RU2833932C2 - Metabiotic composition with sorbent properties and method for preparation thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a metabiotic composition for treating and/or preventing diseases associated with a disturbed composition of the microbiota of the gastrointestinal tract and/or intoxication of the body, including 0.1–7.0 wt.% of a complex of metabolites obtained from a culture fluid of at least one probiotic strain of Bacillus subtilis bacteria and/or bacteria of the Lactobacillaceae family and 93.0–99.9 wt.% aluminosilicate sorbent; and a method for production thereof, comprising growing at least one probiotic strain of bacteria Bacillus subtilis and/or bacteria of the Lactobacillaceae family in a liquid nutrient medium, centrifuging the culture suspension to obtain a culture liquid and a precipitate, ultrafiltration of the culture liquid. Obtained concentrate is dried and mixed with an aluminosilicate sorbent or dried on an aluminosilicate sorbent.

EFFECT: inventions provide a wider range of agents for treating and/or preventing diseases associated with intestinal dysbacteriosis and/or body intoxication.

22 cl, 1 dwg, 7 tbl, 9 ex

Description

Field of technology

[0001] The present invention relates to biotechnology and medicine, namely to a method for producing compositions based on metabolites of probiotic microorganisms immobilized on a mineral sorbent for the treatment and prevention of diseases associated with a disruption of the quantitative and qualitative composition of intestinal microflora, as well as with intoxication of the body. The present invention can be used in the food industry, veterinary science and medicine.

State of the art

[0002] Disturbances in the qualitative and/or quantitative composition of the intestinal microflora are associated with the development of various diseases, such as acute infectious diseases or chronic diseases, such as Crohn's disease or acute ulcerative colitis. The intestinal microflora provides colonization resistance, the formation of an immune response, digestion of food, regulation of intestinal motor function, and detoxification. 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 increased likelihood of the occurrence of infectious diseases of bacterial and viral origin (Bondarenko et al., 2003; Petrovskaya, 1982; Bukharin, 1999), the development of endogenous infections and superinfections of various localizations (Lenzner, 1987; Yushkov, 2002; Minushkin et al., 1999).

[0003] Disruption of the intestinal microflora can be caused by factors such as stress, physical and psycho-emotional stress, smoking, unbalanced diet, and taking antibiotics (Shah et al., 2021). Treatment of dysbacteriosis in such cases is associated with the use of various immunobiological drugs.

[0004] The most popular immunobiological preparations used for the treatment and prevention of dysbacteriosis are probiotic compositions containing live probiotic microorganisms, or probiotics. Several species of bacteria Lactobacillus , Lactococcus , Bacillus , Streptococcus , Bifidobacterium , Pediococcus , and Propionibacterium are well-known probiotics. Yeasts such as Saccharomyces cerevisiae , S. carisbergensis , and S. boulardii , and fungi such as Aspergillus niger and A. oryzae are also considered as probiotics (KS Yoha et al., 2022). Probiotic microorganisms exhibit antagonistic activity against pathogenic and opportunistic microflora. They are capable of synthesizing substances with antimicrobial properties, competing with pathogenic microorganisms for nutrients and sites of microbial adhesion, and modifying toxins or toxin receptors (Vorobeychikov et al., 2005).

[0005] Currently, the following drugs are popular for the treatment and prevention of dysbacteriosis: Linex (Lek, Slovenia) and Bifiform (Ferrosan International A/S, Denmark). Linex contains live lactobacilli Lactobacillus acidophilus , bifidobacteria Bifidobacterium infantis , and enterococci Enterococcus faecium. Bifiform contains bifidobacteria Bifidobacterium longum (strain ATCC 15707) and enterococci Enterococcus faecium (strain SF68). The compositions of these drugs include bacterial strains with a fairly high level of antibiotic resistance, and their use is often recommended simultaneously with antibiotics. However, experience of using the above-mentioned drugs in clinical practice with a wide range of antibiotics in the recommended regimens of their administration has shown that most bacteria die with such combined administration, and the activity of those that enter the intestine in a viable state is extremely low. In addition, drugs that include lactic acid bacteria are not stable during long-term storage, which leads to a decrease in the effectiveness of therapeutic and prophylactic procedures.

