RU2840322C2 - Composition using bacillus subtilis metabolites for treating urinary tract diseases - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: disclosed is a metabiotic composition for treating and preventing urinary tract infectious diseases and a method for preparing it. Composition contains 0.1–7.0 wt.% of a complex of Bacillus subtilis VKM V-3536D or Bacillus subtilis VKPM V-2335 metabolites and 93.0–99.9 wt.% of D-mannose, cranberries or a mixture thereof. Metabolite complex represents substances contained in a mixture of the culture fluid, vegetative cells and spores of the said probiotic Bacillus subtilis strain.

EFFECT: composition is compatible with taking antibiotics, promotes elimination of pathogenic microorganisms from urinary tract, has broad antagonistic action with respect to pathogenic and opportunistic microorganisms, stimulating effect on normal intestinal microbiota, immunomodulatory potential.

21 cl, 1 dwg, 14 tbl, 6 ex

Description

AREA OF TECHNOLOGY

[0001] The present invention relates to biotechnology and medicine, namely to compositions for the treatment and prevention of urinary tract diseases using metabolites of probiotic microorganisms. The present invention can be used in the food industry, veterinary science and medicine.

LEVEL OF TECHNOLOGY

[0002] Urinary tract infection (UTI) is the most common infection seen in humans, affecting 150 million people worldwide each year. While UTIs can be caused by numerous bacterial species, including Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabilis), and Staphylococcus aureus (S. aureus), the opportunistic pathogen Escherichia coli (E. coli) accounts for the majority (>65%) of reported cases (O'Connor et al., 2019). E. coli is a member of the gut microbiota.

[0003] On the one hand, the intestinal microbiota provides colonization resistance and participates in the formation of the immune response. The absence of colonization resistance leads to a significant weakening of the body's anti-infective defenses, an increase in the number and spectrum of pathogenic and opportunistic microorganisms, and, consequently, to an increase in the body's susceptibility to the occurrence of infectious diseases of bacterial and viral origin (Bondarenko et al., 2003; Petrovskaya, 1982; Bukharin, 1999), the development of endogenous infections, superinfections of various localizations (Lenzner, 1987; Yushkov, 2002; Minushkin et al., 1999). On the other hand, many representatives of the normal intestinal microbiota can be classified as opportunistic microorganisms. Getting into organs or organ systems that are not typical for them, these microorganisms can cause serious infectious diseases. For example, when excreted in the feces, E. coli can colonize the periurethral area and enter the urinary tract.

[0004] Individual episodes of UTI are usually well tolerated by patients, but recurrent UTIs can significantly impair their quality of life. Recurrent infections occur in approximately 35-53% of women after one year of therapy (Domenici et al., 2016). Substances that help eliminate pathogens from the urinary tract are used to treat and prevent recurrence of genitourinary infections. The most popular in this regard are cranberry and D-mannose (e.g. US 9895341 B2, published 20.02.2018, IPC: A61K 31/352, A61K 39/00, A61K 45/06, A61K 31/353, A61K 31/7048; US 8153166 B2, published: 10.04.2012, IPC: A01N 65/00; EP 3558459 A1, published 30.10.2019, IPC: A61K 31/70, A61K 36/45, A61K 36/63, A61P 13/10, A61P 31/04). However, the use of cranberry and D-mannose without additional medications may not be sufficiently effective against severe urinary tract infection (Jepson et al., 2012; Barbosa-Cesnik et al., 2011).

[0005] Antibiotics are most often used to treat an established urinary tract infection. However, antibiotics taken orally can cause intestinal dysbiosis, which is associated with a disruption in the qualitative and quantitative composition of the intestinal microflora (Shah et al., 2021). In addition to digestive problems, intestinal dysbiosis causes suppression of general immunity, which leads to the risk of recurrence of infectious diseases. Including urinary tract diseases. It has been shown that the frequency of UTI recurrence returns to baseline after interruption of antibiotic therapy. For example, 60% of women experience a relapse within the next 3 months (Domenici et al., 2016).

[0006] One way to break this vicious circle is to use probiotic formulations that include live probiotic microorganisms. Compositions aimed at the treatment and prevention of urinary tract diseases are known, including probiotic microorganisms (for example, FR 2906109 B1, published 09/22/2006, IPC: A23L 33/00, A61K 31/375, A61K 31/70, A61K 35/74, A61K 35/741, A61K 36/45, A61P 13/02, A61P 31/00; US 20090226548 A1, published 09/10/2009; IPC: A61K 36/45, A61P 31/04). However, such compositions lose effectiveness when taken simultaneously with antibiotics, because The live probiotic microorganisms contained in them are partially or completely destroyed by antibiotics.

