistic microorganisms.

1 cl, 4 dwg, 19 tbl

Description

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

The urgent problem of modern medical science is connected with the study of possible violations of human microecology, and, first of all, violations of a very important body system - 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, the torpid course and the chronicity of inflammatory processes as developing or already occurring microbiota disorders that accompany the underlying 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 that affect people's health [3-5].

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

One of the most important functions of normal microflora is to ensure colonization resistance - a set of mechanisms that ensure the ability of microbiota and macroorganisms, cooperatively interacting, protecting the ecosystem from the effects of pathogenic microflora, stabilizing the normal microflora, preventing the body from being populated by extraneous microorganisms and providing its anti-infection protection [5-7] .

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

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

Therefore, the colonization resistance of intestinal microbiocenosis is mainly associated with the state of the biotope microbiocenosis and the mechanisms of local nonspecific 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 intestinal microbiocenosis. Protective reactions against viral and bacterial pathogens can be enhanced by activation of the immune system [13], when the stimulating signal is nonspecific in nature, directed not selectively against the pathogen that caused the infection, but increases 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. With dysbiotic disorders of 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.

A variety of exogenous and endogenous factors contribute to the development of intestinal dysbiosis [4, 5, 15]. Currently quite common are: stress and overwork, physical and psycho-emotional stress, smoking, unbalanced diet, eating disorders, overeating or snacking on the run, environmental dysfunction, many chronic diseases and a number of other pathological conditions. In such cases, due to the fact that microbiocenosis is an evolutionarily developed and clearly integrated system that ensures the coordinated operation of all organs and systems and is based to a certain extent on the principle of self-regulation, the occurring changes in the symbiotic microflora in most cases are restored spontaneously.

Everything develops completely differently after the irrational use of antibiotics. In clinical practice, a similar situation, in which, first of all, dysbiotic disorders of intestinal microbiocenosis occur, is currently common. Treatment with prescription of antibiotics is often the standard solution for a wide variety of diseases, including those common such as tonsillitis, bacterial infections of the upper and lower respiratory tract (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 the well-known basic schemes for emergency prevention and treatment of bacterial infections of the infectious diseases [16-19].

It should be noted that in order to achieve an adequate antibacterial effect, such drugs are usually taken often, for a long time and in large doses. Antibiotics, on the one hand, can effectively inhibit the excessive growth of pathogenic and opportunistic microorganisms, but on the other hand, with the commonly used dosage regimens, they often cause 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 microbiocenosis of the human intestine due to the lack of selectivity of their action on microorganisms. Under the influence of antibiotics, as a rule, 2/3 of patients have relatively long-term (within 2-3 months) disturbances of intestinal microbiocenosis and, above all, a balance of the qualitative and quantitative composition of microflora, as a result of which there are symptoms of dysbiosis.

It should be borne in mind that antibiotics are toxic, have a nonspecific 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 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 not in doubt. When correcting dysbiotic disorders of the intestinal microflora, a set of measures is carried out taking into account the clinical manifestations of the underlying disease and the severity of dysbiotic disorders of the intestinal microbiocenosis, as well as laboratory data on the biotope microflora and the state of reactivity of the body

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

- selective decontamination of pathogenic and conditionally pathogenic microflora;

- normalization of indigenous intestinal microflora;

- correction of the immunological reactivity of the body.

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 severity. In cases of dysbacteriosis of the 2nd and 3rd degree of severity of the arisen violations, the correction begins with the appointment at the first stage of the funds with selective antibacterial activity and providing selective decontamination of pathogenic and conditionally pathogenic microflora. These are various bacteriophages - staphylococcal, klebsiellosis, pyobacteriophage, intestinal bacteriophage, Pseudomonas aeruginosa and others. With low sensitivity to bacteriophages, antibacterial drugs (furazolidone, chlorophyllipt, metronidazole, nifuroxazide, intetrix and others) and antifungal agents (ketoconazole, fluconazole, natamycin and others) are used. At the second stage, it is mandatory to recommend a course of replacement therapy to normalize their own indigenous intestinal microflora. A significant role in this is played by the differentiated use of various immunobiological preparations.

The range of immunobiological preparations currently recommended for the correction of dysbiotic disorders of intestinal microbiocenosis is quite wide. Of these, probiotics are most widely used, including specially selected strains of living microorganisms or specific substances of microbial, plant or animal origin that exhibit antagonistic activity against pathogenic and conditionally pathogenic microflora, stimulate reparative processes in the intestinal mucosa and increase the body's immunological reactivity 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 areas of microbial adhesion, modify toxins or toxin receptors. Certain types of microorganisms non-pathogenic for humans and substances of microbial origin, with the natural method of administration, have beneficial effects on physiological functions, biochemical and behavioral reactions of the body through optimization and stabilization of its microecological status [21]. These include, first of all, bifidobacteria and lactic agents for regulating the equilibrium of intestinal microflora.

Bifidobacteria and lactobacilli are the basis of intestinal microflora and play a decisive role in ensuring the normal functioning of the biotope. Bifidobacteria, being saccharolytic microbes, secrete a large amount of acidic products. The resulting lactic and acetic acids enhance the absorption of calcium, iron, and vitamin D. The production of lysozyme bifidobacteria, bacteriocins, alcohols and high antagonistic activity against various pathogens prevent their penetration into the upper gastrointestinal tract (GIT). Bifidobacteria are highly capable of synthesizing amino acids, proteins, and many B vitamins, which are subsequently absorbed in the intestine. Therefore, with persistent, severe violations of the functions 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, the pathogens translocate to the upper intestines and their excess growth.

The main lactobacilli colonizing the gastrointestinal tract are Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus fermentum. The 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 several 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 conditions occur, the qualitative and quantitative composition of anaerobic bifidobacteria and lactobacilli undergoes disturbances 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, the drugs of choice are the probiotic complexes Linex and Bifiform [22, 23], the active principle of which are live bacteria. So, the Linex, manufactured by the pharmaceutical company Lek (Slovenia), includes a balanced complex of lactic acid bacteria: lactobacillus Lactobacillus acidophilus, bifidobacteria Bifidobacterium infantis and enterococcus Enterococcus faecium. In contrast to Linex, the Bifiform, produced by the Danish pharmaceutical company Ferrosan International A / C, includes, in addition to Bifidobacterium longum bifidobacteria (ATCC 15707 strain) and Enterococcus faecium enterococci (SF68 strain) from the Christian Hansenaya nutritional laboratory (Denmark) environment for their nutrition, growth and reproduction. In addition, Bifiform does not contain lactose, as a result of which it is indicated for patients with its intolerance. The bacteria included in the preparations are designed to regulate the physiological balance of the intestinal microflora through the production of lactic acid, to a lesser extent acetic and propionic acids. The acidic environment created as a result is unfavorable for the development of pathogenic and conditionally pathogenic microorganisms. Useful bacteria are freeze-dried and packaged in special protective capsules, which ensures their reliable protection against destruction under the influence of gastric juice and allows you to convey it in a slightly modified form directly to the intestine and release it there.

