RU2835358C2 - Metabiotic composition based on lactobacillus reuteri and bacillus subtilis - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: group of inventions relates to biotechnology and medicine, namely to compositions based on metabolites of probiotic organisms, aimed at treating and preventing gastrointestinal diseases caused by Helicobacter pylori. Disclosed is a metabiotic composition containing inactivated Lactobacillus reuteri cells, Bacillus subtilis metabolites and a filler, wherein the components ratio, wt.%: inactivated cells of Lactobacillus reuteri: 16–70; Bacillus subtilis metabolite complex: 1–7; filler: 23–83. Bacillus subtilis metabolite complex represents substances contained in a mixture of culture fluid, vegetative cells and spores of the probiotic strain Bacillus subtilis. Also disclosed are the use of said composition for treating and preventing gastrointestinal diseases associated with Helicobacter pylori, and a method for preparing said composition. Disclosed composition has antagonistic action on pathogenic microorganisms Helicobacter pylori, as well as immunomodulatory action.

EFFECT: administration of the proposed composition has a positive effect on the state of the gastrointestinal tract and, in combination with antibiotic therapy, enables to increase the effectiveness of Helicobacter pylori eradication.

20 cl, 3 dwg, 16 tbl, 9 ex

Description

Field of technology

[0001] The present invention relates to the field of medicine and concerns a metabiotic composition having antagonistic activity with respect to Helicobacter pylori. In particular, this composition is aimed at treating and preventing gastrointestinal disorders caused by Helicobacter pylori, as well as improving digestion and maintaining healthy microflora in the patient's digestive system.

State of the art

[0002] Gram-negative spiral-shaped Helicobacter pylori (H. pylori) causes a wide range of gastrointestinal tract (GIT) diseases, such as gastritis, gastric and duodenal ulcers, irritable bowel syndrome, stomach cancer and others. Approximately half of the world's population is infected with this bacterium [1]. It is known that when H. pylori bacteria enter the gastrointestinal tract, colonization of the gastric mucosa occurs and subsequent production of ammonia with an increase in pH values. This leads to increased secretion of hydrochloric acid and pepsin, which begin to corrode the damaged gastric mucosa with the development of acute gastritis [2]. Preventive treatment of patients can reduce the risk of most gastrointestinal pathologies, including preventing the occurrence of stomach cancer [3]. In some cases, the direct link between the presence of H. pylori and the disease is not fully understood, as is the case with irritable bowel syndrome.

[0003] In the treatment of the body from H. pylori invasion, standard triple eradication therapy is used, which uses a combination of three targeted drugs - a proton pump inhibitor to suppress the production of hydrochloric acid, and antibiotics, such as clarithromycin and amoxicillin, aimed at eradicating (destroying) the bacteria in the body [4]. Since broad-spectrum antibiotics are used for treatment, treatment can be accompanied by intestinal dysbacteriosis. In addition, eradication therapy contributes to an increase in antibiotic resistance of pathogens, which leads to the emergence of bacterial diseases that are difficult to treat with standard treatment [5]. For H. pylori, cases of resistance to clarithromycin, metronidazole and levofloxacin are known, due to which the effectiveness of eradication therapy is reduced worldwide [6].

[0004] The gastrointestinal tract is home to a variety of microorganisms, such as bacteria, fungi, and protozoa, which form the so-called intestinal microflora (microbiota) [7]. Intestinal microflora has a variety of functions and plays an important role in maintaining homeostasis. When treated with antibiotics, the body experiences dysbacteriosis - a violation of the qualitative and quantitative composition of the gastrointestinal microflora, which leads to disruption of the normal functioning of the digestive tract. In addition, intestinal microflora plays an important role in the functioning of the immune system by forming colonization resistance, i.e. protecting the body from pathogenic microflora. Thus, as a result of disruption of the natural individual quantitative and qualitative composition of intestinal microflora, the immunity of the entire body may be weakened [8].

[0005] Probiotics and prebiotics are used to treat and prevent dysbacteriosis. Most often, live bacteria belonging to the genera Bifidobacterium, Lactobacillus and Bacillus are used as probiotics for the treatment and prevention of human intestinal dysbacteriosis [9]. Various oligo- and polysaccharides are often used as prebiotics - for example, inulin, fiber, lactulose and others. Due to their properties, prebiotics enhance the effect of probiotics, so they are often prescribed in combination [10].

[0006] Probiotics, being components of normal intestinal microflora, can exhibit antimicrobial properties against pathogenic microorganisms. Antimicrobial properties can be due to the release of substances that directly suppress the growth of pathogenic microorganisms. In addition, probiotics can affect the acidity of the environment, thereby creating unfavorable conditions for the reproduction of pathogenic microorganisms [11, 12].

[0007] Probiotic compositions are actively used to treat a number of gastrointestinal diseases caused by or associated with H. pylori. Thus, a composition is known that includes bacteria of the Lactobacillus family that inhibit the growth of pathogenic bacteria, including H. pylori (US2 0200069749 A1; published 05.03.2020; IPC A61K 35/747, A61K 35/66, A61K 35/74, A61K 35/744, C12N 1/20, A61K 2035/115, Y02A 50/30).

[0008] The said composition is aimed at reducing gastric acidity caused by taking a proton pump inhibitor during the eradication of H. pylori. The composition includes several probiotic strains of bacteria from the Lactobacillus family. In particular, in one embodiment, the said composition comprises at least one strain of Lactobacillus reuteri (L. reuteri) (EP 2753343 B1, published 16.07.2014; IPC A23L 33/135, A61K 35/747, A23L 33/40, A61K 31/198, A61K 35/74, A61K 35/741, A61K 35/744, A61K 35/745, A61K 38/47, A61K 45/06, A61P 1/00, Y02A 50/30).

[0009] Probiotic strains of L. reuteri are known for their antagonistic properties against H. pylori. It has been shown that L. reuteri specifically recognizes and binds to proteins on the surface of H. pylori, which promotes the elimination of H. pylori from the gastrointestinal tract due to peristalsis. This feature helps to reduce the load of pathogenic flora on the stomach [13]. When combined with triple therapy, the use of L. reuteri improves the efficiency of H. pylori eradication by up to 20%, and the overall picture of symptoms in patients is alleviated [14]. It is also noted that standard antibiotic treatment in combination with these probiotic bacteria shows fewer side effects.

[0010] However, both of the above compositions include live bacteria of the Lactobacillus family. These compositions may significantly lose their effectiveness when combined with antibiotics due to the death of probiotics. In addition, the use of only one probiotic culture in the above compositions increases the chance of developing resistance in the body and with prolonged use of probiotic preparations, competition between the introduced microorganisms and the patient's own microflora, which is unique to each patient, may develop. Synbiotic treatment using prebiotics and metabiotics (bacterial waste products and non-living cells) acts as an alternative to known probiotic compositions, which is especially important in the treatment of H.pylori.

[0011] A method using the probiotic strain Lactobacillus paracasei (L. paracasei) is described for the treatment of irritable bowel syndrome (RU 2490325 C2; published 20.08.2013; IPC A61K 35/74, A61K 35/747, A23L 33/135, A61K 35/745, A61P 1/00, A61P 1/06, A61P 1/12, A61P 29/00, A61P 31/00). The said method describes the possibility of using not live bacteria, but also "material obtained from the probiotic." However, the said "material" is described only in general terms without specifying the composition or method of obtaining. In addition, although H. pylori has been mentioned as a possible pathogen causing irritable bowel syndrome, there is no data on the effectiveness of the said composition against H. pylori.

[0012] A composition based on inactivated (i.e. incapable of reproduction) probiotics is known, which has an antagonistic effect against H. pylori (CN 113577218 A; published 02.11.2021; IPC: A61K 35/747, A61K 36/9064, A61K 8/9789, A61K 8/9794, A61K 8/99, A61P 1/02, A61P 11/04, A61P 31/04, A61Q 11/00). However, this composition, in addition to several probiotic strains, includes nine more components related to traditional Chinese medicine. Such a complex and specific composition complicates the production of the composition.

[0013] A composition based on inactivated L. reuteri cells is known, which has an antagonistic effect with respect to H. pylori. This composition is a dried strain of L. reuteri with known coaggregation activity with respect to H. pylori, in one embodiment this is the strain DSM 17648 (EP 2717890 B1; published 08.06.2012; A61K 35/747, A61P 1/00, A61P 1/04, A61P 1/06, A61P 1/08, A61P 1/12, A61P 1/14, A61P 31/04, A61P 35/00). However, this composition includes only one probiotic strain, which makes the composition more narrowly specific with respect to the pathogen. Thus, there is currently a need to create a metabiotic composition with demonstrated efficacy in inhibiting H. pylori, promoting the prevention of intestinal dysbacteriosis, and available for production.

Terms and abbreviations

[0014] Dysbacteriosis is a qualitative and/or quantitative change in the bacterial microflora in the body, the movement of its various representatives to habitats that are not typical for them, accompanied by metabolic and immune disorders. Moreover, these disorders do not disappear after the elimination of the unfavorable factor that caused dysbacteriosis. Most often, dysbacteriosis is understood as a violation of the intestinal microflora [15,16].

[0015] Culture fluid is a liquid medium obtained during the cultivation of various pro- and eukaryotic cells in vitro and containing residual nutrients and metabolic products of these cells [17].

[0016]Culture suspension- a liquid medium obtained by culturing various pro- and eukaryotic cells in vitro and containing cultured cells and/or spores.

[0017] Metabiotics are substances that are structural components of probiotic microorganisms and/or their metabolites, which are capable of optimizing physiological functions, metabolic, epigenetic, informational, regulatory, transport, immune, neurohormonal, and/or behavioral reactions associated with the activity of the symbiotic (indigenous) microflora of the host organism [18].

[0018] Microflora (microbiota) is a collection of different types of microorganisms that inhabit a particular environment. Microorganisms that constantly live in symbiosis with the host are called normal, or symbiotic, microflora [19].

[0019] Prebiotics are chemical compounds that can stimulate the growth and development of groups of bacteria that are part of the body's natural microflora [10].

[0020] Probiotics are viable microorganisms that help improve digestive functions by restoring and maintaining the normal quantitative and qualitative composition of microflora [11].

[0021] FA - fatty acids.

[0022] GIT - gastrointestinal tract.

[0023] kDa - kilodalton.

[0024] CFU - colony forming unit.

[0025] B. subtilis - Bacillus subtilis.

[0026] E. coli - Escherichia coli.

[0027] L. reuteri - Lactobacillus reuteri.

[0028] CD64 is a membrane protein Fc-gamma receptor 1, a monocyte marker, and also a proinflammatory marker of macrophages.

[0029] CDX-2 - caudal homeobox protein type 2, a marker of intestinal differentiation.

[0030]COX-2- cyclooxygenase-2 protein, an inflammation marker protein.

[0031] IL1b - interleukin 1 beta, a proinflammatory cytokine.

[0032] IL6 - interleukin 6, pro- and anti-inflammatory cytokine, regeneration factor, macrophage activating factor, lymphocyte stimulating factor.

[0033] IL10 - interleukin 10, an anti-inflammatory cytokine.

[0034] IL-23 - interleukin 23, a proinflammatory cytokine.

[0035] MRSA - methicillin-resistant strains of S. Aureus.