[0007] A composition is known for the treatment and prevention of urinary tract diseases, including metabolites of Bacillus subtilis (B.subtilis) (US 6156355 A; published: 05.12.2000; IPC: A23K 1/10, A23K 1/14, A23K 1/16, A23K 1/175, A23K 1/18, A23K 1/175). However, in the said composition proposed for feeding animals, an extract of culture fluid (Bacillus subtilis Fermentation Extract) is used, including only a part of the metabolites of B.subtilis. The selectivity of metabolites can reduce the effectiveness and range of application of this composition.

[0008] Thus, there is a need to develop effective agents for the treatment and/or prevention of urinary tract infections that do not lose their effectiveness when taken concomitantly with antibiotics and modulate the intestinal microbiota.

TERMS AND ABBREVIATIONS

[0009] 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 means a violation of the intestinal microflora (Khursa et al., 2017; Zhuchkova, 2015).

[0010] Pathogen isolator - a component that promotes the removal of pathogenic and/or opportunistic microorganisms from the urinary tract.

[0011] 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 (Tarantula, 2005).

[0012] Culture suspension is a liquid medium obtained during the cultivation of various pro- and eukaryotic cells in vitro and containing cultured cells and/or spores.

[0013] 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 (Shenderov et al., 2018).

[0014] Microbiota (microflora) 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 (Firsov, 2006).

[0015] Probiotics are a functional food ingredient in the form of non-pathogenic and non-toxicogenic live microorganisms that are beneficial to humans and that, when systematically consumed in food in the form of preparations or as part of food products, have a beneficial effect on the human body as a result of normalizing the composition and/or increasing the biological activity of normal intestinal microflora (GOST R 52349-2005).

[0016] HPLC - high performance liquid chromatography.

[0017] HPLC-MS2 - high performance liquid chromatography with tandem mass-selective detection.

[0018] HPLC-MS2 HR-ESI - high-performance liquid chromatography with high-resolution tandem mass-selective detection with electrospray ionization.

[0019] HPLC-MS2HR - high-performance liquid chromatography with high-resolution tandem mass-selective detection.

[0020] FA - fatty acids.

[0021] GIT - gastrointestinal tract.

[0022] kDa - kilodalton.

[0023] CFU - colony forming unit.

[0024] B. subtilis - Bacillus subtilis.

[0025] E. coli - Escherichia coli.

[0026] CD64 is a membrane protein Fc-gamma receptor 1, a monocyte marker, and also a proinflammatory marker of macrophages.

[0027] IL1b - interleukin 1 beta, a proinflammatory cytokine.

[0028] IL6 - interleukin 6, pro- and anti-inflammatory cytokine, regeneration factor, macrophage activating factor, lymphocyte stimulating factor.

[0029] IL10 - interleukin 10, an anti-inflammatory cytokine.

[0030] MRSA - methicillin-resistant strains of S. Aureus.

[0031] RI - retention index.

[0032] RT - retention time.

[0033] TNFa - tumor necrosis factor alpha, macrophage activating factor.

[0034] TGFb - transforming growth factor beta, blocks the activation of macrophages, suppresses the inflammatory immune response.

ESSENCE OF THE INVENTION

[0035] The objective of the present invention is to create a composition for the treatment and prevention of urinary tract diseases that are compatible with the use of antibiotics and prevent the occurrence of intestinal dysbacteriosis.

[0036] This problem is solved by the claimed invention by achieving such a technical result as the creation of a composition that promotes the removal of pathogenic microorganisms from the urinary tract, has a broad antagonistic effect on pathogenic and opportunistic microorganisms, has a stimulating effect on the normal intestinal microbiota, and has immunomodulatory potential.

[0037] The claimed technical result is achieved due to the fact that the claimed composition includes biologically active substances, including a complex of metabolites of the probiotic strain B. subtilis, in an amount of 0.1-7% of the mass of all biologically active substances. In addition, the biologically active substances include a pathogen isolator, which facilitates the removal of pathogenic and/or opportunistic microorganisms from the urinary tract. In this case, the content of the pathogen isolator can be 93.0-99.9% of the mass of all biologically active substances.