However, in addition to the beneficial effects of Linex and Bifiform preparations, there are a number of significant drawbacks that limit the possibility of their widespread use in clinical practice. First of all, it cannot be considered that the bacteria that make up them 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. Individual biocompatibility of microorganisms introduced as part of these preparations and their survival are not guaranteed. Accordingly, the probability of the fulfillment by the indicated bacteria, introduced into the macroorganism from the outside, of their main function (substitution), and even more so in a short period of time, is practically excluded.

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

Linex and Bifiform are recommended for the prevention and treatment of dysbiosis of various etiologies. At the same time, they are used mainly in the correction schemes for intestinal dysbiosis only at stage 2 for the purpose of replacement therapy. The compositions of these drugs included bacterial strains with a fairly high level of antibiotic resistance and often recommend their use simultaneously with antibiotics. However, the experience of using these agents in clinical practice with a wide range of antibiotics with the recommended regimens for their administration has shown that most of the bacteria with this combined purpose die and the activity of those who enter the intestines in a viable state is extremely low.

At the same time, it is now known that one of the promising directions for overcoming the negative effect 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 use with antibiotics, priority should be given to such probiotics that contain non-living microorganisms (Bifidobacteria, Lactobacterin, Sporobacterin, Laminolact, Linex, Bifiform and others), often alien to the natural indigenous microflora of the human gastrointestinal tract, which, moreover, can be destroyed by antibiotics, having a negative effect on the immune system and the macroorganism as a whole, and metabolites of probiotic strains of microorganisms are metabiotics [4, 24, 25].

The modern representative of such metabiotic agents is the composition, which in composition, mechanism of action on the body and achieved beneficial effect is closest to the claimed metabiotic composition and adopted as a prototype composition [26].

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

sterilized culture fluid containing metabolites Bacillus subtilis bacterial strain VKPM No. B-2335 - 1.6-2.3; zeolite - 43.0-46.0; polysaccharide complex (β-glucan and mannan) - 51.0-54.0; calcium stearate or aerosil - 0.5-1.5.

In the composition of the prototype composition, the metabolic agent is represented by a sterilized culture fluid (SCF) containing metabolites obtained during the cultivation and sterilization of bacteria of the species Bacillus subtilis. An integral component of the composition of the prototype, providing its therapeutic and prophylactic properties, are biologically active substances (BAS), synthesized by Bacillus subtilis VKPM No. B-2335 in deep growth, including various natural antibacterial substances (bacteriocins, lysozyme, rolled up basics), which 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 significant quantities in the SCR. This circumstance for the composition of the prototype is important, 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 prototype composition and acts as an adsorbent and carrier of Bacillus subtilis bacillus metabolites, is to immobilize the metabolites synthesized by the bacilli during cultivation and concentrated in the SCR, 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 prototype composition.

The composition of the prototype composition introduced a highly active immunomodulating agent, which is a complex of polysaccharides - 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 action of these easily digestible BAS in the body consists, first of all, in the fact that β-glucan activate the main cells of the immune system - macrophages.

To improve the manufacturability of the process of obtaining the prototype composition in solid dosage form for oral administration, an auxiliary additive — calcium stearate or aerosil — was introduced into its composition.

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

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

- limited biological activity, 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 (Bacillus subtilis) in its composition;

- providing the ability to effectively correct violations of only one component of the indigenous intestinal microflora - lactobacilli;

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

The noted disadvantages significantly limit the possibilities of widespread use of the prototype composition in clinical practice with the aim of correcting colonization resistance of the intestine, 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 increase the correction efficiency of dysbiotic disorders of intestinal microbiocenosis due to the use of antibiotics and leading to a decrease in the colonization resistance of the biotope due to the use of a multicomponent metabiotic composition based on a balanced complex of biologically active substances that provide a beneficial effect on the main components of indigenous intestinal microflora, specific antagonistic activity against a wide range of pathogenic and opportunistic microorganisms, nonspecific immunological resistance of the body by stimulating the functional activity of immunocompetent cells that do not contain substances hazardous to health, non-toxic, non-sensitizing, ability to cumulate, toxic effects on reproductive function, mutagenic effect and long-term negative effects, stable during storage.

Achieving this goal is possible by creating a multicomponent metabiotic composition, the qualitative and quantitative composition of which will normalize the intestinal microbiocenosis as through selective stimulation of the growth and reproduction of lactobacilli and bifidobacteria, the indigenous component of the biotope normoflora and providing specific antagonistic activity against a wide range of pathogenic and conditionally pathogenic microorganisms and by stimulating effects on the system of non-specific immunolo resistance and increase nonspecific resistance of the body.

In the formation of the qualitative and quantitative composition of the claimed metabiotic composition intended to ensure the 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;

- ensure the colonization resistance of the biotope microbiocenosis, exerting 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 a bacterial nature, with which antibiotic therapy is usually associated, ensuring their selective decontamination;

- increase the antiviral defense of the body by activating the production of macrophage system cells such cytokines as interferon;

- to influence the factors of nonspecific resistance of the body and stimulate the functional activity of immunocompetent cells to eliminate dysfunctional changes in the immune system, developing under the influence of pathogens, as well as due to the appointment of antibiotics.

To this end, the composition of 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 immunomodulating activity, an adsorbent carrier and an auxiliary technological additive.

In contrast to the prototype composition, in providing a comprehensive effect on the state of the intestinal microbiocenosis of the intestine due to the beneficial effect on the indigenous component of the microflora of the biotope and to provide a specific antagonistic effect in relation to a wide range of pathogens, biologically active substances are not one type of microorganism (Bacillus subtilis), but an additional three more - Enterococcus faecium, Lactobacillus delbrueckii and Lactobacillus fermentum. The metabiotic component is a rational combination of metabolites of probiotic bacterial strains of Bacillus subtilis VKPM No. B-2335, Enterococcus faecium L-3, Lactobacillus delbrueckii TS1-06, Lactobacillus fermentum TS3-06.

In this case, the bacterial strain Bacillus subtilis VKPM No. B-2335 was introduced in the form of a sterilized dried culture fluid, since it contains a unique set of biologically active substances synthesized by the bacilli during their growth. Of 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 factors of nonspecific resistance of the body and stimulate the functional activity of immunocompetent cells.