[0006] Probiotics that include spore-forming bacteria are more stable during storage. Spore-forming probiotic strains of Bacillus subtilis (B. subtilis) are popular for creating probiotics. For example, a food supplement is known that includes the probiotic strain B. subtilis subsp. inaquosorum NRRL B-67989 (DE111) (US20210315808A1, published 14.10.2021 IPC: A61K9/00, A61K35/742, A61P1/14). However, this composition includes live microorganisms, which means that when combined with antibiotics, it may lose its effectiveness.

[0007] One of the promising areas of treatment and prevention of dysbacteriosis, especially caused by antibiotics, is the use of metabiotics - drugs that contain not live probiotic microorganisms, but their metabolites (Shenderov, 2019). For example, a composition is known that includes sterilized dried culture fluids containing metabolites of probiotic strains Bacillus subtilis VKPM No. B-2335, Enterococcus faecium L-3, Lactobacillus delbrueckii TS1-06, Lactobacillus fermentum TS3-06 bacteria (RU2589818C1, published 10.07.2016; IPC: A61K35 / 02; A61K35 / 74; A61K36 / 899; A61K47 / 48; A61K9 / 48). The specified composition normalizes the intestinal microflora by creating favorable conditions for the reproduction of bifido- and lactobacilli, as well as suppressing the growth of pathogenic and opportunistic microorganisms.

[0008] Also known is a composition comprising a culture fluid and inactivated cells of Lactobacillus paracasei strain B111H (CN113322216A, published 08/23/2021; IPC: A23L33/135; A61K35/747; A61P1/16; A61P3/04; A61P3/06; A61P37/02; A61P7/00; C12N1/20; C12R1/225). This composition has the effect of reducing blood lipid levels, the ability to inhibit the accumulation of lipids in liver cells, and prevents obesity caused by a high-fat diet. However, in addition to the beneficial metabolites of probiotic microorganisms, the culture fluids included in the said metabiotic compositions may include substances that are harmful or, at least, useless for the symbiotic microflora. This may reduce the effectiveness of metabiotic preparations based on culture fluids.

[0009] In addition to probiotics, enterosorbents are used to prevent and treat various pathological conditions of the intestine. Enterosorbents are sorbents with highly selective sorption capacity, non-toxic for animals, including humans, and, accordingly, suitable for oral administration. The mechanism of therapeutic action of enterosorbents is associated with such phenomena as adsorption of exotoxins, adsorption of poisons secreted with secretions of the mucous membranes of the gastrointestinal tract, liver, pancreas, adsorption and hydrolysis of endogenous secretion products, adsorption of pathogenic bacteria and viruses; binding of gases irritating areas of the gastrointestinal tract, as well as prevention or weakening of toxic-allergic reactions, reduction of the metabolic load on the excretory and detoxifying organs, correction of metabolic processes; restoration of the integrity and permeability of the mucous membranes, improvement of blood supply; stimulation of intestinal peristalsis. Therefore, enterosorbents are widely used in gastroenterology, toxicology, treatment of infectious diseases, allergology, dermatology, surgery, oncology, narcology, hepatology and nephrology.

[0010] The use of enterosorbents reduces the metabolic load on the excretory and detoxifying organs (liver, kidneys, etc.), helps to normalize the motor, evacuation and digestive functions of the gastrointestinal tract, helps to restore the integrity and permeability of the intestinal mucosa, blood circulation. Enterosorbents are successfully used not only as a pathogenetic, but also as an etiotropic mono- and combination therapy for intestinal infections. This is especially important in connection with the growth of multi-resistance of microorganisms to antibiotics and chemotherapeutic drugs.

[0011] Using detoxification therapy for patients with various digestive system pathologies caused by heavy metals and organochlorine pesticides, tests were conducted to evaluate the effectiveness of using enterosorbents to reduce the level of these toxins. After a course of treatment, patients showed an effective reduction in the level of such toxic heavy metals as lead and cadmium, as well as organochlorine pesticides.

[0012] The detoxifying properties of enterosorbents can be enhanced when used together with metabolites of probiotic microorganisms. In this case, the enzymatic activity of metabolites can be increased due to their immobilization on the sorbent, which ultimately increases the effectiveness of the use of metabolites.