[0036] RI - retention index.

[0037] RT - retention time.

[0038] TNFa - tumor necrosis factor alpha, macrophage activating factor.

[0039] TGFb - transforming growth factor beta, blocks the activation of macrophages, suppresses the inflammatory immune response.

The essence of the invention

[0040] The objective of the present invention is to create a metabiotic composition for the treatment and prevention of gastrointestinal tract diseases associated with the presence, as well as the eradication of H. pylori.

[0041] This problem is solved by the claimed invention by achieving such a technical result as the creation of a metabiotic composition that has antagonistic properties in relation to pathogenic and opportunistic microorganisms, including H. pylori, a stimulating effect in relation to normal intestinal microflora, and also has immunomodulatory potential.

[0042] The claimed technical result is achieved due to the fact that the claimed composition includes inactivated L. reuteri cells, a complex of B. subtilis metabolites and a filler. In this case, the ratio of components, wt. %:

inactivated L. reuteri cells 16-70 B. subtilis metabolite complex 1-7 filler 23-83

[0043] Within the framework of the claimed invention, the inactivated cells of L. reuteri are dried cells that are not capable of reproduction. The inactivated cells of the probiotic strain of L. reuteri do not compete with the normal microflora of the patient, while performing an antagonistic role with respect to a number of pathogenic bacteria. In a preferred embodiment, the inactivated cells of the probiotic strain of L. reuteri are non-living, for example, killed by heat treatment. As a probiotic strain of L. reuteri, strains of L. reuteri that have a co-aggregation effect with respect to H. pylori can be used, for example, DSM-17648, SALR01 or ATCC-55730. In a preferred embodiment, the probiotic strain of L. reuteri is L. reuteri GMNL-0902. In a preferred embodiment, the inactivated cells of the probiotic strain of L. reuteri are dried.

[0044] The complex of B. subtilis metabolites is substances contained in a mixture of culture fluid, vegetative cells and spores of the probiotic strain of B. subtilis. The following strains can be used as the probiotic strain of B. subtilis: B. subtilis VKPM No. B-2335 (3), B. subtilis 534, B. subtilis TPI 13 - 07.06.21 - DEP/VGNKI, B. subtilis VKPM No. B4759 (3), B. subtilis pBMV105, B. subtilis TPI 9; B. subtilis pBMV 105. In a preferred embodiment, the probiotic strain of B. subtilis is B. subtilis VKM B-3536D and/or VKPM No. B-2335 (3).

[0045] B. subtilis metabolites are capable of selectively suppressing the growth of a wide range of pathogenic and opportunistic microorganisms. In addition, the complex of B. subtilis metabolites also has anti-inflammatory properties, which may further alleviate the condition of patients with gastrointestinal tract diseases. The combination of vegetative cells and metabolites of the probiotic strain B. subtilis allows for a comprehensive effect on pathogenic microorganisms without loss of effectiveness during antibiotic therapy, which allows it to be used for the treatment and prevention of dysbacteriosis for a wide range of patients.

[0046] The filler is one or more substances that can help maintain the integrity of the composition, maintain the biological activity of the said metabolites and/or can have a beneficial physiological effect, enhancing the beneficial effect of the composition.

[0047] In a preferred embodiment, the claimed composition includes one or more prebiotics as a filler, which may be di-, oligo- and polysaccharides. In an even more preferred embodiment, one or more prebiotics may be of plant origin. For example, prebiotics may be represented by polyfructosans such as inulin, oligofructose or a mixture thereof. Prebiotics stimulate the growth of symbiotic intestinal microflora, and have an immunomodulatory and hepatoprotective effect.

[0048] In a preferred embodiment, the claimed composition includes a zinc source in the filler. The zinc source may be a zinc salt, preferably an organic zinc salt, such as zinc citrate.

[0049] The technical result is also achieved by developing an accessible method for obtaining the claimed composition. The method includes the following steps:

a) cultivation of the probiotic strain B. subtilis in a liquid nutrient medium.

In a preferred embodiment, the medium has an initial pH of 7.0±0.2. Cultivation can be carried out by deep cultivation in a fermenter with a filling factor of 60-70% at a temperature of 36-38°C with constant stirring and aeration. In this case, cultivation can be completed when the pH of the culture suspension reaches 8.5-8.9 and/or until the culture reaches a maturity of at least 90%. This stage of cultivation corresponds to the maximum concentration of B. subtilis metabolites in the culture suspension. This cultivation method allows for the most efficient use of equipment for obtaining B. subtilis metabolites;

b) by centrifuging the culture suspension in a flow centrifuge, vegetative cells and spores of the probiotic strain B. subtilis are separated from the culture fluid;

c) the resulting culture liquid is dried. In a preferred embodiment, the culture liquid is dried by lyophilization;

d) inactivate vegetative cells and spores of B. subtilis separated from the culture fluid;

d) mix lyophilized culture fluid, inactivated vegetative cells and B. subtilis spores in the following ratio;

e) inactivated L. reuteri cells and filler are added to the obtained complex of B. subtilis metabolites.

[0050] The claimed method provides for obtaining a composition that includes both L. reuteri metabolites and virtually the entire spectrum of B. subtilis metabolites. Due to this, the claimed composition promotes the eradication of H. pylori, enhances immunity, and makes it possible to avoid or reduce the effect of dysbacteriosis, which can be caused by antibiotics used in the treatment of gastrointestinal diseases associated with H. pylori.

[0051] The method may further comprise drying the inactivated vegetative cells and spores of B. subtilis. In a preferred embodiment, the inactivated cells and spores of B. subtilis are dried by lyophilization.

[0052] In a preferred embodiment, all components are used in dry form to obtain the composition. This increases the shelf life of the composition without losing its effectiveness. This is due to the preservation of the activity of the metabolites and the minimization of the risk of contamination of the composition with any active microorganisms.

Description of drawings

[0053] Fig. 1 shows the results of a study of the immunomodulatory properties of a complex of B. subtilis metabolites.

[0054] Fig. 2A shows the results of a study of the expression of inflammatory markers in a culture of human gastric cells.

[0055] Fig. 2B shows the results of a study of the expression of inflammatory markers in a culture of human intestinal cells.

[0056] Fig. 3 shows the results of a study to evaluate the effectiveness of using the claimed composition in the treatment of Helicobacter pylori infection using a Mongolian gerbil model.

DETAILED DESCRIPTION OF THE INVENTION

[0057] In the following detailed description of the embodiment of the invention, numerous implementation details are set forth in order to provide a clear understanding of the present invention. However, it will be obvious to one skilled in the art how the present invention may be used with or without these implementation details. In other instances, well-known methods, procedures, and components have not been described in detail in order not to unnecessarily obscure the features of the present invention.

[0058] Furthermore, it is clear from the above disclosure that the invention is not limited to the embodiment provided. Numerous possible modifications, changes, variations and substitutions that retain the essence and form of the present invention are obvious to those skilled in the art.

[0059] The claimed composition includes metabolites of L. reuteri cells, a complex of B. subtilis metabolites and a filler. The ratio of components, wt. %:

inactivated L. reuteri cells 16-70 B. subtilis metabolite complex 1-7 filler 23-83

[0060] In the claimed composition, the inactivated L. reuteri cells are dried cells that are not capable of multiplication. Inactivated cells are cells that are not capable of multiplication. In a preferred embodiment, the cells of the probiotic strain of L. reuteri are non-living, for example, killed by heat treatment. The probiotic strain of L. reuteri can be L. reuteri strains that have a co-aggregation effect with respect to H. pylori - SALR01, DSM-17648 or ATCC - 55730. In a preferred embodiment, the probiotic strain of L. reuteri is L. reuteri GMNL-0902.

[0061] The use of inactivated L. reuteri cells at a concentration of less than 16 wt. % leads to a significant decrease in the effectiveness of the composition. Increasing the content of metabolites to more than 70 wt. % is irrational, since it increases the cost of production and does not have a significant effect on the effectiveness of the composition.

[0062] The complex of B. subtilis metabolites is the substances contained in the culture suspension of the probiotic strain B. subtilis. In a preferred embodiment, the complex of metabolites is the substances contained in the mixture of the culture liquid, vegetative cells and spores of the probiotic strain B. subtilis. The said mixture includes substantially the entire spectrum of metabolites of the probiotic strain B. subtilis. Experimental data show that B. subtilis metabolites are capable of selectively suppressing the growth of a wide range of pathogenic and opportunistic microorganisms, such as: Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus epidermidis, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Streptococcus salivarius, Streptococcus mitis, Escherichia coli (Ex. 2 and 4).

[0063] B. subtilis metabolites include a variety of biologically active substances that have antimicrobial, probiotic, and immunomodulatory effects. For example, experimental data (Ex. 3) show that B. subtilis metabolites include amikumacins A and B, which belong to the class of naturally occurring dihydroisocoumarin products and exhibit antibacterial, antifungal, antitumor, and anti-inflammatory activity. Their mechanism of action is to stop ribosome translocation, thereby blocking protein synthesis in cells. Amikumacin A has antagonistic activity against H. pylori [20]. In addition, amikumacins are active against antibiotic-resistant strains of S. aureus (MRSA - methicillin-resistant strains of S. aureus), and are therefore of particular interest for combating the growing resistance of pathogen strains to antibiotics. Amikumacin A is active against gram-positive bacteria Salmonella sp. and Shigella sp. [20, 21, 22, 23, 24, 25].

[0064] Also, the metabolites of B. subtilis include cyclic dipeptides (2,5-diketopiperazines) exhibit antitumor, neuroprotective and antihyperglycemic activity. In addition, cyclic dipeptides exhibit antiviral, antifungal and antibacterial activity. In particular, cyclo(leucylprolyl) (L-Phe-D-Pro lactam) and cyclo(D-phenylalanine-L-prolyl) exhibit antagonistic activity against S. aureus, E. coli, P. aeruginosa and C. albicans. At the same time, P. aeruginosa is one of the most common pathogens of nosocomial infections, acquiring resistance to commonly used antibiotics, and C. albicans is a frequent cause of fungal infections [26].

[0065] Lactic acid is also a metabolite of B. subtilis and has an anti-inflammatory effect by stimulating the anti-inflammatory cytokine IL-10 and suppressing the activity of the pro-inflammatory cytokine IL-12. Also, lactic acid and its derivatives (e.g., 3-phenyllactic acid, 4-hydroxyphenyllactic acid) have an antibacterial and antifungal effect. For example, 3-phenyllactic acid (alone or in combination with lactic acid) exhibits - suppresses the growth of pathogens such as L. monocyotgenes, E. coli, S. typhimurium, B. cereus, E. faecalis [27, 28, 29, 30,31, 32, 33].

[0066] a-Hydroxyisocaproic acid has a broad antibacterial activity, including bacteria such as MRSA (methicillin-resistant S. aureus), which are resistant to many systemically used antimicrobial drugs [34].

[0067] Indole is a heterocyclic condensed aromatic compound formed by the degradation of the amino acid tryptophan. Indole has probiotic properties and maintains the normal quantitative and qualitative composition of the intestinal microbiota. Indole also supports the function of the epithelial barrier and promotes immune tolerance to maintain microbial commensalism during protection against pathogenic infections. In addition, indole has anticarcinogenic, antioxidant and anti-inflammatory effects. The latter is probably due to stimulation of the production of the cytokine IL-22 by intestinal immune cells [35,36].