The ability of Bacillus subtilis to increase the phagocytic activity of macrophages and thereby exhibit an immunomodulating effect is associated with the synthesis of nitrogen bases and their derivatives (adenine, guanine, thymine, uracil, cytosine), which represent a separate group of immunomodulatory substances and are contained in significant quantities in the SCR (Table 1). For the claimed metabiotic composition, this is of great importance, since these biologically active substances contribute to the restoration of the functional activity of a single macrophage system of the body, inhibited due to exposure to pathogens and antibiotics, and a significant increase in the effectiveness of non-specific protection.

It is especially significant that the metabolites of Bacillus subtilis VKPM No. B-2335 represent a lactogenic factor and exhibit a lactogenic effect, which is expressed in stimulating the growth and reproduction of lactobacilli of indigenous intestinal microflora. This leads to favorable consequences due to the normalization of the intestinal microflora and helps to ensure the colonization resistance of the biotope.

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

Probiotic bacterial strains of Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06 actively synthesize metabolites (bacteriocins) with a protein component, lactocin B, during cultivation [28, 29]. The composition of the claimed metabiotic composition of the metabolites introduced in the form of sterilized dried culture fluids and contribute significantly to the 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 macroorganism cells, and exhibits immunomodulating activity.

Oat flakes are able to optimally ensure the restoration of intestinal microflora, since the proportion of dietary fiber in them is 11%, which, in addition, contains 35% of β-glucans very valuable for the human body. In the correction of intestinal dysbiotic conditions, the role of agents that are distinguished by a high content of dietary fiber and allowing optimizing the conditions of symbiont vegetation is undeniable. Dietary fibers (a combination of various water-soluble polysaccharides) are not digested (not broken down into monosaccharides) by endogenous human gastrointestinal secretions and reach the colon in unchanged form, where they are metabolized by anaerobic symbiont microflora to short chain fatty acids. In turn, short-chain fatty acids (acetate, propionate, butyrate, valerate) are the main energy substrate for colon mucosa epithelial cells [30], which stimulates proliferation, cell differentiation, and mucin mucus formation. In addition, dietary fiber, fixing symbiont microorganisms on its vast surface, contributes to a sharp increase in their content per unit of the intestinal volume and an increase in the metabolic activity of the intestinal 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 lymph, in which it interacts with key cells of the immune system (macrophages) by attaching to specific receptors located on their cellular membranes. By acting on macrophages, β-glucan enhances their phagocytic activity, namely, motility and the ability to find and eliminate alien, including pathogenic and conditionally pathogenic microorganisms. In addition, β-glucan stimulates cells such as neutrophils NK (natural killer cells) and LAK (lymphokine-activated killer cells), due to which it further increases the antibacterial activity of the claimed metabolic composition. Also, β-glucan allows you to activate the 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 inventive metabiotic composition is intended primarily for the immobilization of metabolites produced by Bacillus subtilis during their cultivation and concentrated in the SCR. The feasibility of using zeolite as a component of the claimed metabiotic composition is determined by the unique chemical structure of its molecules, as a result of which the zeolite has a high sorbing activity with respect to the metabolites of the bacteria Bacillus subtilis. Optimum conditions arise for the zeolite to perform its main functions - the adsorbent and the carrier of metabolites Bacillus subtilis. Without being absorbed into the digestive tract and passing in transit, the zeolite carries out the transportation of metabolites immobilized on it throughout the intestine. The release of metabolites occurs gradually and allows a sufficiently long period of time (up to 1 day) to maintain the necessary level of activity of the claimed metabiotic composition. The particles of the zeolite used are no more than 500 microns in size and have an oval crystal shape, which excludes the formation of microtraumas in the intestine and is completely safe [31-33].

To improve the manufacturability of the process of obtaining the inventive metabiotic composition as an auxiliary technological additives using SC or Aerosil.

The action of the claimed metabiotic composition, made in capsule form, begins already in the stomach, where the protective capsule dissolves under the influence of gastric juice. The metabolites of probiotic bacterial strains included in the composition of the claimed metabiotic composition, the degradation of which during gastrointestinal transit is practically eliminated, enter the intestine, where and their beneficial effect is manifested.

As you can see, the ingredients are selected 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 the 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 body, and their number in one capsule infuses, wt.%:

sterilized dried culture fluid containing metabolites of the probiotic strain bacteria Bacillus subtilis VKPM No. B-2335 - 1.9; sterilized dried culture fluid containing metabolites of the probiotic strain Enterococcus faecium L-3 bacteria - 4.5; sterilized dried culture fluid containing metabolites of the probiotic strain bacteria Lactobacillus delbrueckii TS1-06 - 4.5; sterilized dried culture fluid containing metabolites of the probiotic strain bacteria Lactobacillus fermentum TS3-06 - 4.5; zeolite - 64.3; cereals - 20.0; calcium stearate or aerosil - 0.3.

The ability to achieve the objectives of the invention is confirmed by the results of the studies presented in the following examples.

Example 1. Obtaining the inventive metabiotic composition in the form of an encapsulated solid dosage form 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 of its constituent BAS from the effects of factors that can cause their degradation.

The claimed metabiotic composition is first obtained in the form of a powder, for which the following algorithm of actions is performed. Microorganisms Bacillus subtilis VKPM No. B-2335 are grown by the method of deep cultivation in a biological reactor. After the cultivation process is completed, the SCR with microorganisms is centrifuged to separate and subsequently remove living cells of the microorganisms, and then sterilized. Before sterilization, SKZh is mixed with an adsorbent - zeolite, previously crushed to particles no larger than 500 microns in size. The resulting mass is subjected to lyophilization, in which the biologically active metabolites of the producer are immobilized on zeolite particles. Next, a mixture is added to the mass, including oat flakes and sterilized dried culture liquids containing metabolites of the probiotic bacteria strains Lactobacillus delbrueckii TS1-06, Lactobacillus delbrueckii TS1-06 and Enterococcus faecium L-3, then CK or aerosil are added and placed in the tank of the granulation unit for the purpose of mixing and drying. Then the mass is sieved on a vibrating screen and transferred to the encapsulation stage. Capsule filling is carried out on a capsule filling machine.

Sterilized dried culture liquids containing metabolites of the probiotic bacterial strains Lactobacillus delbrueckii TS1-06 and Lactobacillus delbrueckii TS1-06 are administered as the dried product Avena-L (Avena LLC, St. Petersburg) [34].