[0013] A combination of yeast cell walls with metabolites of spore-forming microorganisms immobilized on natural enterosorbents of the glauconite type is known (RU 2475254 C1, published 20.03.2013; IPC: A61K 35/74, A61P 1/00). This combination significantly expands the range of sorption properties of the sorbent with a prebiotic component, which is a mixture of metabolites of bacteria of the genus Bacillus and bacteria of the genus Halobacterium , as well as fragments of the membranes of yeast of the genus Saccharomyces , and methods of using this composition, including in combination with antibiotics. Both the whole membranes of yeast cells and their components, the polysaccharides themselves and their conjugates with protein, have an effect on the body. However, due to the presence of fragments of yeast cell walls in the composition, which have strong immunogenic properties, the patient may develop an allergic reaction.

[0014] The drug "Baktistatin" is known, which includes sterilized culture fluid containing metabolites of Bacillus subtilis (RU 2287335 C1, published 20.11.2006; IPC: A61K35/74; A61P1/00), a sorbent - zeolite and additional active substances. The said drug normalizes the quantitative and qualitative composition of the intestinal microflora due to selective stimulation of the growth and reproduction of bifido- and lactobacteria, the detoxifying properties of the sorbent, as well as specific antagonistic activity with respect to a wide range of pathogenic and opportunistic microorganisms. However, in addition to the beneficial metabolites of Bacillus subtilis, the whole culture liquid may also contain substances that are harmful or at least useless to the symbiotic microflora, which reduces the effectiveness of the metabiotic preparation based on the whole culture liquid.

[0015] Thus, there is a need to develop effective means for the treatment and/or prevention of gastrointestinal diseases associated with disruption of the quantitative and qualitative composition of intestinal microflora, which do not lose their effectiveness when taken simultaneously with antibiotics and have detoxifying activity.

Terms and abbreviations

[0016] Biomass is the totality of cellular components of the filtrate; it also contains spores.

[0017] 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 refers to a violation of the intestinal microflora (Khursa et al., 2017; Zhuchkova, 2015).

[0018] 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).

[0019] 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.

[0020] 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).

[0021] 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 (Firsov, 2006). In this text, the term "microflora" refers to the intestinal microflora.

[0022] Permeate is the phase that has passed through the membrane in the process of membrane separation, based on the preferential permeability of one or more components of a liquid or gas mixture, as well as a colloidal system, through the separating membrane. In this case, the delayed phase is called a concentrate (Chemical Encyclopedia, 1988).

[0023] Prebiotics are physiologically functional food ingredients that provide a beneficial effect on the human body as a result of selective stimulation of growth and/or increased biological activity of normal intestinal microflora (GOST R 52349-2005, Gibson et al., 2017).

[0024] 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).

[0025] Tangential ultrafiltration is an ultrafiltration process in which the liquid being filtered moves parallel to the membrane.

[0026] Ultrafiltration is a process of membrane separation, as well as fractionation and concentration of substances, carried out by filtering a liquid under the action of a pressure difference before and after the membrane. In this case, the size of the separated particles (i.e. particles of what size do not pass through the filter, but remain in the concentrate) is approximately 0.001-0.05 µm (5-500 kDa).

[0027] Enterosorbents are medicinal products of various structures that bind exogenous and endogenous substances in the gastrointestinal tract by adsorption.

[0028] GIT – gastrointestinal tract.

[0029] kDa – kilodalton.

[0030] B. subtilis - Bacillus subtilis.

[0031] E. coli - Escherichia coli .

[0032] L. reuteri - Lactobacillus reuteri.

[0034] CD64 is a membrane protein Fc-gamma receptor 1, a monocyte marker, and also a proinflammatory marker of macrophages.

[0035] IL1b – interleukin 1 beta, a proinflammatory cytokine.

[0036] IL6 – interleukin 6, pro- and anti-inflammatory cytokine, regeneration factor, macrophage activating factor, lymphocyte stimulating factor.

[0037] IL10 – interleukin 10, an anti-inflammatory cytokine.

[0038] MRSA – methicillin-resistant strains of S. aureus.