[0038] The metabolites can be substances contained in the culture suspension of the probiotic strain B. subtilis. In a preferred embodiment, the metabolites are substances contained in a mixture of the culture fluid, vegetative cells and spores of the probiotic strain B. subtilis. The said mixture includes virtually the entire spectrum of metabolites of the probiotic strain B. subtilis. In this case, the culture fluid can be dried to such a state that the moisture content of the dry culture fluid is no more than 6% by weight. In this case, the vegetative cells and spores of the probiotic strain B. subtilis can be inactivated (non-living). In this case, the vegetative cells and spores of the probiotic strain B. subtilis can also be dried so that their moisture content is no more than 6% by weight. In a preferred embodiment, the probiotic strain B. subtilis is B. subtilis VKM B-3536D. Metabolites of the probiotic strain B. subtilis are capable of selectively suppressing the growth of a wide range of pathogenic and opportunistic microorganisms. Due to the absence of active live microorganisms, the claimed composition does not lose its effectiveness when taken in combination with antibiotics, which allows it to be used to treat and prevent urinary tract diseases in a wide range of patients. The use of dried culture fluid, as well as the use of dried vegetative cells and spores of the probiotic strain B. subtilis allows for the creation of a dry composition suitable for long-term storage and convenient for transportation.

[0039] The pathogen isolate may include D-mannose, cranberry, or a mixture thereof. In this case, the content of D-mannose in the mixture may be 27.0-98.0% of the weight of the biologically active substances, and the content of cranberry may be 1.9-66.0% of the weight of the biologically active substances. In a preferred embodiment, cranberry is included in the composition in the form of a powder obtained from whole berries.

[0040] The claimed composition may further comprise zinc. In a preferred embodiment, the zinc is included in the composition as an organic salt. In an even more preferred embodiment, the zinc salt is zinc citrate.

[0041] Due to its composition, the claimed composition normalizes the quantitative and qualitative composition of the microflora in the intestine, selectively inhibiting the growth of pathogenic and opportunistic microorganisms and creating conditions for the growth of symbiotic microflora. This ensures the immunostimulating effect of the claimed composition on the patient's body. In addition, the claimed composition promotes the removal of pathogenic and opportunistic microorganisms from the urinary tract.

[0042] The technical result is also achieved by developing an accessible method for obtaining the claimed composition. The method includes the following steps:

a) growing the probiotic strain B. subtilis 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 with a filling factor of 60-70% at a temperature of 36-38°C with constant stirring and aeration. In this case, cultivation is 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 cultivation stage corresponds to the maximum concentration of B. subtilis metabolites in the culture suspension. The specified 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) drying the culture liquid. In a preferred embodiment, the culture liquid is dried by lyophilization;

d) inactivate vegetative cells and spores;

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

e) a pathogen isolator is added to the obtained complex of metabolites.

[0043] The claimed method provides for obtaining a composition that includes virtually the entire spectrum of metabolites of the probiotic strain B. subtilis. Due to this, the claimed composition 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 urinary tract diseases.

[0044] The method may further comprise drying the inactivated vegetative cells and spores. In a preferred embodiment, the inactivated cells and spores are dried by lyophilization.

[0045] The method may further comprise adding a zinc salt to a composition comprising a complex of metabiotics of a probiotic strain of B. subtilis and a pathogen isolator. This allows for the creation of a composition with an enhanced immunostimulating effect.

[0046] 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

[0047] Fig. 1 shows the results of a study of the immunomodulatory properties of a complex of B. subtilis metabolites.

DETAILED DESCRIPTION OF THE INVENTION

[0048] 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.

[0049] Furthermore, it is clear from the above disclosure that the invention is not limited to the embodiment shown. 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.

[0050] The claimed composition promotes the treatment and prevention of urinary tract diseases. The claimed composition includes biologically active substances, including a complex of B. subtilis metabolites and a pathogen isolate. The ratio of biologically active substances, wt. %:

Bacillus subtilis metabolite complex 0.1-7.0 pathogen isolator 93.0-99.9

[0051] The complex of metabolites is substances contained in a culture suspension of the probiotic strain B. subtilis. In a preferred embodiment, the complex of metabolites is substances contained in a mixture of culture liquid, vegetative cells and spores of the probiotic strain B. subtilis. The said mixture includes virtually 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 (Examples 2 and 4).