A sterilized dried culture fluid containing metabolites of the probiotic bacterial strain Enterococcus faecium L-3 is administered as a dried product Avena (Avena LLC, St. Petersburg) [35].

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

As an adsorbent, natural zeolite is used — natural microporous silicate mineral of the Kholinsky deposit (Buryatia) (KRAFT LLC, St. Petersburg) [37, 38].

SK [39] or Aerosil [40] are used as an auxiliary additive.

As you can see, the process of obtaining the inventive metabiotic composition in an encapsulated solid dosage form for oral administration is simple, and the ingredients used are available under industrial conditions, and affordable.

Example 2. Safety assessment of the claimed metabiotic composition.

The safety of the claimed metabiotic composition was assessed by sanitary-chemical analysis, microbiological studies, as well as determination of toxicological characteristics, including acute toxicity with intragastric administration, chronic toxicity and the ability to cumulate, irritating and sensitizing effects, 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 to identify 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 the determination of arsenic. " Mercury was determined according to the "Guidelines for the detection and determination of total mercury 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 residues of organochlorine pesticides” (No. 1766-77) and the official methods of analysis of AC AC (“Official methods of analysis of the AO AC”, 1984, 14th ed., Chapter 29 , P. 537-538).

The studies were carried out using laboratory scales VLR-500, gas-liquid chromatograph "Carlo Erba" (model HRGC 5700), exchange cells of the Italian company "Texnoplast".

The results of the measurements are presented in table 2 and indicate that the content of toxicants (organochlorine pesticides HCTs and DLT) and hazardous substances (lead, cadmium, mercury, arsenic) in the claimed metabiotic composition does not exceed the allowable levels established by the Technical Regulations of the Customs Union TP TS 021/2011 "On the safety of food products" (Appendix 3, Section 10) [41]. The same pesticides as aldrin and heptachlor were 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 baby food, therapeutic nutrition and their components" and MUK 2.3.2.721-98 "Determining the safety and effectiveness of biologically active food additives", GOST R 52814 -2007 “Food products. Method for the identification of bacteria of the genus Salmonella ", GOST R 52816-2007" Food products. A method for identifying and determining the number of bacteria of the group of Escherichia coli (coliform bacteria) ", GOST 10444.12-88" Food. Method for determination of yeast and mold fungi ”, GOST 30726-2001“ Food products. Methods for the identification and quantification of bacteria of the species Escherichia coli. "

The sterility and purity of the metabolites of probiotic bacterial strains (Bacillus subtilis VKPM No. B-23 35, Enterococcus faecium L3, Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06) included in the inventive metabiotic composition are evaluated. When assessing the sterility of metabolites, cultivation was carried out at a temperature of 37 ° C in liquid and solid nutrient media. For this, 1 g of the starting material was diluted in 9 ml of water, phosphate buffer or physiological solution of sodium chloride.

In the study of the sterility of Bacillus subtilis metabolites, the presence of Bacillus subtilis in them was checked. Bacillus subtilis metabolites were inoculated into a liquid nutrient medium. For this, 2 g of metabolite powder was added to 20 ml of broth and cultured for 24 hours at a temperature of 37 ° C. Turbidity of the broth and the appearance of sediment (from the powder) were noted. When seeding turbid broth on tryptose agar, no bacilli were detected.

The purity of the metabolites of Bacillus subtilis was also checked (allowable total seeding - less than 10 ² CFU / g). A weighed portion of Bacillus subtilis metabolites weighing 10 g was introduced into 90 ml of a sterile physiological solution of sodium chloride, thoroughly mixed for 5 minutes. The supernatant was sterilely taken in 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, no growth of extraneous microflora was observed.

It was found that the metabolites of Bacillus subtilis adsorbed on the zeolite do not contain live bacilli. Extraneous microflora also did not grow (less than 10 ² CFU / g).

When studying the sterility of Enterococcus faecium L3 metabolites, they checked for the presence of Enterococcus faecium L3 in them. For this purpose, Enterococcus faecium L3 metabolites were inoculated into Luria broth liquid nutrient medium. For this, 2 g of metabolite powder was added to 20 ml of broth and cultured for 24 hours at a temperature of 37 ° C. When the broth was seeded on enterococcal agar, enterococci were not found.

When checking the purity of Enterococcus faecium L3 metabolites (permissible total seed count is less than 10 ² CFU / g), a weighed sample of Enterococcus faecium L3 metabolites weighing 10 g was introduced into 90 g of sterile physiological sodium chloride solution, 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 for 24 hours at 37 ° C. In both cases, no growth of extraneous microflora was observed.

It was found that the metabolites of Enterococcus faecium L3 do not contain live enterococci. Extraneous microflora growth was also absent (less than 10 ² CFU / g).

When studying the sterility of the metabolites of Lactobacillus delbrueckii TS1-06, they checked for the presence of Lactobacillus delbrueckii TS1-06 in them. For this purpose, Lactobacillus delbrueckii TS1-06 metabolites were inoculated into Luria broth liquid nutrient medium. The metabolite powder in an amount of 2 g was added to 20 ml of broth and cultured for 24 hours at a temperature of 37 ° C. When seeding the broth on a selective solid nutrient medium, live lactobacilli Lactobacillus delbrueckii TS1-06 were not detected.

When checking the purity of the metabolites of Lactobacillus delbrueckii TS1-06 (permissible total seeding is less than 10 ² CFU / g), a weighed portion of Lactobacillus delbrueckii TS1-06 metabolites weighing 10 g was added to 90 g of sterile physiological saline 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 for 24 hours at 37 ° C. The growth of extraneous microflora was not observed.

It was established that the metabolites of Lactobacillus delbrueckii TS1-06 do not contain live lactobacilli. Extraneous microflora growth was also absent (less than 10 ² CFU / g).

When studying the sterility of the metabolites of Lactobacillus fermentum TS3-06, they checked for the presence of Lactobacillus fermentum TS3-06 in them. For this, Lactobacillus fermentum TS3-06 metabolites were inoculated into Luria broth liquid nutrient medium, 2 g of metabolite powder was added to 20 ml of broth, and cultured for 24 h at 37 ° С. When sowing the broth on a selective solid nutrient medium, live Lactobacillus fermentum TS3-06 lactobacilli were not detected.

When checking the purity of the metabolites of Lactobacillus fermentum TS3-06 (permissible total seeding is less than 10 ² CFU / g), a weighed portion of Lactobacillus fermentum TS3-06 metabolites weighing 10 g was added to 90 g of sterile physiological sodium chloride solution, 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 for 24 hours at 37 ° C. The growth of extraneous microflora was not observed.