[0068] Long-chain monounsaturated fatty acids (so-called omega-9 fatty acids), such as myristoleic (C14:1), palmitoleic (C16:1), oleic (C18:1), and elaidic (C18:1) acids, increase the diversity of intestinal microbiota [37]. In addition, myristoleic, palmitoleic, and oleic acids have antagonistic activity against H. pylori.

[0069] Some long-chain saturated fatty acids (LCFAs) have antimicrobial and anti-inflammatory effects. For example, palmitic acid has antagonistic activity against S.mutans, S.gordonii, S.sanquis, P.ginbivalis, F.nucleatum. Pentadecanoic and margaric acids have anti-inflammatory effects [38,39].

[0070] Medium chain saturated fatty acids, such as caprylic (C8), capric (C10), undecyl (C11) and lauric (C12) acids, improve the function of the intestinal epithelium, increasing the activity of enzymes of the microvilli of the mucous membrane in the small intestine. These acids have antibacterial, antifungal, and anti-inflammatory effects. For example, lauric acid is most effective against Campylobacter jejuni, capric and lauric acids have antibacterial effects against H. pylori [37,40, 41, 42].

[0071] Vitamin B5 (pantothenic acid) inhibits bacterial growth through regulation of innate immunity and adaptive immunity in mice infected with Mycobacterium tuberculosis [43].

[0072] Amino acids support the function of the intestinal barrier and the endocrine function of the intestine. Amino acids entering the intestine from the outside are metabolized by the intestinal microbiota, which uses them to synthesize its own proteins and catabolic pathways. It has been shown that dietary amino acids can regulate the composition of the microbiota. For example, lysine supports the commensal (probiotic) microbiota in the intestine. In addition, lysine helps maintain the immune system [44, 45].

[0073] The culture fluid of the probiotic strain B. subtilis in the composition of the claimed composition can be dried to such a state that the moisture content of the dry culture fluid is 4-6 wt. % In this case, the vegetative cells and spores of the probiotic strain B. subtilis can be inactivated, i.e. incapable of reproduction, preferably non-living. In one embodiment, the vegetative cells and spores of the probiotic strain B. subtilis can be inactivated by heating.

[0074] The following strains can be used as the probiotic strain of B. subtilis: B. subtilis VKPM No. B-2335 (3), B. subtilis 534, B. subtilis TPI 13 - 07.06.21 - DEP/VGNKI, B. subtilis VKPM No. B4759 (3), B. subtilis pBMV105, B. subtilis TPI 9; B. subtilis pBMV 105. In a preferred embodiment, the probiotic strain of B. subtilis is B. subtilis VKM B-3536D and/or VKPM No. B-2335 (3).

[0075] The use of a complex of B. subtilis metabolites in a concentration of less than 1 wt. % leads to a significant decrease in the effectiveness of the composition. Increasing the content of metabolites to more than 7 wt. % is irrational, since it increases the cost of production and does not have a significant effect on the effectiveness of the composition.

[0076] Due to the absence of live microorganisms, the claimed composition does not lose its effectiveness when taken in combination with antibiotics, which allows it to be used for the treatment and prevention of gastrointestinal diseases in a wide range of patients. In addition, inactivated vegetative cells and spores of B. subtilis, as well as inactivated cells of L. reuteri, do not create competition for the intestinal microflora and do not disrupt its balance, which is unique for each patient.

[0077] The claimed composition includes a filler, which is one or more substances that can help maintain the integrity of the composition, maintain the biological activity of the said metabolites and/or can have a beneficial physiological effect, enhancing the beneficial effect of the composition. The filler can be, for example, but not limited to: maltodextrin, dietary fiber, starch derivatives, pectins, cellulose, microcrystalline cellulose and other cellulose derivatives, as well as other technologically acceptable fillers (for example, selected from the list provided on the website https://lexparency.org/eu/32008R1333/ANX_III/). In addition, one or more prebiotics can be used as a filler, which can be di-, oligo- and polysaccharides that are resistant to hydrolysis by human digestive enzymes and are not absorbed in the upper digestive tract. For example, these may be dietary fibers of cereals, vegetables, fruits, herbs, such as inulin and/or oligofructose, or synthetic polysaccharides such as lactulose, or combinations thereof. In an even more preferred embodiment, one or more prebiotics may be of plant origin. For example, polyfructosans (fructans), such as inulin and/or oligofructose, may be used as prebiotics. In this case, polyfructosans may be obtained, for example, from chicory, Jerusalem artichoke, dahlia, artichoke, beetroot, and other plants.

[0078] The filler may also include a zinc source. Zinc plays an important role in the development and maintenance of the immune system. The use of zinc suppresses the growth of pathogenic microorganisms, such as Mycobacterium, and also prevents the development of a viral infection [46]. The zinc source in the composition may be at least 3 wt. % In a preferred embodiment, the zinc source is an organic zinc salt. This is due to the fact that organic salts have greater bioavailability than inorganic zinc salts. In one embodiment, zinc can be included in the composition in chelate form, for example, in the form of a compound with an amino acid. However, such compounds are relatively expensive on the market, which significantly increases the cost of the composition. In a preferred embodiment, the zinc source is zinc citrate. Zinc citrate is widely available on the market and provides an optimal cost of production of the composition without loss of its effectiveness.

[0079] The claimed composition can be used to manufacture a food supplement or a pharmaceutical preparation in the form of a powder, tablet or capsule.

[0080] The combination of bacterial metabiotics L. reuteri and B. subtilis helps treat gastrointestinal diseases associated with H. pylori. In addition, the combination helps prevent intestinal dysbacteriosis caused by the use of antibiotics during H. pylori eradication. Prevention of dysbacteriosis, in turn, helps prevent recurrent infection.

[0081] An important part of the claimed invention is a method for producing the claimed composition, which includes the following steps. In step a), submerged cultivation of the probiotic strain B. subtilis is carried out in a liquid nutrient medium having an initial pH of 7.0±0.2. In a preferred embodiment, submerged cultivation is carried out in a fermenter. In this case, the filling factor of the fermenter can be from about 60% to about 70%. This filling factor, on the one hand, allows to avoid undesirable foaming, and on the other hand, allows to use the equipment for obtaining a culture suspension saturated with metabolites of the probiotic strain B. subtilis as efficiently as possible. Submerged cultivation is carried out at a temperature of 36-38°C with constant stirring and aeration until the pH of the culture suspension reaches 8.5-8.9 and/or until the culture reaches a maturity of at least 90%. At this stage of cultivation, the culture transitions to a stationary phase, when the cells stop dividing, i.e. the amount of cell mass does not increase. This stage of cultivation corresponds to the maximum concentration of B. subtilis metabolites in the culture liquid.

[0082] In step b), the culture fluid is separated from the cells and spores of the probiotic strain B. subtilis. In one embodiment, the culture fluid is separated from the vegetative cells and spores by filtration. In a preferred embodiment, the culture fluid is separated from the vegetative cells and spores by centrifuging the culture suspension in a flow centrifuge until a clear supernatant is obtained, which is the culture fluid. The sediment mainly contains vegetative cells and spores of the probiotic strain B. subtilis.

[0083] In step c), the culture liquid obtained in step b) is dried. The culture liquid can be dried by any suitable method known from the prior art. In a preferred embodiment, the culture liquid is dried by lyophilization. In this case, the culture liquid is dried to such a state that the moisture content of the dry culture liquid is 4-6 wt. %

[0084] In step c), the vegetative cells and spores of the probiotic strain B. subtilis are inactivated by heating the sediment obtained in step b) to a temperature of 60-140°C. The sediment is incubated at the said temperature for 5-120 minutes. Inactivation by heating requires the separation of the vegetative cells and spores from the culture fluid. The fact is that the said inactivation temperature can critically reduce the biological activity of at least some of the B. subtilis metabolites contained in the culture fluid.

[0085] After this, the dried culture liquid of B. subtilis is mixed with inactivated vegetative cells and spores.

[0086] Next, inactivated cells of the probiotic strain L. reuteri, as well as a filler, are added to the obtained complex of B. subtilis metabolites.

[0087] The claimed method provides for obtaining a composition comprising a wide range of metabolites of the probiotic strains L. reuteri and B. subtilis. Separating the vegetative cells and spores of B. subtilis from the culture fluid before heating the vegetative cells and spores of B. subtilis allows for obtaining a composition with the widest possible range of biologically active metabolites of B. subtilis - both secreted by the vegetative cells into the culture fluid and contained in the vegetative cells and spores themselves. In this case, heating the vegetative cells and spores of B. subtilis in the above mode allows for effective inactivation of the vegetative cells and spores.

[0088] In one embodiment, the method further comprises drying the inactivated vegetative cells and spores of the probiotic strain B. subtilis. In this case, the inactivated vegetative cells and spores of B. subtilis can be dried by any suitable method known in the art. In a preferred embodiment, the inactivated vegetative cells and spores of B. subtilis are dried by lyophilization. In one embodiment, the inactivated vegetative cells and spores of B. subtilis are first mixed with a culture liquid, and then the resulting mixture is dried, for example, lyophilized using a suitable carrier, for example, maltodextrin. In a preferred embodiment, the inactivated vegetative cells and spores of B. subtilis are dried separately from the culture liquid. In any case, the inactivated cells and spores of B. subtilis are dried to a state that their moisture content is 6-8 wt. % The use of dried vegetative cells and spores of B. subtilis allows obtaining the composition in dry form.

[0089] In a preferred embodiment, the composition is obtained in dry form, for example, in the form of a powder. This increases the shelf life of the composition without losing its effectiveness. This is due to both the preservation of the activity of the metabolites and the minimization of the risk of contamination of the composition with any active microorganisms. In addition, the dry composition is less demanding in terms of storage conditions and is easier to transport, which increases its availability to a wide range of users.

DESCRIPTION OF THE IMPLEMENTATION OF THE INVENTION

Example 1. Obtaining a complex of B. subtilis metabolites

[0090] In this example, metabolites of the B. subtilis strain VKM B-3536D were obtained. The submerged cultivation process was carried out in a P1000 fermenter from Bioengineering (Switzerland) with a volume of 1000 ^dm3 and a filling factor of 0.6. During the cultivation, a temperature of 37±1°C was maintained. The stirring speed was 250 rpm. The initial aeration speed was 0.2 rpm, after 16 hours of growth the speed was changed to 0.5 rpm. The submerged cultivation process was carried out for 32 hours.

[0091] Approximately 30 L of the B. subtilis VKM B-3536D culture suspension was centrifuged in a Sharpless AS-26 flow supercentrifuge to obtain approximately 20 L of clear supernatant. The supernatant was the culture liquid, and the sediment mainly included vegetative cells and spores of B. subtilis.

[0092] The resulting culture liquid was lyophilized with the addition of 2.5 wt. % maltodextrin as a carrier. As a result, 560 g of powder was obtained, which was a lyophilized culture liquid.

[0093] The sediment, including vegetative cells and spores, was incubated at 118°C for 40 minutes. In this way, the vegetative cells and spores of B. subtilis were inactivated. Then, the inactivated vegetative cells and spores were lyophilized with the addition of 2.5 wt. % maltodextrin as a carrier.