[0039] RI – retention index.

[0040] RT – retention time.

[0041] TNFa – tumor necrosis factor alpha, macrophage activating factor.

[0042] TGFb – transforming growth factor beta, blocks the activation of macrophages, suppresses the inflammatory immune response.

The essence of the invention

[0043] The objective of the present invention is to obtain a means for the treatment and/or prevention of diseases associated with intestinal dysbacteriosis and/or intoxication of the body.

[0044] This problem is solved by the claimed invention by achieving such a technical result as the creation of a composition that modulates the intestinal microflora, has detoxifying activity and immunomodulatory potential, and does not lose its effectiveness when taken simultaneously with antibiotics. Also an important part of the technical result is the development of an accessible method for obtaining the claimed composition.

[0045] The claimed technical result is achieved due to the fact that the claimed composition includes a complex of metabolites of at least one probiotic strain of B. subtilis bacteria or bacteria of the Lactobacillaceae family, as well as a sorbent. In this case, the ratio of components in wt. %:

- a complex of metabolites of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family – 0.1-7.0;

- sorbent – 93.0-99.9.

[0046] The metabolite complex includes all substances obtained from the culture fluid of one or more probiotic strains of B. subtilis bacteria and/or one or more probiotic strains of the Lactobacillaceae family.

[0047]In one embodiment, the metabolite complex further comprises inactivated vegetative cells and spores of the probiotic strain.B. subtilis. In another embodiment, the metabolite complex additionally includes inactivated cells of bacteria of the familyLactobacillaceae.In another embodiment, the metabolite complex additionally includes inactivated vegetative cells and spores of the probiotic strain.B. subtilisand inactivated cells of bacteria of the familyLactobacillaceae.In a preferred embodiment, the inactivated vegetative cells and spores of the probiotic strainB. subtilisand inactivated cells of bacteria of the familyLactobacillaceaeare dried. This allows the composition to be created in dry form, which facilitates its storage and transportation.

[0048] Metabolites of B. subtilis are capable of selectively suppressing the growth of a wide range of pathogenic and opportunistic microorganisms, have immunomodulatory potential, and metabolites of bacteria of the Lactobacillaceae family create conditions of the natural environment of normal intestinal microflora.

[0049] In this case, the presence of metabolites of at least one of the said probiotic microorganisms is sufficient in the claimed composition, since the metabiotic components necessary for the effectiveness of the composition have, in general, a similar effect on the intestinal microflora. A combination of metabiotics of several strains will allow the effect of the composition to be expanded depending on the purposes of a specific drug, which will be based on the described composition.

[0050]As probiotic strainsB. subtilisstrains can be usedB. subtilisVKPM No. B-2335 (3),B. subtilis534,B. subtilis TPI 13 - 06/07/21 - DEP/VGNKI, B. subtilisVKPM No. B4759 (3),B. subtilisr VMV105,B. subtilisTPI 9;B. subtilisrBMV 105. In a preferred embodiment, the probiotic strainB. subtilisrepresentsB. subtilisVKM B-3536D or VKPM No. B-2335 (3). As probiotic strains bacteria of the familyLactobacillaceaestrains can be usedL. reuteriDSM-17648 or ATCC-55730, SALR01, GMNL-0902, and strainL. plantarum -VKPM B-11342 or SALP01.

[0051] The sorbent is one or more silicate and/or aluminosilicate minerals that can help maintain the integrity of the composition, maintain the biological activity of the said metabolites and/or can enhance the beneficial effect of the composition due to its own sorption properties. The sorbent used in this composition acts as an enterosorbent - a substance capable of binding exogenous and endogenous compounds in the gastrointestinal tract, as well as supramolecular structures and cells, thereby providing the detoxifying activity of the composition. In a preferred embodiment, silicate or aluminosilicate sorbents such as smectite, bentonite, zeolite or a mixture thereof are used as the sorbent.

[0052] In a preferred embodiment, the described complex of metabolites is immobilized on a sorbent. This ensures a gradual release of metabolites from the sorbent surface, which allows maintaining the activity of the composition in the gastrointestinal tract for a long time.