It was found that metabolites of Lactobacillus fermentum TS3-06 do not contain live lactobacilli. Extraneous microflora growth was also absent (less than 10 ² CFU / g).

Thus, it has been experimentally established that, according to microbiological safety indicators, the claimed metabiotic composition complies with the requirements established by the Technical Regulation of the Customs Union TP 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 tube, slowly immersing it to the stomach. The studies were carried out using Litchfield-Wilcoxon probit analysis in modification 3. Rota [42]. During the experiment, two groups of 10 animals were formed each: experimental (introduced the inventive metabiotic composition twice a day in a maximum tolerated dose of 15 g / kg) and control (introduced equivalent volumes of distilled water). Animals were randomly assigned to groups using randomization. The criteria for the acceptability of randomization were considered to be the absence of external signs of diseases and the homogeneity of groups by body weight (± 10%).

Experimental animals were monitored for 14 days with registration of indicators: mortality, time of death of animals, symptoms of poisoning, results of daily monitoring 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 studies (euthanasia was carried out by an overdose of ether), the results of determining the mass coefficients (MK) of internal organs.

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

The results of studying the dynamics of changes in body weight of experimental animals receiving the inventive metabiotic composition during 14 days of observation made it possible to establish that the dynamics of body weight of animals of the experimental and control groups practically did not differ (table 4).

Analysis of the MK values of organs of white rats made it possible to establish that the acute administration of the claimed metabiotic composition 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 observation of experimental animals within 14 days after acute administration allow us to classify the claimed metabiotic composition as non-toxic (relatively harmless according to S.D. Zagolnikov) and low-hazard (hazard class IV according to GOST 12.1.007).

The study of chronic toxicity and the ability to cumulate.

Studies of chronic toxicity were carried out on white non-linear male rats weighing 180-200 g with the introduction of the inventive metabiotic composition intragastrically through an atraumatic metal probe for 30 days at a daily dose of 1.5 g / kg. Animals of the control group received distilled water in the same volume.

Measurement of basic physiological parameters was carried out after 30 days of chronic daily administration of the inventive metabiotic composition.

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

The results of the study of integral indicators of general toxicity, presented in tables 6 and 7, indicate that no statistically significant differences were found in any of the analyzed parameters with the control group of animals treated with distilled water (p> 0.05 at a 95% probability level) or pathological deviations beyond the variation of the physiological norm.

The autopsy of rats euthanized the day after the last injection of the claimed metabiotic composition showed that the size, shape and color of their internal organs had no macroscopic changes compared to the control group. The mucous membranes of the stomach and small intestine were shiny, pale pink, with no signs of irritation or inflammation.

During a histological examination of preparations of the lungs, myocardium, liver, kidneys and gastric mucosa of experimental animals that received the claimed metabiotic composition, dystrophic, inflammatory, or necrobiotic organ changes were not detected.

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

Investigation of a possible local irritant effect.

The local irritating effect of the claimed metabiotic composition was evaluated on a model of the ciliated epithelium of the frog's esophagus. The cytotoxic effect was studied on swamp frogs Rana temporaria weighing 25 g. After immobilization by breaking the spinal cord and fixing the animals on a cork table, the chest was opened, the esophagus was isolated and dissected in the caudal direction. Two rods were installed over the pharyngeal part of the esophagus. During the experiment, the transit time of the cork crumb of the esophagus 10 mm long, limited by rods, before and after application of distilled water or the claimed metabiotic composition in the form of a powder in the amount of 50 mg to the mucous membrane of the esophagus was recorded. The exposure duration was 1 min.

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

Research on 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 were placed for 2/3 for 4 hours in a pulp, which was obtained by mixing the inventive metabolic composition in powder form with cold water in amounts of 90% and 10%, respectively. 1 hour and 16 hours after the end of a single application, erythema or edema of the skin of the tails was not observed (the edema was estimated by measuring the thickness of the tail in the middle part using a thickness gauge of the type TR-1-10). It was established that a 20-fold application did not cause the death of experimental animals and did not reveal an irritating effect on the skin during the subsequent 5-day observation period.

Morphologically, the skin of the tails at the place of application of the powder had no changes. The epidermis and appendages of the skin were also unchanged. The layers of the epidermis are distinct, the basement membrane is preserved. The epithelial cells of the external and internal root sheaths of the hair follicles and the connective tissue bag were well defined.

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

A study of the 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 inventive metabiotic composition does not have an irritating effect on the skin and skin-resorptive effect, that is, it is non-toxic when it comes into contact with skin.

The irritating effect of the claimed metabolic composition on the mucous membrane of the rabbit eye was also investigated. During the experiment, it was found that 1 min after a single application of the inventive metabiotic composition in the form of a powder in the conjunctival sac of the rabbit eye in an amount of 50 mg, moderate hyperemia and lacrimation appeared, accompanied by short blepharospasm, which can be regarded as a reaction to an indifferent mechanical foreign body. Vascular congestion lasted no more than 20 minutes, and after 40-45 minutes all the phenomena of irritation that occurred completely disappeared. Therefore, the claimed metabiotic composition has a weak 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 inventive metabiotic composition in the form of a powder on white female rats weighing 130-150 g was performed for 2 hours in standard B.A. chambers. Courland 200 liters at a temperature of 20 ° C and an air supply rate of 60 l / min. The maximum possible dust concentrations, which were determined by the gravimetric method during the experiment, were achieved using dust sprayers. Air samples were taken every 30 minutes. During the experiment, the average dust concentration was 53.4 ± 3.1 g / m ³ .

Both during and after exposure, experimental animals did not have any lethal outcomes, 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, no changes were detected, except for moderate plethora of the internal organs. In the airways, powder particles of the claimed metabiotic composition were determined.

Therefore, 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.

Sensitizing study.

The study of the sensitizing effect of the claimed metabiotic composition was carried out on guinea pigs. For this purpose, 0.02 ml of aqueous dilution of the inventive metabiotic composition at a dose of 10 mg / kg was injected once into the skin of the outer surface of the ear of animals with a tuberculin syringe, and after 10 days, the claimed metabiotic was applied and fixed on pre-trimmed sections of the skin of the lateral back surface of 2 × 2 cm composition in the form of a powder at the rate of 20 mg / cm ² . Blood sampling in animals was carried out 3 hours after the staging of skin samples.

The results of evaluating the effect of the claimed metabiotic composition on the sensitizing effect 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 leukocyte lysis (RSLL) compared with the control. Examination of the back skin of experimental animals showed no signs of irritation. Therefore, the claimed metabiotic composition with epicutaneous contact does not have a sensitizing effect, that is, it does not provoke the development of allergies.