[0094] Lyophilized culture fluid was mixed with lyophilized inactivated vegetative cells and spores of B. subtilis. As a result, the entire complex of metabolites present in the B. subtilis culture suspension was obtained.

Example 2. Study of the composition of the culture liquid of B. subtilis

[0095] In this example, the composition of the culture fluid of the probiotic strain B. subtilis VKM B-3536D was quantitatively studied. The study was carried out at the Federal State Unitary Enterprise “Research Institute of Hygiene, Occupational Pathology and Human Ecology” of the Federal Medical and Biological Agency (FSUE “Research Institute of Hygiene, Occupational Pathology and Human Ecology” of the Federal Medical and Biological Agency of Russia).

[0096] The culture fluid sample was divided into portions after mixing and stored frozen at -18°C until analysis. The portions were then divided into aliquots, in which the mass concentrations of amino acids, vitamins, free and esterified fatty acids were measured. The contents of macro/micro elements and heavy metals were determined. The results of the study are presented in Tables 2-5.

[0097]

Table 2. Amino acid content in the culture fluid of the probiotic strain B. subtilis Amino acid name Amino acid concentration, ng/ml Sample 1 Sample 2 Average Alanine 84.4 88.6 86.5 Arginine 13.1 13.2 13.2 Asparagine 1.7 1.7 1.7 Aspartic acid 8.5 7.1 7.8 Valin 67.9 68.8 68.4 Hydroxyproline 44.8 46.2 45.5 Histidine 2,3 2.6 2.5 Glycine 70.9 69.9 70.4 Glutamine 0,0 0,0 0,0 Glutamic acid 25.3 23.2 24.3 Isoleucine 46.8 47.0 46.9 Lysine 266.8 273.5 270.2 Methionine 3.9 3.9 3.9 Leucine 50.5 50.0 50.2 Ornithine 1.5 1.6 1.5 Proline 5.5 5.5 5.5 Serene 18.2 18.3 18.2 Tyrosine 7.1 7.4 7.2 Threonine 32.4 33.7 33.1 Tryptophan - 2,2 2,2 Phenylalanine 60.6 63.1 61.9 Cystine 0.7 0.5 0.6 Citrulline 0.7 0.7 0.7

[0098]

Table 3. Vitamin content in the culture fluid of the probiotic strain B. subtilis Vitamin name Vitamin concentration, mcg/ml B5 14.5 PP 0.4 B2 0.4 B1 0.05 C 0.025 B6 0.05

[0099]

Table 4. Content of free and bound (esterified) fatty acids in the culture fluid of the probiotic strain B. subtilis, determined by chromatograph mass spectrometry in samples of culture fluids Name of fatty acid Free fatty acids, ng/ml Esterified fatty acids, ng/ml Caprylic, C8:0 406 10 Capric, C10:0 980 37 Undecyl, C11:0 570 58 Lauric, C12:0 1361 1621 Tridecanoic, C13:0 485 61 Myristoleic, C14:1 695 444 Myristic, C14:0 3355 1342 cis-10-Pentadecene, C15:1 BUT ^* BUT ^* Pentadecanoic, C15:0 5907 1041 Palmitoleic, C16:1n-7 5842 2365 Palmitic, C16:0 41442 10350 cis-10-Heptadecenoic, C17:1 BUT ^* BUT ^* Margarine, C17:0 1220 294 γ-Linolenic, C18:3n6 BUT ^* BUT ^* Linoleic, C18:2n6c BUT ^* BUT ^* Oleic, C18:1n9c 202 93 Linoelaidic, C18:2t BUT ^* BUT ^* Linolenic, C18:3n3 BUT ^* BUT ^* Elaidic, C18:1n9t 15 59 Stearic, C18:0 6459 773 Arachidonic, C20:4n6 BUT ^* BUT ^* cis-5,8,11,14,17-Eicosapentaenoic, C20:5 BUT ^* BUT ^* cis-8,11,14-Eicosatrienoic, C20:3n8 BUT ^* BUT cis-11,14-Eicosadienoic, C20:2 BUT ^* BUT ^* cis-11-eicosenoic, C20:1 BUT ^* BUT ^* cis-11,14,17-Eicosatrienoic, C20:3n11 BUT ^* BUT ^* Arachidic, C20:0 188 11 Heneicosanoic, C21:0 17 BUT ^* cis-4,7,10,13,16,19-Docosahexaenoic, C22:6n3 BUT ^* BUT ^* cis-13,16-Docosadienoic, C22:2 BUT ^* BUT ^* Erucic acid, C22:1n9 BUT ^* BUT ^* Behenic, C22:0 66 BUT ^* Tricosanovaya, C23:0 6 BUT ^* Nervonovaya, C24:1n9 BUT ^* BUT ^* Lignocerol, C24:0 185 BUT ^* BUT* - not determined

[00100]

Table 5. Content of microelements and heavy metals in the culture liquid of the probiotic strain B. subtilis, determined by ICP-MS analysis Element / m/z Content in sample, ppb (µg/kg) without helium Na / 23 705050 Mg/24 10647 Al / 27 112.1 K / 39 4228 Ca / 43 38987 Ca / 44 41447 V / 51 4.58 Cr/52 23.0 Mn / 55 9976 Fe / 56 687 Co / 59 49.2 Ni/60 69.9 Cu/63 1050 Zn/66 1186 As / 75 1.17 Se / 82 7.75 Mo/95 32.4 Ag/107 0.26 Cd/111 0.41 Sb/121 0.55 Ba / 137 4.59 Pb/206 78.2

[00101] A general screening was also performed to identify compounds not predicted a priori in the culture fluid of the probiotic strain B. subtilis. The general screening was performed by gas chromatograph mass spectrometry using mass spectra and retention index databases in the automatic chromatograph mass spectrometry data interpretation mode. The groups of organic compounds identified during the chromatograph mass spectrometry screening of the samples are presented in Tables 6–10.

[00102]

Table 6. Quantitative assessment of the content of low-molecular organic acids (except fatty acids) and inorganic acids in the culture fluid of the probiotic strain B. subtilis RT, min RI (exp) RI (lit.) Identified connection Content in sample, ng/ml 6,220 987 978 Methylbutanoic acid or Pentanoic acid 241.8 7,726 1083 1066 Lactic acid 244.0 8,598 1140 1252 Hydroxyisocaproic acid 202.9 8,702 1147 1139 Oxalic acid 287.4 10,813 1294 1286 Phosphoric acid 1490.7 11,011 1308 1298 Phenylacetic acid - 12,624 1427 1414 Hydrocinnamic acid + Methacryloyl glycine 2191.6 14,291 1560 1610 3-Phenyllactic acid 1326.9 14,511 1578 1610 3-Phenyllactic acid isomer 568.8 14,567 1582 1563 Hydroxyphenylacetic acid - 17,881 1885 1935 4-Hydroxyphenyllactic acid 936.3 18,047 1902 1935 Hydroxyphenyllactic acid isomer 545.8 18,655 1962 - 2-Hydroxysebacic acid X 18,917 1987 1981 3-Indoleacetic acid* X RT - Retention time, RI - Retention index, exp. - in experiment, lit. - according to literature data

[00103]

Table 7. Quantitative assessment of the content of phenolic compounds identified in the culture fluid of the probiotic strain B. subtilis RT, min RI (exp) RI (lit.) Identified connection Content in sample, ng/ml 7,664 1079 1056 Phenol 22584.4 11,355 1333 1330 Catechol (1,2-dihydroxybenzene, pyrocatechin) 2382.3 12,185 1393 1410 Hydroquinone 704.5 12,332 1403 1399 3-Methylcatechol 368.7 12,442 1412 1410 Hydroquinone isomer + unidentified compound 759.3

RT - retention time, RI - retention index, exp. - in experiment, lit. - according to literature data;

x - quantitative assessment of the content of the identified compound is not possible.

[00104]

Table 8. Quantitative assessment of the content of alcohols and glycols identified in the culture fluid of the probiotic strain B. subtilis RT, min RI (exp) RI (lit.) Identified connection Content in sample, ng/ml 6,220 991 990 Ethylene glycol 1620.2 6,764 1022 1011 Propylene glycol 970.3 8,114 1108 - Methionol* - 8,977 1165 1153 Benzyl alcohol 1799.4 9,279 1185 - Unidentified compound 607,0 20,650 2181 2165 1-Octadecanol X 24,021 2563 2532 Heptaethylene glycol 1489.3 32,278 >3000 3052 Nonaethylene glycol 229.5 41,630 >3000 3316 Decaethylene glycol 386.7

RT - retention time, RI - retention index, exp. - in experiment, lit. - according to literature data;

x - quantitative assessment of the content of the identified compound is not possible.

[00105]

Table 9. Quantitative assessment of the content of nitrogen-containing compounds identified in the culture fluid of the probiotic strain B. subtilis RT, min RI (exp) RI (lit.) Identified connection Content in sample, ng/ml 8,814 1155 1143 2-Pyrrolidinone
(2-pyrrolidone) X 10,483 1271 1266 Caprolactam 218.2 11,629 1353 1332 Uracil* - 13,609 1504 1488 Dihydrouracil - 12,985 1455 - Indole** 12419.9 12,483 1415 1412 Timin*** - 15,285 1644 1636 Phenylalanine - 15,354 1650 1653 Tyramine**** - 16,754 1776 1809 5-Hydroxy-1H-indole 1269.4 17,265 1825 1812 9H-Purin-6-ol - 18,230 1920 - Tryptophol***** X 26,604 2754 2625 13-Docosenamide
(Erucic acid amide) 344.0

RT - retention time, RI - retention index, exp. - in experiment, lit. - according to literature data;

x - quantitative assessment of the content of the identified compound is not possible.

[00106]

Table 10. Quantitative assessment of the content of peptides identified in the culture fluid of the probiotic strain B. subtilis RT, min RI (exp) RI (lit.) Identified connection Content in sample, ng/ml 16,392 1743 - DL-Alanyl-L-leucine* 1063.4 17,371 1835 1908 Unidentified cyclic dipeptide.
Cyclo(leucylprolyl) (L-Phe-D-Pro lactam)** 456.9 17,614 1859 1908 Unidentified cyclic dipeptide.
Cyclo(leucylprolyl)** 793.1*** 18,550 1951 1908 Unidentified cyclic dipeptide.
Cyclo(leucylprolyl) (L-Phe-D-Pro lactam)** 1236.8 18,714 1968 - N-valeryl-L-proline tetradecyl ester X 20,650 2181 - Unidentified cyclic dipeptide.
3-Methyl-6-(phenylmethyl)-2,5-piperazindione (Cyclo(Ala-Phe))** X 22,322 2369 - Unidentified cyclic dipeptide.
3-Benziohexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Cyclo(D-phenylalanine-L-prolyl))** 786.7 22,682 2409 - Unidentified cyclic dipeptide.
3-Benziohexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Cyclo(D-phenylalanine-L-prolyl))** 744.5

RT - retention time, RI - retention index, exp. - in experiment, lit. - according to literature data;

x - quantitative assessment of the content of the identified compound is not possible.