[0052] B. subtilis metabolites include a variety of biologically active substances that have antimicrobial, probiotic, and immunomodulatory effects. For example, experimental data (Example 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. Amikumacins are active against antibiotic-resistant strains of the pathogen 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 the gram-positive bacteria Salmonella sp. and Shigella sp. (Maksimova et al., 2021; Kaspar et al., 2019; Park et al., 2016; Prokhorova et al., 2016; Tosco et al., 2015; Hamdache et al., 2011).

[0053] 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) show 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 common cause of fungal infections (Zhao et al., 2020).

[0054] 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 growth inhibition effects against pathogens such as L. monocyotgenes, E. coli, S. typhimurium, B. cereus, E. faecalis (Ning et al., 2021; Liu et al., 2020; Zheng et al., 2019; Wang et al., 2018; Ning et al., 2017; Sun et al., 2017; Narayana et al., 2007).

[0055] a-Hydroxyisocaproic acid has a broad antibacterial activity, including bacteria such as MRSA (methicillin-resistant S. aureus), which are resistant to multiple systemically used antimicrobials (Sakko et al., 2012).

[0056] 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 likely due to the stimulation of the production of the cytokine IL-22 by intestinal immune cells (Guimarães et al., 2018; Zhang and Davies, 2016).

[0057] Long-chain monounsaturated fatty acids (aka omega-9 fatty acids), such as myristoleic (C14:1), palmitoleic (C16:1), oleic (C18:1), and elaidic (C18:1) acids, increase the diversity of gut microbiota (Machate et al., 2020).

[0058] 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 (Venn-Watson et al., 2020; Huang et al., 2011).

[0059] Medium-chain saturated fatty acids, such as caprylic (C8), capric (C10), undecyl (C11), and lauric (C12) acids, improve intestinal epithelial function by increasing the activity of microvilli enzymes in the small intestine. These acids have antibacterial, antifungal, and anti-inflammatory effects. For example, lauric acid is most effective against Campylobacter jejuni, while capric and lauric acids have antibacterial effects against Helicobacter pylori (Machate et al., 2020; Bae and Rhee 2019; Jung et al., 2016; Omura et al., 2011).

[0060] Vitamin B5 (pantothenic acid) inhibits bacterial growth via regulation of innate immunity and adaptive immunity in mice infected with Mycobacterium tuberculosis (He et al., 2018).

[0061] Amino acids support intestinal barrier function and intestinal endocrine function. Amino acids entering the intestine from the outside are metabolized by the intestinal microbiota, which uses them to synthesize their own proteins and catabolic pathways. It has been shown that dietary amino acids can regulate the composition of the microbiota. For example, lysine supports commensal (probiotic) microbiota in the intestine. In addition, lysine helps maintain the immune system (Levine and Lohinai, 2021; Lin et al., 2017).

[0062] The culture liquid 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 liquid is no more than 6% by weight. 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 a preferred embodiment, the probiotic strain B. subtilis is B. subtilis VKM B-3536D.

[0063] The use of a complex of B. subtilis metabolites in a concentration of less than 0.1% by weight leads to a significant decrease in the effectiveness of the composition. Increasing the content of metabolites to more than 7% by weight is irrational, since it increases the cost of production and does not have a significant effect on the effectiveness of the composition.

[0064] 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.

[0065] Due to the absence of living 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 urinary tract diseases in a wide range of patients. In addition, non-living vegetative cells and spores of B. subtilis do not create competition for the intestinal microflora, which is unique to each patient, and, accordingly, do not disturb its balance.

[0066] The use of dried culture liquid, as well as the use of dried vegetative cells and spores of the probiotic strain B. subtilis, makes it possible to create a dry composition suitable for long-term storage and convenient for transportation.

[0067] The pathogen isolator is designed to bind to the fimbriae or other adhesive structures of pathogenic microorganisms, and accordingly, suppress the adhesive activity of bacteria in the urinary tract. D-mannose, cranberry (Vaccinium macrocarpon) or a mixture thereof can be used as a pathogen isolator. In this case, the content of D-mannose in the mixture can be 27.0-98.0% of the mass of biologically active substances. The content of cranberry in the mixture can be 1.9-66.0% of the mass of biologically active substances. The specified content of D-mannose and cranberry is most effective for removing pathogenic and opportunistic microorganisms from the urinary tract.