Investigation of possible long-term effects

The study of a possible gonadotoxic effect was carried out in accordance with the guidelines for indicators required in the initial assessment of chemicals [43,44]. Mutagenic activity was studied by the frequency of micronuclei formation in polychromatophilic bone marrow erythrocytes (from the femur) of white mice in accordance with the guidelines [45, 46].

The results of a study of the possible gonadotoxic and mutagenic effects of the inventive metabiotic composition when administered for 10 days at a dose of 1 g / kg, carried out 15 days after the end of its administration, indicate the absence of significantly significant changes in gonadotoxicity and mutagenicity indicators (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 claimed metabiotic composition during storage.

We investigated the possibility of maintaining the activity and safety of the claimed metabiotic composition in the form of a powder and established a guaranteed shelf life in case of storage at various 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 ° С, 10 ± 3 ° С, 25 ± 5 ° С, 35 ± 6 ° С (according to 25 packs for each temperature storage mode). Every 6 months of storage, 5 packs were taken from the corresponding temperature regime and the characteristics of the powder were determined: moisture, amylolytic and proteolytic activities, antagonistic activity, and microbiological purity. To assess 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 at the selected storage temperature conditions for testing (from 5 to 40 ° C), the estimated indicators practically do not change 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 - 1.0 ± 0.12 units. PA / g The microbiological purity of the claimed metabiotic composition, assessed by the absence of pathogenic microorganisms (Pseudomonas aeruginosa, Entero-bacteriaceae, Staphylococcus aureus and Bacillus cereus) in it, was maintained for 2.5 years, i.e. the entire study period.

Thus, the guaranteed shelf life of the claimed metabiotic composition, made in solid form in the form of a powder, is at least 2.5 years at a temperature of no higher than 40 ° C, subject to the preservation of the packaging. Special temperature storage conditions or the use of special equipment is not required.

Example 4. The study of the influence of metabolites of Bacillus subtilis VKPM No. B-2335 on the growth and reproduction of the probiotic lactobacilli Lactobacillus plantarum 8R-A3.

In the course of the experiment, the effect of Bacillus subtilis metabolites VKPM No. B-2335 on the growth of Lactobacillus plantarum 8R-A3 lactobacilli, close to the normal component of the indigenous microflora of the human intestine, was studied in vitro

Studies were performed using:

- direct antagonism method by applying sterile metabolites of the bacterial strain Bacillus subtilis VKPM No. B-2335 to the wells or to the surface of a solid nutrient medium (agar MPC-4) immediately after plating of Lactobacillus plantarum 8R-A3 lactobacilli into it, followed by incubation for 24 hours at a temperature of 37 ° C;

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

The metabolites of Bacillus subtilis VKPM No. B-2335 were administered at concentrations of 1 g or 0.1 g in 10 ml of agar.

Lactobacilli were seeded in various amounts of 7 lg CFU / ml -2 lg CFU / ml.

After applying sterile metabolites of Bacillus subtilis VKPM No. B-2335 to wells or to the surface of an agar seeded with Lactobacillus plantarum 8R-A3 lactobacilli, experimentally, using the direct antagonism method, it was found that metabolites of Bacillus subtilis VKPM No. B-2335 do not show antagonistic activity against the specified strain of lactobacilli, zones of inhibition of growth of lactobacilli are absent.

Using the double-layer agar method, it was confirmed that metabolites of Bacillus subtilis VKPM No. B-2335, when administered at the indicated concentration, do not have an inhibitory effect on the growth of Lactobacillus plantarum 8R-A3 lactobacilli. Moreover, it was experimentally established that the metabolites of Bacillus subtilis VKPM No. B-2335 stimulate the growth of probiotic lactobacilli Lactobacillus plantarum 8R-A3 (table 12). In the presence of these metabolites in the accepted concentrations, a larger number of Lactobacillus plantarum 8R-A3 lactobacilli grew (four or more times).

It was also established that lactobacilli colonies, when 50 colonies were sown on a Petri dish and grew in the presence of Bacillus subtilis metabolites VKPM No. B-2335, were 2 times larger, their diameter increased from 1 mm to 2 mm compared to the control (without the addition of metabolites of this strain bacteria).

Thus, the lactogenic effect of Bacillus subtilis metabolites VKPM No. B-2335, which is expressed in stimulating the growth and reproduction of the probiotic lactobacilli Lactobacillus plantarum 8R-A3, was experimentally established. This indicates that the metabolites of Bacillus subtilis VKPM No. B-2335 in the composition of the inventive metabolic composition are lactogenic factor, which is due to their positive effect in the restoration of intestinal flora.

Example 5. The study of the influence of Enterococcus faecium L-3 metabolites on the growth and reproduction of probiotic bifidobacteria.

During the experiment, the effect of Enterococcus faecium L-3 metabolites on the growth and reproduction of probiotic bifidobacteria Bifidobacterium bifidum 1 and Bifidobacterium longum GT15, close to the natural component of the indigenous intestinal microflora, was studied in vitro.

The studies were carried out using the two-layer agar method, as described in Example 4. Enterococcus faecium L-3 metabolites were administered at concentrations of 1 g or 0.1 g in 10 ml of bifidobacteria growth medium. Indicator strains of bifidobacteria Bifidobacterium bifidum 1 and Bifidobacterium longum GT15 were seeded in various amounts of 7 lg CFU / ml -2 lg CFU / ml.

Experimentally, using the two-layer agar method, it was found that the metabolites of the probiotic strain of bacteria Enterococcus faecium L-3 in these concentrations do not inhibit the bifidobacteria Bifidobacterium bifidum 1 and Bifidobacterium longum GT15 and even stimulate their growth and reproduction (tables 13 and 14). In the presence of these metabolites, at the accepted concentrations, a greater number of bifidobacteria Bifidobacterium bifidum 1 (three or more times) and Bifidobacterium longum GT15 (four or more times) grew.

Thus, the bifidogenic effect of the inventive synbiotic composition was experimentally established, which included metabolites of Enterococcus faecium L-3, expressed in stimulating the growth and reproduction of probiotic bifidobacteria Bifidobacterium bifidum 1 and Bifidobacterium longum GT15.

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

During the experiment, comparative in vitro studies of the specific antagonistic activity of the prototype composition, including metabolites of the probiotic bacterial strain of Bacillus subtilis VKPM No. B-2335, and the inventive metabolic composition, including the complex of metabolites of the probiotic bacterial strain of Bacillus subtilis VKPM No. B-2335, Enterococcus faecium L -3, Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06, against opportunistic microorganisms.