[0053] 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 dysbacteriosis for a wide range of patients. Due to the presence of the said metabolites of probiotic microorganisms, 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 intestinal microflora. In addition, the claimed composition has detoxifying properties due to the presence of a sorbent.

[0054] The technical result is also achieved by developing an accessible method for obtaining a composition based on metabolites of at least one probiotic strain of B. subtilis bacteria or bacteria of the Lactobacillaceae family. The method includes the following steps:

a) growing bacteriaB. subtilisand/or bacteria of the familyLactobacillaceaein 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 30-40°C with constant stirring. In this case, the cultivation of bacteria of the genusBacillusare carried out during aeration and finish when the pH of the culture suspension reaches 8.5-8.9. This pH corresponds to the maximum concentration of bacterial metabolitesB. subtilis.Completion of the process of culturing bacteria of the familyLactobacillaceaeconsider the absence of a drop in pH from the set value. pH is stable under optimal conditions for culturing bacteria of the familyLactobacillaceae, when bacteria exit the exponential growth phase, the acidity index decreases. This method of cultivation allows the most efficient use of equipment for obtaining bacterial metabolitesB. subtilisand/or bacteria of the familyLactobacillaceae;

b) centrifuging the culture suspension in a flow centrifuge to obtain a culture fluid and sediment, including vegetative cells and spores of B. subtilis and/or cells of bacteria of the Lactobacillaceae family, or carrying out ultrafiltration to separate the culture fluid from the cell sediment;

c) then ultrafiltration of the obtained culture liquid is carried out with a certain cut-off threshold to concentrate the metabolites, and also, if a fraction of metabolites of a certain molecular weight is required, to obtain a permeate;

d) the obtained metabolites are dried on a sorbent until the metabolite content in the final composition is in the range of 0.1-7.0% by weight.

In a preferred embodiment, the metabolites are dried on a sorbent by lyophilization.

[0055] In some embodiments, the claimed method further comprises mixing the metabolites with inactivated vegetative cells and spores of B. subtilis bacteria and/or inactivated cells of bacteria of the Lactobacillaceae family. In this case, the method comprises several additional steps:

d) inactivate vegetative cells and bacterial sporesB. subtilisand/or bacterial cellsLactobacillaceaeby heating the precipitate obtained in step b). In this case, the bacterial cells and sporesB. subtilisinactivated at a temperature of 60-140°C for 5-120 minutes; cells of a probiotic strain of bacteria of the familyLactobacillaceaeinactivated at 60-120°C for 20-40 minutes;

In some embodiments, the claimed method further comprises mixing the metabolites with inactivated vegetative cells and spores of B. subtilis bacteria and/or inactivated cells of bacteria of the Lactobacillaceae family. In this case, the method comprises several additional steps:

e) mixing the metabolites and inactivated vegetative cells and spores of B. subtilis bacteria and/or inactivated cells of Lactobacillaceae bacteria. The method may further include drying the inactivated vegetative cells and spores of B. subtilis bacteria and/or inactivated cells of Lactobacillaceae bacteria. In a preferred embodiment, the inactivated cells are dried by lyophilization on a sorbent. For this, the same sorbent is used as for drying the metabolites. In this case, the sorbent may be smectite, bentonite, zeolite, or a mixture thereof. In one embodiment, the inactivated vegetative cells and spores of B. subtilis bacteria and/or inactivated cells of Lactobacillaceae bacteria are dried separately from the metabolites with a molecular weight of 5-300 kDa. In a preferred embodiment, the vegetative cells and spores of B. subtilis bacteria and/or inactivated cells of Lactobacillaceae bacteria are first mixed with the metabolites obtained after concentration by ultrafiltration in step c), and then the obtained metabolites are jointly dried on a sorbent until the metabolite content in the final composition is in the range of 0.1-7.0% by weight.

[0056] The claimed method provides for obtaining a composition comprising a wide range of metabolites of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family, as well as a sorbent. The claimed method provides for obtaining metabolites in quantities necessary for the production of the claimed composition on an industrial scale.

[0057] In a preferred embodiment, all components are used in dry form to obtain the composition. This increases the shelf life of the composition without loss of 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 unwanted living microorganisms.