[0068] D-mannose is a simple sugar, a monosaccharide structurally related to glucose. D-mannose is rapidly absorbed and reaches the peripheral organs within 30 minutes, then is excreted from the urinary tract. It cannot be converted into glycogen and does not accumulate in the human body. In the urinary tract, pathogenic and opportunistic microorganisms, such as E. coli, Salmonella spp., Proteus mirabilis, can bind to mannose in the protein uromodulin on the surface of cells lining the urothelium. Binding of microorganisms to the urothelium contributes to the development of urinary tract infections. By binding to free exogenous D-mannose in the urine, rather than to proteins on the cell surface, microorganisms are eliminated with the urine stream (Domenici et al., 2016).

[0069] Substances contained in cranberry (Vaccinium macrocarpon) may reduce the incidence of bacteriuria. In particular, proanthocyanidins have been shown to inhibit the adherence of uropathogenic P-fimbriae of E. coli. Complex phenolic polymers such as ellagitannins have also been identified as potent antibacterial agents (Puupponen-Pimia et al., 2005). In addition, proanthocyanidins exhibit anti-inflammatory and immunoregulatory functions, hypoglycemic and hypolipidemic effects, metabolic regulation, DNA repair, and anticancer effects (Qi et al., 2022). Anthocyanins contained in cranberry have pronounced antioxidant activity both in vitro and in vivo. Anthocyanins are characterized by high bioavailability when taken orally, which determines their high therapeutic and prophylactic effect (Alekseenko and Azarova, 2018). In a preferred embodiment, the pathogen isolator includes ellagitannins, proanthocyanidins and/or polyphenols. These substances can bind to the fimbriae of microorganisms resistant to mannose, which determines the effectiveness against pathogens that are not affected by D-mannose alone.

[0070] In addition to removing pathogens from the urinary tract, cranberries have prebiotic properties. Cranberry-derived salicylate has been shown to modulate the composition of the gut microbiota by decreasing Enterobacteriaceae and increasing Bacteroidaceae. Reducing the abundance of potentially pathogenic microorganisms belonging to the Enterobacteriaceae family (Shigella/Escherichia) has a beneficial effect on gut function and reduces the risk of diarrhea.

[0071] In one embodiment, the cranberry is included in the composition as an extract. For example, as an extract enriched with proanthocyanidins. In a preferred embodiment, the cranberry is included in the composition as a powder obtained from whole berries. This is due to the fact that the use of whole berries allows the use of the entire spectrum and volume of biologically active substances contained in cranberries. For example, studies of the localization of anthocyanins in cranberries have shown that there are 5.8-10.8 times more of them in the skin than in the pulp (Alekseenko and Azarova, 2018). In addition, the use of powder from whole berries provides an optimal ratio of cost and effectiveness of the claimed composition, which makes it more accessible to a wide range of consumers.

[0072] Biologically active substances may include zinc. Zinc plays an important role in the development and maintenance of the immune system. The use of zinc suppresses the growth of pathogenic microorganisms, for example, Mycobacterium, and also prevents the development of viral infections (Read et al., 2019). The claimed composition may include zinc in the form of a compound with other chemical elements. In this case, the zinc content in the composition can be 0.2-3% of the weight of all biologically active substances. In a preferred embodiment, zinc can be included in the composition as 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, as 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, zinc can be included in the composition as zinc citrate. Zinc citrate is widely available on the market and provides an optimal cost of production of the composition without losing its effectiveness. In this case, the content of the remaining components in the claimed composition is reduced in total by the amount of mass % that corresponds to the content of zinc salt. In this case, the content of each component, except for the zinc salt, is reduced in proportion to the initial content of each component in the composition.

[0073] The claimed composition may include 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 following can be used as a filler, 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.

[0074] Due to its composition, the claimed composition enhances the axis: gastrointestinal tract - urinary tract. Due to the action of the described complex of metabolites of the probiotic strain B. subtilis, the quantitative and qualitative composition of the individual intestinal microbiota of the patient is normalized, immunity is strengthened, the efficiency of removing pathogens from the urinary tract increases. At the same time, even when taking antibiotics, intestinal dysbacteriosis does not develop, which could reduce the patient's overall immunity and lead to relapses of an infectious disease of the urinary tract. Thus, the claimed composition contributes not only to the treatment, but also to the prevention of infectious diseases of the urinary tract.