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

Gram-positive (enterococci, staphylococci, listeria) and gram-negative bacteria (Escherichia, Klebsiella, pseudomonads) were used as indicator test cultures (table 15). The cultures of microorganisms used as indicators correspond to those that are usually detected in the diagnosis of dysbiotic disorders of the gastrointestinal microbiocenosis.

The indicator test cultures of microorganisms were seeded during the experiment on a nutrient medium in various amounts of 7 lg CFU / ml - 2 lg CFU / ml.

As a nutrient medium used solid agar medium MPC-4.

The studies included the determination of growth retardation of indicator test cultures of opportunistic microorganisms when exposed to the prototype composition or the claimed metabiotic composition and were carried out using:

- the method of direct antagonism by direct application of the prototype composition or the inventive metabiotic composition to wells or to the surface of a solid nutrient medium immediately after inoculation of indicator test cultures, followed by incubation for 24 hours at a temperature of 37 ° C;

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

Using the direct antagonism method, it was found that the prototype composition and the claimed metabiotic composition exhibit a specific antagonistic effect with respect to 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 that of the prototype composition

Using the method of two-layer agar, the antagonistic activity of both the prototype composition and the claimed metabiotic composition was evaluated. For this, 1 g of the prototype composition or the claimed metabiotic composition was added to the lower agar layer. In this case, test cultures of opportunistic microorganisms were introduced into the upper agar layer.

Also, the prototype composition and the claimed metabiotic composition show significant antagonistic effects against staphylococci and Escherichia (tables 18 and 19), the growth of these indicator test cultures is absent when 1 g of each of the studied compositions is added to the lower agar layer. Moreover, 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 was experimentally established that the claimed metabiotic composition has a sufficient level of specific antagonistic activity against a wide range of opportunistic microorganisms, which is associated with a high level of activity of Enterococcus faecium L-3 bacteria metabolites (bacteriocins A and B), as well as Lactobacillus delbrueckii TS1 06 and Lactobacillus fermentum TS3-06 (lactocin B). The most reliably observed effects were demonstrated on the example of test cultures of pseudomonads (frequent pathogens of gaioinflammatory diseases) and listeria (pathogens of intestinal and urogenital infections).

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

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

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 animals each: control (received isotonic sodium chloride solution) and two experimental animals in which the prototype composition or the claimed metabiotic composition was administered to animals.

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

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

The prototype composition and the claimed metabiotic composition were administered for 7 days. Evaluation of the effect of their effects was carried out after 1, 5 and 7 days. For this, before the administration of the preparations (control), as well as 1, 3, and 7 days after the administration, animals of each group were sampled for the study. At each stage of the study, material was collected from 10 animals from the group, individually from each mouse after its decapitation.

The dynamics of changes in non-specific resistance factors was evaluated by determining the functional state of the cells of the phagocytic system (the digesting activity of granular leukocytes), as well as the concentration in the blood serum of lysozyme and myeloperoxidase, which have not only a direct bactericidal effect, but also mediate intercellular interaction in the development of an immune response to any antigenic impact.

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

The phagocytic index (Phi), which represents the ratio of the severity of absorbing 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. lysodeicticus) grown on meat and peptone agar in a thermostat at a temperature of 37 ° C within 24 hours. The index of digesting activity of granular leukocytes (PI), representing the ratio of the number of phagocytic cells in a smear after 30 min of incubation of blood with a phagocytosis test object to the number of in phagocytic cells in the smear after 15 min incubation with the blood of the test subject phagocytosis. The IP values were determined by the formula:

where A is the number of phagocytic cells per 100 cells, calculated in a smear of granular leukocytes, which absorbed the test microbe within 15 minutes of incubation;

B - the number of phagocytic cells per 100 cells, calculated in a smear of granular leukocytes, which absorbed the test microbe within 30 minutes of incubation.

The value of PI was expressed in arbitrary units (Fig. 1). Experimental data indicate that the use of the inventive metabiotic composition as well as the prototype composition significantly increases the functional activity of granular peripheral blood leukocytes, since the IP values significantly exceed the level of the control group (differences with the control group are significant at p <0.05). Moreover, the effect of the claimed metabiotic composition is higher than that of the prototype composition (differences with the prototype composition group are significant at p <0.05).

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

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

The level of myeloperoxidase (MPO) in blood serum was determined by the method developed on the basis of generally accepted approaches to detect this substrate in blood serum and immunocompetent cells (Gell P.G.H. et al. (1968); Page R.C. et al. (1978)). To determine the concentration of MPO in serum in 96-well plates for immunological reactions with a flat bottom, starting from the second well of row A, 0.1 ml of sera from experimental animals were poured. Then, 0.1 ml of the reagent, which is a mixture of benzidine and 3% hydrogen peroxide, was added to them. Instead of serum, 0.1 ml of physiological saline was poured into the first well of row A of the panel (control). After adding the reagent, the panels were shaken, placed in a thermostat and kept at a temperature of 37 ° C for 60 minutes. After the incubation time, the panels were removed from the thermostat and viewed on a vertical Dynatech spectrophotometer (Germany) at a wavelength of 495 nm. The concentration of the enzyme in blood serum was obtained in units of extinction and expressed in arbitrary units.

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

The introduction of both the prototype composition and the inventive metabiotic composition for 1, 3 and 7 days leads to a significant increase in the activity of non-specific resistance factors compared to the control. This is manifested both in an increase in the index of digesting activity of granular leukocytes, and in an increase in the concentration of lysozyme and myeloperoxidase. A sufficiently significant increase in the functional activity of these factors is observed after a single injection of the studied compositions within 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 from 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 microflora of the gastrointestinal tract, 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 containing both the prototype composition of β-glucan (as part of oatmeal), almost the same effectiveness in the established effects was observed in comparison with the prototype composition. So, the claimed metabiotic composition had a similar effect on the cells of the phagocytic system of blood and their oxygen-dependent bactericidal mechanism (increased concentration of myeloperoxidase in blood serum). At the same time, it is known that an increase in the concentration of serum myeloperoxidase indicates not only the activation of non-specific immunological resistance, but also an increase in the likelihood of its activating effect on immunocompetent cells, since their membrane has a corresponding specific receptor.

It was experimentally established that the stimulating effect of the claimed metabiotic composition on the mechanisms of non-specific resistance is practically the same as that of the prototype composition. The concentration levels of lysozyme and myeloperoxidase in the blood serum of experimental animals receiving the inventive metabolic 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 receiving the prototype composition, the levels of lysozyme and myeloperoxidase concentrations increased after 1 day, respectively, by 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 interferonogenic properties of the composition of the prototype and the claimed metabiotic composition.