[00107] Screening was also performed using high-performance liquid chromatography-mass spectrometry (HR-HPLC-MS). As a result of screening by HR-HPLC-MS, the compounds presented in Table 11 were identified. The difference in theoretical mass from the practical mass is 0.7 ppm, thus, it can be stated that the identification is reliable.

[00108]

Table 11. Compounds in the culture fluid of the probiotic strain B. subtilis identified by HR-HPLC-MS Identified compounds Chemical formula M+H M+2H RT Sample, µg/ml Tyramine (Phenylethanolamine) _{C8H11NO ​} 138,091 6.95 15,315 Phenylethylamine _{C8H11N ​} 122,096 8.03 12,727 Uracil _{C4H4N2O2 ​ ​ ​} 113,0345 5.5 11,563 Dihydrouracil _{C4H6N2O2 ​ ​ ​} 115,0502 5.42 0.793 Timin _{C5H6N2O2 ​ ​ ​} 126,1133 7.73 4,049 Amikumacin B _{C20H28O8N2 ​ ​ ​} 425,1918 8.75 133,463 Amikumacin A _{C20H29N3O7 ​ ​ ​} 424,2078 8.65 0.509 Fengycin A C15-LC C ₇₁ H ₁₀₈ N ₁₂ O ₂₀ 1449,788 725,3977 10.75 0,000 Fengycin A C16 --LC C ₇₂ H ₁₁₀ N ₁₂ O ₂₀ 1463,803 732,405 11.55 0,000 Fengycin A C17 --LC C ₇₃ H ₁₁₂ N ₁₂ O ₂₀ 1477,819 739,413 12.74 0,000 Fengitsin n B C16 --LC C ₇₄ H ₁₁₄ N ₁₂ O ₂₀ 1491,835 749.42 13.11 0,000 Fengycin B C17 --LC C ₇₅ H ₁₁₆ N ₁₂ O ₂₀ 1505,85 753,428 13.62 0,000 Fengycin B C18 --LC C ₇₆ H ₁₁₈ N ₁₂ O ₂₀ 1519,866 760,436 15.12 0,000

RT - Retention time, FA - fatty acid.

Example 3. Study of the effect of B. subtilis culture suspension on inhibition of growth of pathogenic and opportunistic microflora

[00109] In this example, the antagonistic activity of a culture suspension of the probiotic strain B. subtilis was studied using the perpendicular streak method. The B. subtilis strain VKM B-3536D was used as the probiotic strain. Streaks of the culture suspension of the B. subtilis strain VKM B-3536D were applied to the surface of the agarized Luria-Bertani medium in a Petri dish. Then, the said Petri dish was placed in a thermostat at a temperature of 37°C for 24 hours. After the B. subtilis metabolites had diffused into the agarized medium, test cultures were seeded with streaks perpendicular to the grown streak, starting from the edges of the Petri dish. Then, the said Petri dish was placed in a thermostat at a temperature of 37°C for 48 hours. The presence of antagonistic activity was recorded by measuring the growth inhibition zones of the test cultures.

[00110]

Table 1. Results of the study of the antagonistic activity of the culture suspension of B. subtilis Object of study Growth inhibition zone, mm Staphylococcus aureus Listeria monocytogenes Escherichia coli Pseudomonas aeruginosa Salmonella typhimurium B. subtilis 10 17 25 30 28

As a result of the study, a pronounced ability of the culture suspension of the probiotic strain B. subtilis to suppress the growth of pathogenic and opportunistic microorganisms was established.

Example 4. Study of the effect of B. subtilis metabolites on the inhibition of the growth of pathogenic and opportunistic microflora

[00111] In this example, the ability of metabolites of the probiotic strain B. subtilis VKM B-3536D with a molecular weight of more than 5 kDa to suppress the growth of indicator cultures was studied. The study was conducted using an adapted standard disk method for determining the sensitivity of microorganisms to antibiotics. The indicator cultures used in this example are represented by microorganisms pathogenic and opportunistic for humans. To implement the method, the indicator cultures were placed on the surface of an agarized Luria-Bertani nutrient medium in a Petri dish in an amount of 10 ⁷ CFU. After solidification, the Petri dishes were dried for 15 minutes at 37 °C. Then, 10 μl of solutions including B. subtilis metabolites with a molecular weight of more than 5 kDa were applied to the surface of the nutrient medium with the indicator cultures in the form of drops with a diameter of 5 ± 1 mm. These solutions were obtained by tangential ultrafiltration. In this case, solution No. 1 included metabolites with a molecular weight of more than 100 kDa, and solution No. 2 included metabolites with a molecular weight of 5-100 kDa.

[00112] Petri dishes with applied solutions were left at room temperature until the drops were completely absorbed and then incubated at 37°C for 24-48 hours. The results were detected by measuring the growth inhibition zone of indicator cultures around the area of application of solutions containing B. subtilis metabolites. The appearance of indicator culture-free zones around the area of application of the said solutions indicates the antagonistic activity of B. subtilis metabolites with respect to the indicator cultures indicated in Table 2. The size of the indicator culture-free zones indicates the effectiveness of the antagonistic action of B. subtilis metabolites.

[00113]

Table 12. Growth retardation of indicator cultures exposed to B. subtilis metabolites Indicator culture Zone of growth inhibition outside a drop of solution containing B. subtilis metabolites Solution No. 1
(> 100 kDa) Solution No. 2
(5-100 kDa) Escherichia coli DH5α 1 mm* - Staphylococcus aureus 3.2 2 mm 2 mm Staphylococcus aureus 4.1 3 mm 2 mm Staphylococcus epidermidis 3.1 4 mm 4 mm Streptococcus agalactiae 42 1 mm 2 mm Streptococcus agalactiae 30 1 mm 1 mm Streptococcus agalactiae 9177 1 mm 1 mm Enterococcus faecium 24 1 mm 2 mm Enterococcus faecalis 4387 1 mm 1 mm Enterococcus faecalis 4661 1 mm 1 mm Streptococcus salivarius 1453 0.5mm* 0.5mm* Streptococcus gordonii 221 - - Streptococcus mitis cl. is. 1 1 mm* 1 mm*

* - bacteriostatic action;

“-” - growth retardation was not observed.

[00114] Table 12 shows that both high-molecular metabolites weighing more than 100 kDa (solution No. 1) and metabolites weighing 5-100 kDa (solution No. 2) have the ability to suppress growth. At the same time, there is a significant difference in the effect of these two variants of metabolites: high-molecular metabolites weighing >100 kDa cause a delay in the growth of E. coli, while metabolites weighing 5-100 kDa do not. At the same time, a culture suspension of the B. subtilis strain VKM B-3536D, including all metabolites, also caused a significant delay in the growth of E. coli (Ex. 3). It is worth noting that E. coli is one of the opportunistic microorganisms often found in the gastrointestinal tract during the development of inflammatory diseases. Thus, the use of the entire spectrum of B. subtilis metabolites ensures the effectiveness of the declared composition against the widest possible range of pathogenic and opportunistic microorganisms.

Example 5. Study of cytotoxicity of B. subtilis metabolites in relation to mammalian cells

[00115] In this example, the potential cytotoxicity of metabolites of the probiotic strain B. subtilis was investigated. Cytotoxicity was assessed according to the recommendations of the European Food Safety Authority (EFSA) for the characterization of microorganisms used as feed additives or producer strains [47]. The B. subtilis strain VKM B-3536D was grown in brain heart broth at 30°C for 6 h. The cells were separated from the culture fluid by centrifugation.

[00116] Cytotoxicity analysis on Vero cells (green monkey renal epithelial cell line) was carried out according to the method [48] with modifications. Vero cells were incubated with culture fluid. The degree of damage to Vero cells was assessed by the release of the enzyme lactate dehydrogenase by the cells. Cytotoxicity was expressed as a percentage of the toxicity of the detergent Triton-X100, which causes the release of lactate dehydrogenase from the cells due to perforation of the cell membranes. Cytotoxic and non-cytotoxic strains of the genus Bacillus were used as control strains. Table 3 presents the average values for the strain VKM B-3536D and two control strains. Each experiment was carried out in duplicate.

[00117]

Table 13. Cytotoxicity of B. subtilis metabolites to Vero cells Strain Cytotoxicity, %* Toxicity ** B. subtilis VKM B-3536D 4.6 Non-toxic Non-toxic strain of Bacillus 2.0 Non-toxic Toxic strain of Bacillus 84.1 Toxic *% cytotoxicity was calculated taking the amount of lactate dehydrogenase secreted by Triton-X100-treated cells as 100%.
** Interpretation of results: 0-20% - non-toxic, 20-100% - toxic.

[00118]

The presented results show that the cytotoxicity of the culture fluid of the B. subtilis VKM B-3536D strain does not exceed the limit of 20%. Thus, the metabolites of the B. subtilis VKM B-3536D strain contained in the studied culture fluid are not toxic to Vero cells and can be considered safe for mammalian cells.

Example 6. Evaluation of the immunomodulatory properties of B. subtilis metabolites

[00119] In this example, the immunomodulatory properties of a metabolite complex including inactivated vegetative cells and spores of the probiotic strain B. subtilis VKM B-3536D, as well as substances from the culture fluid with a molecular weight of more than 5 kDa were studied. The said substances with a molecular weight of more than 5 kDa were obtained by tangential ultrafiltration of the culture fluid of the probiotic strain B. subtilis VKM B-3536D, mixed with inactivated cells and spores, and then lyophilized on maltodextrin as a carrier. The said metabolite complex was added to a culture of human monocytes THP-1 cells. The effect of adding metabolites was compared with the control without metabolites 1) at the stage of differentiation of monocytes into macrophages using phorbol myristate acetate and 2) at the stage of polarization of previously differentiated monocytes. The effect was assessed using the reverse transcription-PCR method, based on the expression of mRNA marker genes encoding interleukins and other factors: IL1b, IL6, IL10, TGFb, TNFa and CD64.

[00120] Fig. 1 shows the expression values of the indicated marker genes upon addition of metabolites, normalized to the control without addition of metabolites. The average values of two experiments, with three repetitions in each experiment, are shown. Statistically significant (p≤0.05) quantitative changes (at least twofold compared to the control) in both experiments are marked with an asterisk (*). Light gray bars reflect the expression level of the corresponding gene at the differentiation stage, dark gray bars reflect the expression level at the polarization stage.

[00121] It is evident that at the differentiation stage, the studied metabolites significantly increased the expression of the TNFa gene, and at the same time significantly decreased the expression of the CD64 gene. This indicates an increase in the process of differentiation of monocytes into macrophages. The expression of the cytokine IL1b also significantly decreased, which indicates an anti-inflammatory effect. At the polarization stage, the studied metabolites significantly increased the expression of the cytokines IL6 and IL10. IL10 is a classic anti-inflammatory cytokine, and an increase in its expression indicates the activation of anti-inflammatory polarization of macrophages. IL6 is an important regulatory cytokine, high concentrations of which are also associated with an anti-inflammatory effect. At the same time, the metabolites did not have a reliably significant effect on the expression of the anti-inflammatory cytokine TGFb, which indicates the selectivity of their action.

[00122] The obtained data indicate that the studied metabolites have immunomodulatory properties. They can promote the differentiation of monocytes into macrophages, as well as promote the anti-inflammatory polarization of macrophages, which is necessary for the termination of inflammation and tissue regeneration.