[0075] The claimed composition can be used to manufacture a food supplement or a pharmaceutical preparation in the form of a powder, tablet or capsule.

[0076] The method for producing the claimed composition 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 producing 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.

[0077] 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.

[0078] 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 no more than 6% by weight.

[0079] 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.

[0080] d) mix the culture liquid with inactivated vegetative cells and spores.

[0081] e) a pathogen isolator is added to the obtained complex of metabolites. In a preferred embodiment, D-mannose and/or cranberry are used as the pathogen isolator. If both of the said pathogen isolators are used, they can be added to the composition separately or as a mixture.

[0082] The claimed method provides for obtaining a composition that includes the entire spectrum of metabolites of the probiotic strain B. subtilis. Due to this, the claimed composition 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 diseases of the urinary system.

[0083] 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 can be dried by any suitable method known in the art. In a preferred embodiment, the inactivated vegetative cells and spores are dried by lyophilization. In one embodiment, the inactivated vegetative cells and spores are first mixed with the 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 are dried separately from the culture liquid. In any case, the inactivated cells spores are dried to such a state that the moisture content of the dried vegetative cells and spores is no more than 6% by weight. The use of dried vegetative cells and spores makes it possible to obtain a composition in dry form.

[0084] In a preferred embodiment, the method further comprises adding a zinc salt to a composition comprising metabiotics of the probiotic strain B. subtilis, as well as cranberry and/or D-mannose. This allows for the creation of a composition with an enhanced immunostimulating effect.

[0085] In a preferred embodiment, the composition is obtained in dry form, for example, in the form of a powder. Firstly, this is due to the low solubility of the biologically active substances contained in cranberries. Secondly, in dry form, the shelf life of the composition is increased without loss of its effectiveness. This is due both to the preservation of the activity of the metabolites and to the minimization of the risk of contamination of the composition with any active microorganisms.

DESCRIPTION OF THE IMPLEMENTATION OF THE INVENTION

Example 1. Obtaining a complex of B. subtilis metabolites

[0086] 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 rate was 250 rpm. The initial aeration rate was 0.2 rpm; after 16 hours of growth, the rate was changed to 0.5 rpm. The submerged cultivation process was carried out for 32 hours.

[0087] 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.

[0088] The resulting culture liquid was lyophilized with the addition of 2.5% by weight of maltodextrin as a carrier. As a result, 560 g of powder was obtained, which was a lyophilized culture liquid.

[0089] 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% by weight maltodextrin as a carrier.

[0090] 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 effect of B. subtilis culture suspension on inhibition of growth of pathogenic and opportunistic microflora

[0091] 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.

[0092]

Table 1. Results of the study of the antagonistic activity of the cultural 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

[0093] 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 3. Study of the composition of the culture liquid of B. subtilis

[0094] 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).

[0095] 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.

[0096]

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

[0097]

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

[0098]

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

[0099]

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

[00100] A general screening was also performed to identify a priori unpredicted compounds 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.

[00101]

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

[00102]

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, pyrocatechol) 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

[00103]

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

[00104]

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-Pyrrolidone (2-pyrrolidinone) 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

[00105]

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

[00106] 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.

[00107]

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 C ₈ H ₁₁ N 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 Fengycin 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 4. Study of the effect of B. subtilis metabolites on the inhibition of the growth of pathogenic and opportunistic microflora

[00108] 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 the tangential ultrafiltration method. 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.

[00109] 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.

[00110]

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
"-" - no growth retardation was observed

[00111] 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 (Example 2). It is worth noting that E. coli is one of the opportunistic microorganisms often found in the urinary system 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

[00112] 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 (Rychen et al., 2018). 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.

[00113] Cytotoxicity analysis on Vero cells (green monkey renal epithelial cell line) was carried out according to the method (Roberts et al., 2001) 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 13 presents the average values for the strain VKM B-3536D and two control strains. Each experiment was carried out in duplicate.

[00114]

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.

[00115] From the presented results it is evident that the cytotoxicity of the culture fluid of the B. subtilis strain VKM B-3536D does not exceed the limit of 20%. Thus, the metabolites of the B. subtilis strain VKM B-3536D contained in the studied culture fluid are not toxic to Vero cells and can be considered safe for mammalian cells.