Since non-specific immunological resistance is designed to provide protection not only for bacterial, but also for viral infections, we studied the extent to which the prototype composition and the inventive metabiotic composition are able to influence interferonogenesis.

The studies were performed on white outbred laboratory mice (Rappolovo nursery, Russian Academy of Medical Sciences) weighing 18-23 g. Experimental groups of animals were formed as described in example 7.

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

To determine the interferonogenic properties of the prototype composition and the claimed metabiotic composition in dynamics, blood was collected from experimental animals from the retroorbital sinus, combining material from three animals in one sample. The serum obtained after centrifugation at 1000 rpm for 10 minutes was aspirated and stored at a temperature of minus 20 ° С. The internal organs of mice were triturated with physiological saline, the homogenate was centrifuged (4000 rpm) for 20 minutes, and a 10% suspension in physiological saline was prepared from the supernatant.

The samples thus obtained were stored at a temperature of minus 20 ° C. Interferon titration (IFN) in the obtained samples was carried out by micromethod in cell culture. The 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 an incubator for 24 hours. Throughout the entire titration process, the cell cultures were incubated under 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 support medium was introduced, in which successive twofold dilutions of the tested samples were made (in paired wells). At the end of the daily incubation, the test samples were removed and the encephalomyocarditis test virus (EMC) was added at a working dilution of 100 CPD ₅₀ (cytopathogenic dose at which 50% of the cells die). After a day, the results were recorded using an inverted microscope. IFN titer is the reciprocal of the last dilution of serum or organ homogenate sample, at which 50% protection against the test virus is observed.

It was experimentally established that a single injection of both the prototype composition and the inventive metabiotic composition at a dose of 150 μg / mouse each has almost the same quite pronounced interferonogenic effect, since already 1 day after the administration of the studied compositions, the level of serum interferon increased, respectively, to 158 , 4 PIECES / ml and 163.7 PIECES / ml, which is 49.5 times (prototype composition) or 51.2 times (the claimed metabiotic composition) exceeds the indicator of the control group of animals (differences in access cores (p <0.05)). Subsequently, a decrease in the level of serum interferon was observed, however, and at subsequent periods of the study, the estimated indicator for the studied compositions significantly exceeded the data of the control group of animals (p <0.05).

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

Thus, the studies showed 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 total effect of the metabolites of lactobacilli Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06, as well as metabolites of the probiotic bacterial strain Enterococcus faecium L-3. Most significantly, these effects are manifested in relation to pseudomonads (frequent pathogens of purulent-inflammatory diseases) and listeria (pathogens of intestinal and urogenital infections).

- shows a lactogenic effect. It has been experimentally shown that the metabolites of Bacillus subtilis VKPM No. B-2335 as part of the inventive metabolic composition represent a lactogenic factor and stimulate the growth and reproduction of Lactobacillus plantarum 8R-A3 lactobacilli, which are close to the indigenous component of the human intestinal microflora;

- exhibits a bifidogenic effect. It was experimentally shown that metabolites of Enterococcus faecium L-3 cause a bifidogenic effect and stimulate the growth and reproduction of probiotic bifidobacteria Bifidobacterium bifidum 1 and Bifidobacterium longum GT15, 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 helps to increase the nonspecific resistance of the body;

- contributes to a sufficiently rapid production of endogenous interferon, which allows already at an early stage to provide colonization resistance and reliable protection of the body from pathogens of a viral nature.

In addition, the claimed metabiotic composition does not contain toxic and hazardous components in quantities exceeding acceptable levels, is a low-hazard substance, does not have a sensitizing effect and the ability to cumulate, undesirable side effects, including toxic effects on reproductive function, mutagenic effect and long-term negative effects, stable during storage for 2.5 years.

The claimed invention meets the criterion of "novelty" , since for the first time to ensure the colonization resistance of human intestinal microbiocenosis, it is proposed to use a metabiotic composition made in a solid encapsulated dosage form for oral administration and representing a balanced complex of biologically active substances that together provide a sufficient level of antagonistic effect against a wide range of pathogens, lactogenic and bifidogenic effects against ind genetic component of the intestinal microflora that enhance non-specific resistance of the organism.

The claimed invention meets the criterion of "inventive step" , since from the available information it did not seem obvious the feasibility of including probiotic bacterial strains of bacteria Bacillus subtilis VKPM No. B-2335 and Enterococcus faecium L-3 in the composition of the claimed metabolic composition and the possibility of providing them, respectively, with 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 exposure to si metabolites of lactobacilli Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06 and production of endogenous interferon.

The compliance of the claimed invention with the criterion of "suitability for use" is confirmed by the above examples, which showed a simple technology for producing the inventive metabiotic composition in a solid encapsulated dosage form for oral administration, the ingredients of which are produced under industrial conditions, are affordable and inexpensive, and the claimed metabiotic composition has high storage stability, as well as the results of numerous studies confirming the safety of the claimed metabiotic th composition and its effectiveness in providing colonization resistance microbiocenosis human intestine.

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Claims (1)

Metabiotic composition for providing colonization resistance of human intestinal microbiocenosis, including metabiotic component, prebiotic component, adsorbent carrier and auxiliary technological additive, in which sterilized dried culture liquid containing metabolites of the probiotic bacterial strain Bacillus subtilis VKPM No. B-23 is used as the metabiotic component as a carrier adsorbent use the natural mineral zeolite, as an auxiliary technology logical additives use calcium stearate or aerosil, characterized in that the metabiotic component is a rational combination of metabolites of probiotic strains of microorganisms and further includes sterilized dried culture fluids containing metabolites of Enterococcus faecium L-3, Lactobacillus delbrueckii TS1-06, and Lactobacillus fermentum TS1-06, and Lactobacillus fermentum TS oat flakes are used as a prebiotic component, in this case, in solid dosage form in the form of capsules, and the number of ingredients in one capsule is em, wt.%:
sterilized dried culture fluid containing metabolites of the probiotic strain bacteria Bacillus subtilis VKPM No. B-2335 1.9 sterilized dried culture fluid containing metabolites of the probiotic strain Enterococcus faecium L-3 bacteria 4,5 sterilized dried culture fluid containing metabolites of the probiotic strain bacteria Lactobacillus delbrueckii TS1-06 4,5 sterilized dried culture fluid containing metabolites of the probiotic strain bacteria Lactobacillus fermentum TS3-06 4,5 zeolite 64.3 cereals 20,0 calcium stearate or aerosil 0.3

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