Example 7. Evaluation of anti-inflammatory and anti-neoplastic properties of B. subtilis metabolites

[00123] In this example, the effect of the purified metabolite fraction of the B. subtilis strain VKPM B-2335 (5-100 kDa) on the expression of inflammation marker proteins: cyclooxygenase 2 (COX-2 ) and tumor necrosis factor α ( TNF-α ) in gastric epithelial cells, and interleukin 23 ( IL-23 ) in primary cell cultures of human intestinal and gastric epithelium was studied. In addition, the effect of B. subtilis metabolites on the expression of the intestinal differentiation marker, caudal homeobox protein type 2 ( CDX-2 ), was studied in intestinal epithelial cells.

[00124] Primary cell cultures were obtained from small intestinal and stomach biopsy tissues. Culture passages were performed every 2 days. In total, the cultures underwent 7-12 passages. B.subtilis metabolites were added to the experimental cultures at a concentration of 0.1 μg/ml. Marker expression was determined by immunocytochemistry (ICC) using primary specific antibodies to the proteins under study. Detection was performed using secondary fluorescently labeled antibodies (Alexa Fluor 647 label) binding to the primary antibodies, followed by visualization on an Olympus FluoView FV1000-IX70 inverted confocal microscope with a 606 UPlan apochromatic objective (Japan) at a magnification of 400×. Four fields of view were recorded for each preparation. The images were evaluated by the parameter of relative expression area in %, which was calculated using the formula: S _rel = S _expr / S _tot * 100%, where S _rel is the relative expression area, S _expr is the area of the image with marker expression, S tot is the total area of the preparation in the image. The significance of differences was assessed using Student's t-test (p≤0.05).

[00125] The results of the expression assessment of the studied markers on the cells of the gastric epithelium are shown in Fig. 2A. The results of the expression assessment of the studied markers on the cells of the intestinal epithelium are shown in Fig. 2B. The control culture is shown in dark gray, and the culture with the addition of B. subtilis metabolites is shown in gray. The abscissa axis shows the passage number of the culture, and the ordinate axis shows the relative area of marker expression in %. Statistically significant (p≤0.05) quantitative changes (at least twofold compared to the control) in the experiments are marked with an asterisk (*).

[00126] B. subtilis metabolites significantly reduced the expression of the inflammatory marker COX-2 after passage 12 of the culture and the expression of TNF-α at passage 3 in gastric cells. IL-23 expression in intestinal cells was reduced by B. subtilis metabolites at both passages 3 and 7. The metaplasia marker CDX-2 was significantly expressed at passage 7 of the intestinal cell culture and was also reduced in the culture with the addition of B. subtilis metabolites.

[00127] The data obtained demonstrate the anti-inflammatory and antimetaplastic effects of B. subtilis metabolites. In the case of COX-2 and TNF-α markers, the effect of B. subtilis metabolites depended on the number of passages passed by the cultures, i.e. on the “maturity” of the culture. Under in vivo conditions, gastrointestinal epithelial cells are at different stages of differentiation (“maturation”), but B. subtilis metabolites can potentially act on these cells simultaneously due to their different molecular targets.

Example 8. Evaluation of the anti-Helicobacter effect of the claimed composition on the Mongolian gerbil model

[00128] In this example, we investigated the effect of the declared composition of metabiotics on the efficiency of H. pylori eradication in the model of Helicobacter pylori infection in Mongolian gerbils. Mongolian gerbils are an effective model for studying the pathogenesis of H. pylori, reflecting many features of gastric tissue inflammation caused by H. pylori in humans [49]. The animals were infected with a double (every other day) intragastric administration of a bacterial suspension of H. pylori. After double infection, the animals were observed for 11 weeks. By the 12th week after infection, the animals developed gastritis and ulcerative lesions of the gastric tissue. From the 12th week, the administration of the studied substances began in accordance with the scheme (Table 14). The studied substances were:

1) a mixture of inactivated cells of the L. reuteri strain DSM17648 (L. reuteri metabolites) and filler;

2) a mixture of inactivated cells of the L. reuteri strain DSM17648 (metabolites of L. reuteri), metabolites of 5-100 kDa of the B. subtilis strain VKPM B-2335 and filler.

The filler was starch in an amount of 1 wt. % As a positive control, drugs from standard eradication therapy for H. pylori in humans were used, which includes the proton pump inhibitor omeprazole and the antibiotics clarithromycin and amoxicillin [50]. As a negative control, just the filler was used. The effect of the indicated substances 1 and 2 in combination with the positive control drugs was also studied.

[00129]

Table 14. Scheme of infection of Mongolian gerbils and characteristics of the obtained model groups Groups Number of animals Substance under study Dose per animal (mg/kg) Introduction scheme Intact 3 - - - Negative control 7 Filler 0 Daily for 28 days until euthanasia Positive control 7 Omeprazole 60 Daily for 14 days (starting 28 days before euthanasia) Clarithromycin 100 Amoxicillin 40 Substance 1 7 Metabolites of L.reuteri + filler 6.6 Daily for 28 days until euthanasia Substance 2 7 Metabolites of L.reuteri + B.subtilis + filler 6.8 Substance 1
+ positive control 7 Omeprazole 60 Daily for 14 days (starting 28 days before euthanasia) Clarithromycin 100 Amoxicillin 40 Metabolites of L.reuteri + filler 6.6 Daily for 28 days until euthanasia Substance 2
+ positive control 7 Omeprazole 60 Daily for 14 days (starting 28 days before euthanasia) Clarithromycin 100 Amoxicillin 40 Metabolites of L.reuteri + B.subtilis + filler 6.8 Daily for 28 days until euthanasia

[00130] The vehicle, positive control preparations and test substances were administered to gerbils intragastrically, since this method is analogous to the oral route of administration in humans. At the end of the experiment, the animals were euthanized and a histological examination of the stomach and duodenum tissues was performed. The condition of the stomach was assessed using the following parameters: area of inflammation; macroscopic changes in the mucosa; morphological changes (degree of inflammation; inflammation activity; atrophy of the gastric glands; metaplasia; colonization of the mucosa with H. pylori). For morphological assessment of the severity of pathology, visual analogue scales proposed in the Sydney system were used [49]. In this case, a point system of assessment was used:

0 - normal;

1 - weakly expressed;

2 - moderate;

3 - pronounced.

[00131] Macroscopic evaluation of the stomach and duodenum tissues in all experimental groups revealed no visible pathological changes. The results of microscopic histological studies are presented in Table 15.

[00132]

Table 15. Summary results of semiquantitative microscopic analysis of gastric tissues Criteria Intact Negative. control Positive. control Substance 1
(metabolites of L.reuteri + filler) Substance 2
(metabolites of L.reuteri, metabolites+ of B.subtilis
+ filler) Substance 1
+
positive control Substance 2
+
positive control Area of inflammation 0,0
(0,0; 0,0) 3.0
(1.0; 3.0) ^a 1.0
(1.0; 1.0) ^b 1.0
(1.0; 1.0) ^b 1.0
(1.0; 1.0) ^b 1.0
(1.0; 1.0) 1.0
(1.0; 1.0) ^b 0 15 7 7 7 8 7 Degree of inflammation 0,0
(0,0; 0,0) 2.0
(2.0; 2.0) ^a 1.0
(1.0; 2.0) ^b 2.0
(1.0; 2.0) 1.0
(1.0; 2.0) ^b 1.0
(1.0; 2.0) ^b 1.0
(1.0; 1.0) ^b 0 15 9 11 9 9 8 Inflammatory activity 0,0
(0,0; 0,0) 2.0
(2.0; 3.0) ^a 1.0
(1.0; 2.0) ^b 1.0
(1.0; 2.0) ^b 1.0
(1.0; 2.0) ^b 1.0
(0.0; 1.0) ^b 1.0
(0.0; 1.0) ^b 0 16 9 10 10 5 6 Atrophy of the gastric glands 0,0
(0,0; 0,0) 2.0
(2.0; 3.0) ^a 1.0
(0.0; 1.0) ^b 1.0
(1.0; 1.0) ^b 0,0
(0.0; 1.0) ^b 0,0
(0.0; 1.0) ^b 0,0
(0.0; 1.0) ^b 0 15 5 6 3 2 2 Degree of colonization of H. pylori 0,0
(0,0; 0,0) 1.0
(1.0; 2.0) ^a 1.0
(0.0; 1.0) ^b 1.0
(0.0; 1.0) ^b 0,0
(0.0; 1.0) ^b 0,0
(0.0; 1.0) ^b 0,0
(0.0; 1.0) ^b

[00133] Median, upper and lower quartile (Me (Q₁; Q₃)) are indicated in white cells, the sum of points in the group is indicated in gray cells; a - reliable difference from the intact group of mice, (p<0.05); b - reliable difference from the negative control group (p<0.05). In the intact group of mice that were not infected or treated, the structure of the stomach wall was normal. In the negative control group, all animals had morphological changes in the gastric mucosa characteristic of catarrhal gastritis of varying severity: from mild to moderate, with a tendency to chronicity of the process. The development of infection in animals of the negative control group led to the formation of predominantly pangastritis (inflammation area 3, average score 15, Table 15). In this group, a reliable increase in the severity of pathological changes, the degree and activity of inflammation, atrophy of the gastric glands and colonization of the gastric mucosa with H. pylori was noted compared to the intact group (p<0.05). In the positive control group, there was a significant decrease in the indicators for all the studied criteria compared to the negative control group (Table 15).

[00134] Fig. 3 shows the range diagrams of the total indices of the severity of pathological symptoms for the substances under study. Box plot 1 corresponds to the negative control. Box plot 2 corresponds to the positive control. Box plot 3 corresponds to substance 1 (L. reuteri metabolites + filler). Box plot 4 corresponds to substance 2 (L. reuteri metabolites + B. subtilis metabolites + filler). The combination of substance 1 and the positive control corresponds to box plot 5. The combination of substance 2 and the positive control corresponds to box plot 6. Along the ordinate axis, the height of the diagrams corresponds to the total severity of pathological symptoms in points of the visual analogue scale. The mean values and median of the experiments are indicated, with seven repetitions in each experiment. Statistically significant (p≤0.05) quantitative changes (at least twofold compared to the negative control) in the experiments are marked with an asterisk (*).

[00135] With monotherapy with substance 1 (L. reuteri metabolites + vehicle), a significant decrease in the area and activity of inflammation, morphological changes in the stomach, and the degree of colonization of the gastric mucosa with H. pylori was observed compared with the negative control. However, for most criteria, monotherapy with substance 1 was inferior in effectiveness to the positive control (Table 15).

[00136] Monotherapy with substance 2 (L. reuteri metabolites + B. subtilis metabolites + vehicle) was not inferior in effectiveness to the positive control, and in some parameters even surpassed it. Thus, with monotherapy with substance 2, atrophy of the gastric glands, as well as the degree of colonization of the gastric mucosa with H. pylori were less than in the positive control group (Table 15).

[00137] Combination therapy with substance 1 together with positive control drugs or substance 2 with positive control drugs was superior to the positive control in some respects. Thus, the addition of the studied substances to the positive control drugs reduced the activity of inflammation, the severity of gastric gland atrophy, and the degree of colonization of the gastric mucosa with H. pylori (Table 15). At the same time, combination therapy with substance 2 with positive control drugs showed the smallest data scatter and mean error (Fig. 3). Thus, combination therapy with substance 2 with positive control drugs most strongly and statistically significantly reduced the overall severity of pathological changes compared to the other studied therapy options.