Example 6. Study of the effectiveness of the composition in suppressing shigella infection

[00116] In this example, the protective efficacy of the claimed composition and its individual components was studied using the example of experimental shigella infection in mice. Bacteria of the genus Shigella and the species E. coli belong to the same family Enterobacteriaceae, and have such a close phylogenetic relationship that some researchers classify Shigella species as E. coli (Devanga Ragupathi et al., 2017). Due to the brightness of manifestation, shigella infections are a convenient model for the pathogenesis of closely related bacterial species, such as E. coli, which are the most common causative agents of infectious diseases of the urinary tract.

[00117] The causative agent of dysentery, the Shigella sonnei II strain, was used for infection. The strain was obtained from the working collection of microorganisms of the Research Center (MBZ) of the State Research Institute of Military Microbiology of the Ministry of Defense of the Russian Federation (St. Petersburg). Modeling of experimental shigella infection in vivo was carried out on white outbred male mice weighing 18.0-20.0 g by intraperitoneal infection with a suspension of spores of the S. sonnei II strain in a volume of 0.5 ml. Preliminary studies showed that 1 LD ₅₀ (half lethal dose) of the pathogen was 5×10 ³ CFU/ml. The infectious dose of the bioagent was 10 LD ₅₀ . The infected animals were observed for 10 days, recording the number of live and dead animals in the experimental and control groups daily. The specificity of animal death was confirmed bacteriologically - by seeding the spleen of dead animals on a special nutrient medium. The effectiveness of the claimed composition and its individual components was determined by comparing the values of the animal survival rates in the experimental group, where the animals received the claimed composition or its individual components, and the control groups. Two control groups were formed: infection control - a group where the animals were infected with the pathogen, but were not given the claimed composition or its individual components, and a health group - a group in which the animals were not infected with the pathogen. The percentage of surviving animals in the experimental and control groups was determined using the tables of V.S. Genes (Genes, 1964).

[00118] When evaluating the effectiveness of individual components of the composition, the following single doses of the components were used:

- D-mannose - 0.5 mg/individual;

- cranberry - 0.1 mg/individual;

- complex of B. subtilis metabolites VKM B-3536D - 0.002 mg/individual.

[00119] An evaluation of the effectiveness of various variants of the composition and combinations of its individual components was also conducted:

- D-mannose and cranberry;

- D-mannose and complex of metabolites;

- cranberry and complex of metabolites;

- D-mannose, cranberry and metabolite complex

[00120] The components of the composition and combination of components were diluted to the required concentration with physiological solution and administered orally to the animal in a volume of 0.5 ml, independently (health group) or simultaneously with infection with a biopathogen. The results of the studies conducted are presented in Table 14.

[00121]

Table 14. Protective efficacy of D-mannose, cranberry and B. subtilis metabolite complex against experimental infection caused by Sh. sonnei II Component Dose
Sh. sonnei II, LD ₅₀ Survival rate, %,
X; (X-tm÷X+t) Protection, %, - 10 0 (0-31) - D-mannose - 100 (69-100) - 10 30 (7-65) 30 Cranberry - 100 (69-100) - 10 0 (0-31) 0 Metabolite complex - 100 (69-100) - 10 40 (12-72) 40 D-mannose + cranberry - 100 (69-100) - 10 30 (7-65) 30 D-mannose + metabolite complex - 100 (69-100) - 10 70 (35-97)* 70* Cranberry + Metabolite Complex - 100 (69-100) - 10 40 (12-72) 40 D-mannose + cranberry + metabolite complex - 100 (69-100) - 10 80 (44-88)* 80* Animals in each group n=10; * - differences with the infection control are significant at p<0.05.

[00122] In the health group, where the pathogen infection was not performed, the survival rate of the animals was about 100% when individual components and their combinations were administered. This indicates that the components of the composition, both individually and in combination, are safe for mice during the selected observation period. At the same time, in the infection group, shigella infection led to 100% mortality of the experimental animals (0% survival). The use of D-mannose and B. subtilis metabolites separately ensured, respectively, a survival rate of 30% and 40% of the infected mice compared to the control. Although these differences were not reliable, a clear positive trend was observed. Against this background, cranberry itself did not have a protective effect.