[00138] The obtained data show that the claimed composition, including metabolites of L. reuteri and B. subtilis, is more effective in the treatment of Helicobacter pylori infection than the composition, including only metabolites of L. reuteri. Moreover, this pattern was observed both with monotherapy with the studied substances 1 and 2, and with a combination of these substances with drugs for standard eradication therapy of H. pylori. This suggests that the addition of B. subtilis metabolites to L. reuteri metabolites determines the effectiveness of the claimed composition with respect to H. pylori and concomitant inflammation. The latter may be associated with the anti-inflammatory and immunomodulatory action of B. subtilis metabolites, shown in Ex. 6 and 7.

Example 9. Evaluation of the effectiveness of using the claimed composition in therapy in patients with chronic gastritis or functional dyspepsia associated with H. pylori infection

[00139] In this example, an open, randomized, prospective, controlled study of the efficacy and safety of the claimed composition was conducted involving patients with functional dyspepsia and/or chronic gastritis associated with H. pylori infection. The composition used in the example included inactivated cells of the probiotic strain L. reuteri GMNL-0902, a complex of metabolites of the probiotic strain B. subtilis VKM B-3536D, zinc citrate trihydrate, and fructooligosaccharides. The study included 40 patients of both sexes aged 18 to 65 years inclusive with a confirmed diagnosis of functional dyspepsia or chronic gastritis, and with a positive analysis for the presence of H. pylori DNA in feces using real-time PCR. A separate requirement for patients was the absence of a history of previously administered eradication therapy less than a year before screening.

[00140] All patients were equally divided into experimental group 1 and control group 2. Patients from experimental group 1 received the claimed composition 2 times a day in the morning and evening after meals for 14 days in addition to the standard three-component first-line eradication therapy (esomeprazole 20 mg orally 2 times a day in the morning and evening after meals, amoxicillin 1000 mg 2 times a day in the morning and evening after meals, clarithromycin 500 mg 2 times a day in the morning and evening after meals). For the next 14 days after the end of therapy, patients in this group received the claimed composition 2 times a day in the morning and evening after meals as a monotherapy. Patients from control group 2 received eradication therapy for 14 days, including esomeprazole 20 mg orally 2 times a day in the morning and evening after meals, amoxicillin 1000 mg orally 2 times a day in the morning and evening after meals, clarithromycin 500 mg orally 2 times a day in the morning and evening after meals. The severity of symptoms characteristic of chronic gastritis or functional dyspepsia was assessed using questionnaires that all patients filled in before the start of the experiment and 28 days after the start of the experiment (Table 16).

Table 16. Dynamics of gastrointestinal complaints in patients of the main and control groups Complaints Moment of observation (beginning/end of therapy course) Number of patients, abs. (%) Significance of differences between groups, p Experimental group, n=20 Control group, n=20 Excessive gas emission start 8 (40) 7 (35) 0.005** ending 0 (0) 10(50) Rumbling in the stomach start 11(55) 11(55) <0.001** ending 2 (10) 14(70) Stomach ache start 15 (75) 16(80) 0.336 ending 4(20) 5 (25) Feeling of heaviness in the stomach start 14(70) 12(60) 0.7150 ending 5(25) 5(25) Belching start 7(35) 9(45) 0.439 ending 3(15) 4 (25) Heartburn start 8(40) 8 (40) 0.234 ending 2 (10) 0 (0) Nausea start 7(45) 6 (30) 0,017 ending 2 (10) 9(45) Vomit start 0 (0) 0 (0) - ending 0 (0) 0 (0) Bad taste in the mouth start 0 (0) 2 (13) - ending 0 (0) 0 (0) Tendency to constipation start 6(30) 7(35) - ending 0(0) 0 (0) Tendency to diarrhea start 8(40) 8 (40) 0.002** ending 4(20) 14(70) Decreased performance start 10(50) 6(30) 0.5 ending 5 (25) 4(20) Weakness start 9(45) 6(30) 0.358 ending 4(20) 6 (30)

[00141] Statistical comparison of the effects of therapy in the experimental and control groups showed that the use of the claimed composition reliably alleviated such symptoms as excessive gas, rumbling in the abdomen and a tendency to diarrhea. At the same time, in the experimental group, the frequency of these symptoms decreased after the course of therapy compared to the start of therapy. At the same time, in the control group, these symptoms began to occur more often after the therapy than at the beginning. This indicates that standard eradication therapy disrupts the functioning of the gastrointestinal tract. Most likely, this is due to dysbacteriosis caused by broad-spectrum antibiotics. At the same time, the use of the claimed composition together with standard eradication therapy drugs reduced the frequency of almost all symptoms after the therapy. This may be due to the relief or prevention of dysbacteriosis, due to the beneficial effect of the claimed composition on representatives of the normal intestinal microflora.

[00142] The present application materials present a preferred disclosure of the implementation of the claimed technical solution, which should not be used as limiting other, particular embodiments of its implementation that do not go beyond the scope of the requested scope of legal protection and are obvious to specialists in the relevant field of technology.

List of references:

1. Brown L.M. Helicobacter pylori: epidemiology and routes of transmission. Epidemiol Rev. 2000; 22 (2): 283-97.

2. Warren J.R, Marshall B. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983 Jun 4; 1 (8336): 1273-5.

3. Watari J., Chen N., Amenta P.S., Fukui H., Oshima T., Tomita T., Miwa H., Lim K.J., Das K.M. Helicobacter pylori associated chronic gastritis, clinical syndromes, precancerous lesions, and pathogenesis of gastric cancer development. World J Gastroenterol. 2014 May 14; 20 (18): 5461-73.

4. Molina-Infante J., Gisbert J.P. Optimizing clarithromycin-containing therapy for Helicobacter pylori in the era of antibiotic resistance. World J Gastroenterol. 2014 Aug 14; 20 (30): 10338-47.

5. Namazova-Baranova Leila Seymurovna, Baranov A.A. "Antibiotic resistance in the modern world". Pediatric pharmacology, vol. 14, no. 5, 2017, pp. 341-354.

6. Akhmetova D.G., Baltabekova A.Zh., Shustov A.V. Antibiotic resistance of Helicobacter pylori: review of epidemiological trends and problems of treatment // RMJ. Medical Review. 2018. No. 7(I). P. 13-18.

7. Shah T., Baloch Z., Shah Z., Cui X., Xia X. “The Intestinal Microbiota: Impacts of Antibiotics Therapy, Colonization Resistance and Diseases” // Int. J. Mol. Sci. 2021, 22, 6597.

8. Bondarenko V.M., Gracheva N.M., Matsulevich T.V. “Intestinal dysbacteriosis in adults” // M.: KMK. - 2003. - 224 p.

9. Sullivan A., Nord C.E. Probiotics and gastrointestinal diseases. J Intern Med. 2005; 257: 78-92.

10. Shenderov B.A. Probiotics and Functional Foods / B.A. Shenderov // Food Engineering, Encyclopedia of Life Support Systems (EOLSS). Developed under the Auspices of the UNESCO. - Eolss Publishers: Oxford, UK, 2011.

11. Vorobeychikov E.V., Vasilenko A.Zh., Sinitsa A.V., et al. “Methodological aspects of the use of probiotics and antibiotics for emergency prevention of infectious diseases” // Collection of scientific papers of the III Congress of the Yu.A. Ovchinnikov Society of Biotechnologists of Russia. - M: MaxPress, 2005. - P. 35-36.

12. Homan M., Orel R. Are probiotics useful in Helicobacter pylori eradication? World J Gastroenterol. 2015 Oct 7; 21 (37): 10644-53.

13. Parth K., Prudhivi R., Palatheeya S., Abbas S.K., Varsha K., Niharika B. et al. (2021). Efficacy of lactobacillus reuteri supplementation in eradication of h. pylori: A comparison study with triple drug therapy. J. Pharm. Res. Int.

14. Lin R., Liu W., Piao M., Zhu H. A review of the relationship between the gut microbiota and amino acid metabolism. Amino Acids. 2017 Dec; 49 (12): 2083-2090. doi:10.1007/s00726-017-2493-3.

15. Bondarenko V.M., Boev B.V., Lykova E.A. et al. Dysbacteriosis of the gastrointestinal tract // Russian journal of gastroenterology, hepatology, proctology. 1999. No. 1. P. 66-70 [Bondarenko V.M., Boev B.V., Lykova E.A. et al. Dysbacteriosis of the gastrointestinal tract // Russian journal of gastroenterology, gepatology, proctology. 1999. No. 1. P. 66-70 (in Russian)].

16. Khursa R.V., Mesnikova I.L., Miksha Ya.S. “Intestinal microflora: role in maintaining health and developing pathology, possibilities of correction” // Minsk BGMU, 2017.

17. Zhuchkova T.V., “Modern concepts of human microflora: what is dysbacteriosis?” // Vidal Medical Encyclopedia: Gastroenterology, 2015.

18. Tarantula V.Z. “Dictionary of biotechnological terms” // Moscow: Rospatent, 2005.

19. Shenderov B.A., Tkachenko E.I., Lazebnik L.B., Ardatskaya M.D., Sinitsa A.V., Zakharchenko M.M. “Metabiotics - a new technology for the prevention and treatment of diseases associated with microecological disturbances in the human body” // Experimental and Clinical Gastroenterology. 2018; 151 (3): 83-92.

20. Firsov N.N., “Microbiology: dictionary of terms” // M: Bustard, 2006.

21. Maksimova E.M., Vinogradova D.S., Osterman I.A., Kasatsky P.S., Nikonov O.S., Milón P., Dontsova O.A., Sergiev P.V., Paleskava A., Konevega A.L. Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation. Front Microbiol. 2021 Feb 12; 12: 618857.

22. Kaspar F., Neubauer P., Gimpel M. Bioactive Secondary Metabolites from Bacillus subtilis: A Comprehensive Review. J Nat Prod. 2019, Jul 26; 82(7):2038-2053. doi: 10.1021/acs.jnatprod.9b00110.

23. Park H.B., Perez C.E., Perry E.K. and Crawford J.M. Activating and Attenuating the Amicoumacin Antibiotics.// Molecules.2016. 21 (7).

24. Prokhorova I.V., Akulich K.A., Makeeva D.S., Osterman I.A., Skvortsov D.A., Sergiev P.V., Dontsova O.A., Yusupova G., Yusupov M.M., Dmitriev S.E. Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome. Sci Rep. 2016 Jun 14; 6: 27720. doi: 10.1038/srep27720.

25. Tosco A., Chobelet A., Bathany K., Schmitter J.-M, Urdaci M. C., Buré C. Characterization by Tandem Mass Spectrometry of Biologically Active Compounds Produced by Bacillus Strains // journal of Applied bioanalysis. 2015. Jan., pp. 19-25

26. Hamdache A., Lamarti A.; Aleu J.; Collado I.G. Non-peptide metabolites from the genus Bacillus // J. Nat. Prod. 2011. V. 74, P. 893-899.

27. Zhao K., Xing R., Yan X. Cyclic dipeptides: Biological activities and self-assembled materials. Peptide Science, 2020, https://doi.org/10.1002/pep2.24202.

28. Ning Y., Fu Y., Hou L., Ma M., Wang Z., Li X., Jia Y. iTRAQ-based quantitative proteomic analysis of synergistic antibacterial mechanism of phenyllactic acid and lactic acid against Bacillus cereus. Food Res Int. 2021 Jan; 139:109562. doi: 10.1016/j.foodres.2020.109562. Epub 2020 Nov 1. PMID: 33509445.

29. Liu F., Sun Z., Wang F., Liu Y., Zhu Y., Du L., Wang D., Xu W. Inhibition of biofilm formation and exopolysaccharide synthesis of Enterococcus faecalis by phenyllactic acid. Food Microbiol. 2020 Apr; 86: 103344. doi: 10.1016/j.fm.2019.103344.

30. Zheng R., Zhao T., Hung Y.C., Adhikari K. Evaluation of Bactericidal Effects of Phenyllactic Acid on Escherichia coli O157:H7 and Salmonella Typhimurium on Beef Meat. J Food Prot. 2019 Dec; 82 (12): 2016-2022. doi: 10.4315/0362-028X.JFP-19-217.

31. Wang F., Wu H., Jin P., Sun Z., Liu F., Du L., Wang D., Xu W. Antimicrobial Activity of Phenyllactic Acid Against Enterococcus faecalis and Its Effect on Cell Membrane. Foodborne Pathog Dis. Oct 2018; 15 (10): 645-652. doi: 10.1089/fpd.2018.2470.

32. Ning Y., Yan A., Yang K., Wang Z., Li X., Jia Y. Antibacterial activity of phenyllactic acid against Listeria monocytogenes and Escherichia coli by dual mechanisms. Food Chem. 2017 Aug. 1; 228: 533-540. doi: 10.1016/j.foodchem.2017.01.112. Epub 2017 Jan 25. PMID: 28317760.

33. Sun S., Li H., Chen J., Qian Q. Lactic Acid: No Longer an Inert and End-Product of Glycolysis. Physiology (Bethesda). 2017 Nov; 32 (6): 453-463. doi: 10.1152/physiol.00016.2017.

34. Narayana K.J., Prabhakar P., Vijayalakshmi M., Venkateswarlu Y., Krishna P.S. Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes. Pol J Microbiol. 2007; 56 (3): 191-7.

35. Sakko M., Tjäderhane L., Sorsa T., Hietala P., Järvinen A., Bowyer P., Rautemaa R. 2-Hydroxyisocaproic acid (HICA): a new potential topical antibacterial agent. Int J Antimicrob Agents. 2012 Jun; 39 (6): 539-40. doi: 10.1016/j.ijantimicag.2012.02.006.

36. Guimarães A., Venancio A., Abrunhosa L. Antifungal effect of organic acids from lactic acid bacteria on Penicillium nordicum. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2018 Sep; 35.(9):.1803-1818. doi: 10.1080/19440049.2018.1500718.

37. Zhang, L.S., Davies, S.S. Microbial metabolism of dietary components to bioactive metabolites: opportunities for new therapeutic interventions. Genome Med 8, 46, 2016. https://doi.org/10.1186/s13073-016-0296-x.

38. Machate D.J., Figueiredo P.S., Marcelino G., Guimarães R.C.A., Hiane P.A., Bogo D., Pinheiro V.A.Z., Oliveira L.C.S., Pott A. Fatty Acid Diets: Regulation of Gut Microbiota Composition and Obesity and Its Related Metabolic Dysbiosis. Int J Mol Sci. 2020 Jun 8; 21 (11): 4093.

39. Venn-Watson, S., Lumpkin, R. & Dennis, E.A. Efficacy of dietary odd-chain saturated fatty acid pentadecanoic acid parallels broad associated health benefits in humans: could it be essential?. Sci Rep 10, 8161, 2020. https://doi.org/10.1038/s41598-020-64960-y.

40. Huang C.B., Alimova Y., Myers T.M., Ebersole J.L. Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms. Arch Oral Biol. July 2011; 56 (7): 650-4. doi: 10.1016/j.archoralbio.2011.01.011.

41. Bae Y.S., Rhee M.S. Short-Term Antifungal Treatments of Caprylic Acid with Carvacrol or Thymol Induce Synergistic 6-Log Reduction of Pathogenic Candida albicans by Cell Membrane Disruption and Efflux Pump Inhibition. Cell Physiol Biochem. 2019; 53 (2): 285-300. doi: 10.33594/000000139.

42. Jung S.W., Lee S.W. The antibacterial effect of fatty acids on Helicobacter pylori infection. Korean J Intern Med. Jan 2016; 31 (1): 30-5. doi: 10.3904/kjim.2016.31.1.30. Epub 2015 Dec 28. PMID: 26767854; PMCID: PMC4712431.

43. Omura Y., O'Young B., Jones M., Pallos A., Duvvi H., Shimotsuura Y. Caprylic acid in the effective treatment of intractable medical problems of frequent urination, incontinence, chronic upper respiratory infection, root canalled tooth infection, ALS, etc., caused by asbestos & mixed infections of Candida albicans, Helicobacter pylori & cytomegalovirus with or without other microorganisms & mercury. Acupunct Electrother Res. 2011; 36 (1-2): 19-64. doi: 10.3727/036012911803860886.

44. He W., Hu S., Du X., Wen Q., Zhong X.P., Zhou X., Zhou C., Xiong W., Gao Y., Zhang S., Wang R., Yang J., Ma L. Vitamin B5 Reduces Bacterial Growth via Regulating Innate Immunity and Adaptive Immunity in Mice Infected with Mycobacterium tuberculosis. Front Immunol. 2018 Feb 26; 9: 365. doi: 10.3389/fimmu.2018.00365.

45. Levine M, Lohinai Z.M. Resolving the Contradictory Functions of Lysine Decarboxylase and Butyrate in Periodontal and Intestinal Diseases. Journal of Clinical Medicine. 2021; 10 (11): 2360. https://doi.org/10.3390/jcm10112360.

46. Read S.A., Obeid S., Ahlenstiel C., Ahlenstiel G. The Role of Zinc in Antiviral Immunity. Adv Nutr. 2019 Jul 1; 10 (4): 696-710. doi: 10.1093/advances/nmz013.

47. Rychen G., Aquilina G., Azimonti G., Bampidis V., Bastos M.L., Bories G., Chesson A., Cocconcelli P.S., Flachowsky G., Gropp J., Kolar B., Kouba M., Lopez-Alonso M., Lopez Puente S., Mantovani A., Mayo B., Ramos F., Saarela M., Villa R.E., Wallace R.J., Wester P., Glandorf B., Herman L., Kärenlampi S., Aguilera J., Anguita M., Brozzi R., Galobart J. “EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed): Guidance on the characterization of microorganisms used as feed additives or as production organisms” // EFSA Journal, 2018, 16 (3), 5206, 24 pp. https://doi.org/10.2903/j.efsa.2018.5206.

48. Roberts P.H., Davis K.C., Garstka W., Bhunia A.K. “Lactate dehydrogenase release assay from Vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains” // Journal of Microbiological, 2001, Methods, 43: 171-181.

49. Noto, Jennifer M., Judith Romero-Gallo, M. Blanca Piazuelo, and Richard M. Peek. 2016. “The Mongolian Gerbil: A Robust Model of Helicobacter Pylori-Induced Gastric Inflammation and Cancer.”, Methods in molecular biology (Clifton, N.J.), 1422, pp. 263-280.

50. Ivashkin V.T., Maev I.V., Lapina T.L. Clinical recommendations of the Russian Gastroenterological Association for the diagnosis and treatment of Helicobacter pylori infection in adults // Russian Journal of Gastroenterology, Hepatology, Coloproctology. - 2018. - No. 28 (1). - P. 55-70.

51. Dixon M.F., Genta R.M., Yardley J.H., Correa P. Classification and grading of gastritis: the updated Sydney system // The American journal of surgical pathology. - 1996. - Vol. 20. - No. 10. - P. 1161-1181.

Claims (28)

1. A metabiotic composition characterized in that it includes inactivated Lactobacillus reuteri cells, Bacillus subtilis metabolites and a filler, wherein the ratio of components, wt. %: inactivated Lactobacillus reuteri cells 16-70 Bacillus subtilis metabolite complex 1-7 filler 23-83 where the complex of Bacillus subtilis metabolites represents substances contained in a mixture of culture fluid, vegetative cells and spores of the probiotic strain Bacillus subtilis. 2. The metabiotic composition according to claim 1, characterized in that the probiotic strain B. subtilis is VKM B-3536D and/or VKPM B-2335. 3. The metabiotic composition according to claim 1, characterized in that the vegetative cells and spores of the probiotic strain B. subtilis are inactivated. 4. The metabiotic composition according to claim 1, characterized in that the filler includes a prebiotic. 5. The metabiotic composition according to claim 4, characterized in that the prebiotic is polyfructosan. 6. The metabiotic composition according to claim 1, characterized in that the filler includes a zinc source in a content of at least 3 wt.% of the weight of the composition. 7. The metabiotic composition according to claim 6, characterized in that the zinc source is zinc citrate. 8. Use of the composition according to claim 1 for the treatment and prevention of gastrointestinal tract diseases associated with Helicobacter pylori. 9. Use of the composition according to claim 8 for the treatment and prevention of chronic gastritis associated with Helicobacter pylori. 10. Use of the composition according to claim 8 for the treatment and prevention of functional dyspepsia associated with Helicobacter pylori. 11. A method for producing a composition according to item 1, comprising the following steps: a) Bacillus subtilis is grown in a liquid nutrient medium; b) centrifuge the Bacillus subtilis culture suspension in a flow centrifuge to obtain the Bacillus subtilis culture fluid and sediment, including vegetative cells and spores of Bacillus subtilis; c) dry the resulting culture liquid of Bacillus subtilis; d) inactivate vegetative cells and spores of Bacillus subtilis; d) mixing the dried culture liquid of Bacillus subtilis and inactivated vegetative cells and spores of Bacillus subtilis to obtain a complex of Bacillus subtilis metabolites; e) add inactivated Lactobacillus reuteri cells and filler to the resulting complex of Bacillus subtilis metabolites. 12. The method according to claim 11, wherein the Bacillus subtilis culture liquid is dried to a moisture content of 4-6% by weight. 13. The method according to claim 11, wherein the Bacillus subtilis culture liquid is dried by lyophilization. 14. The method according to claim 11, wherein the vegetative cells and spores of Bacillus subtilis are inactivated by incubating the sediment obtained in the centrifugation step at a temperature of 60-140°C. 15. The method according to claim 14, wherein the sediment is incubated for 5-120 minutes. 16. The method according to claim 11, wherein after inactivation the vegetative cells and spores of Bacillus subtilis are additionally dried so that their moisture content is no more than 6-8% by weight. 17. The method according to claim 16, wherein the inactivated cells and spores of Bacillus subtilis are dried by lyophilization. 18. The method according to claim 11, wherein a filler is used that includes a prebiotic. 19. The method according to claim 11, wherein a filler is used that includes a zinc source. 20. The method according to claim 19, wherein zinc citrate is used as the source of zinc.