[00123] In the combinations of D-mannose + cranberry and cranberry + metabolite complex, the efficacy was the same as that of D-mannose and the metabolite complex separately, i.e. there was no synergistic effect with cranberry. However, in the combination of D-mannose + metabolite complex, the survival rate increased to 70%, and in the combination of D-mannose + cranberry + metabolite complex - up to 80%, in both cases statistically different from the control. This indicates a synergistic effect between the components in these combinations. In the combination of D-mannose + cranberry + metabolite complex, the efficacy was higher than in the combination of D-mannose + metabolite complex, meaning that cranberry, although it does not have an independent effect, enhances the effect of the combination of D-mannose + metabolite complex. The results of the study show that the claimed composition has anti-infective activity due to the synergistic action of the components.

Example 7. Evaluation of the immunomodulatory properties of B. subtilis metabolites

[00124] 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.

[00125] 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.

[00126] 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 IL1b cytokine also significantly decreased, which indicates an anti-inflammatory effect. At the polarization stage, the studied metabolites significantly increased the expression of the IL6 and IL10 cytokines. 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.

[00127] 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.

[00128] 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.

Literature

Alekseenko E.V., Azarova M.M. Cranberry berries - a promising source of bioactive anthocyanin dyes. Food industry 9/2018.

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

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Claims (30)

1. A metabiotic composition for the treatment and prevention of infectious diseases of the urinary tract, which includes biologically active substances, including a complex of Bacillus subtilis metabolites and a pathogen isolate, wherein the ratio of biologically active substances, wt. %: Bacillus subtilis metabolite complex 0.1-7.0 pathogen isolator 93.0-99.9, where the complex of metabolites is substances contained in a mixture of culture fluid, vegetative cells and spores of a probiotic strain of Bacillus subtilis selected from Bacillus subtilis VKM B-3536D or Bacillus subtilis VKPM B-2335, and the pathogen isolate is D-mannose, cranberry, or a mixture of both. 2. The metabiotic composition according to item 1, characterized in that the culture liquid has a moisture content of no more than 6% by weight. 3. A metabiotic composition according to claim 1, characterized in that the vegetative cells and spores of the probiotic strain Bacillus subtilis are used in a non-living state. 4. The metabiotic composition according to claim 3, characterized in that the vegetative cells and spores of the probiotic strain Bacillus subtilis have a moisture content of no more than 6% by weight. 5. The metabiotic composition according to claim 1, characterized in that the cranberry is a powder obtained from whole berries. 6. The metabiotic composition according to claim 1, additionally comprising a zinc salt. 7. The metabiotic composition according to claim 6, characterized in that the zinc salt is zinc citrate. 8. 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 culture suspension in a flow centrifuge to obtain a culture liquid and sediment, including vegetative cells and spores of Bacillus subtilis ; c) dry the resulting culture liquid; d) inactivate vegetative cells and spores of Bacillus subtilis ; d) mix the dried culture liquid and inactivated vegetative cells and spores to obtain a complex of Bacillus subtilis metabolites; e) adding a pathogen isolator to the resulting metabolite complex, where the pathogen isolator is D-mannose, cranberry, or a mixture thereof. 9. The method according to claim 8, wherein the cultivation of Bacillus subtilis is carried out using the deep method. 10. The method according to item 9, wherein the cultivation is carried out in a fermenter with a filling factor of 60-70%. 11. The method according to item 9, wherein the cultivation is carried out at a temperature of 36-38°C. 12. The method according to claim 10, wherein the cultivation is carried out with constant mixing and aeration. 13. The method according to item 8, in which a nutrient medium with an initial pH of 6.8-7.2 is used. 14. The method according to claim 13, wherein the cultivation of Bacillus subtilis is carried out until the liquid nutrient medium reaches a pH of 8.5-8.9. 15. The method according to claim 8, wherein the culture liquid is dried to such a state that the moisture content is no more than 6% by weight. 16. The method according to claim 15, wherein the culture liquid is dried by lyophilization. 17. The method according to claim 8, wherein vegetative cells and spores are inactivated by incubating the sediment obtained in the centrifugation step at a temperature of 60-140°C. 18. The method according to claim 17, wherein the sediment is incubated for 5-120 minutes. 19. The method according to claim 8, wherein after inactivation the vegetative cells and spores of Bacillus subtilis are additionally dried so that the moisture content is no more than 6% by weight. 20. The method according to claim 19, wherein the inactivated cells and spores are dried by lyophilization. 21. The method of claim 8, further comprising adding a zinc salt to the mixture of the metabolite complex with the pathogen isolator.