Description of drawings

[0058] Fig. 1 shows the results of a study of the immunomodulatory properties of a complex of B. subtilis metabolites.

Detailed description of the invention

[0059] 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.

[0060] 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.

[0061] The claimed composition normalizes the quantitative and qualitative composition of microflora in the intestine, selectively inhibiting the growth of pathogenic and opportunistic microorganisms, has detoxifying properties due to the action of the sorbent and creates conditions for the growth of symbiotic microflora.

[0062] The claimed composition includes a complex of metabolites of probiotic strains of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family, as well as a sorbent . In this case, the ratio of components in wt. %:

- a complex of metabolites of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family – 0.1-7.0;

- sorbent – 93.0-99.9.

[0063] The complex of metabolites includes metabolites obtained from the culture fluid of at least one probiotic strain of B. subtilis bacteria or bacteria of the Lactobacillaceae family. The said probiotic microorganisms produce various enzymes and coenzymes, polypeptides, prebiotic components that promote the growth of symbiotic microflora, affect metabolic processes and have an immunomodulatory effect (Example 6).

[0064] Metabolites of B. subtilis and bacteria of the Lactobacillaceae family include a variety of biologically active substances that have antimicrobial, probiotic, and immunomodulatory effects.

[0065] For example, amikumacins produced by B. subtilis are active against antibiotic-resistant strains of S. aureus (MRSA – methicillin-resistant S. aureus strains), 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 . (Maksimova et al., 2021; Kaspar et al., 2019; Park et al., 2016; Prokhorova et al., 2016; Tosco et al., 2015; Hamdache et al., 2011).

[0066] Also, the metabolites of B. subtilis include cyclic dipeptides (2,5-diketopiperazines), which exhibit antitumor, neuroprotective, and antihyperglycemic activities. In addition, cyclic dipeptides exhibit antiviral, antifungal, and antibacterial activities. 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 . 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).

[0067] 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).

[0068] 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).

[0069] 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).

[0070] 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).

[0071] 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).

[0072] 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).

[0073] 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).

[0074] 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 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 commensal (probiotic) microbiota in the intestine. In addition, lysine helps maintain the immune system (Levine and Lohinai, 2021; Lin et al., 2017).

[0075] Bacteriocins are present in the culture fluid of bacteria of the family Lactobacillaceae as metabolites. They are cationic and amphipathic small molecules with antagonistic activity against microorganisms closely related to the producer strain. These metabolites belong mainly to the families of organic acids, ketones, alcohols, amino acids and monosaccharides. Among the most common bacterial metabolite molecules are lactate, mannitol, glycine, betaine, acetate, ethanol, phenylalanine, formate, uridine and isoleucine, as well as alanine and valine. Some of these molecules are initially present in the culture medium; others are natural metabolites of lactobacilli (e.g. acetate and ethanol), while some are secondary products of metabolism.

[0076] When dried cells are added to the composition, all possible metabolites contained in the cells, including substances that make up the bacterial cell walls, are additionally introduced into the final product in small quantities. In some embodiments, the metabolite complex includes narrower fractions of metabolites isolated from the culture fluids of the said probiotic microorganisms. For example, in some embodiments, the metabolite complex is represented by substances with a molecular weight of 5-100 kDa.

[0077] In one embodiment, the metabolite complex consists of metabolites of at least one probiotic strain of B. subtilis bacteria or bacteria of the Lactobacillaceae family. In another embodiment, the metabolite complex consists of metabolites of more than one probiotic strain of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family. In a preferred embodiment, the metabolite complex includes metabolites of one or more probiotic strains of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family, and also includes inactivated vegetative cells and spores of a probiotic strain of B. subtilis bacteria and/or inactivated cells of bacteria of the Lactobacillaceae family.

[0078] In this case, the inactivated vegetative cells and spores of the probiotic strain of bacteria B. subtilis , as well as the inactivated cells of bacteria of the Lactobacillaceae family, are non-living, for example, killed by heat treatment. Accordingly, the inactivated vegetative cells and spores of the probiotic strain of bacteria B. subtilis , as well as the inactivated cells of bacteria of the Lactobacillaceae family, are not capable of reproduction and do not compete with the normal microflora of the patient, while performing an antagonistic role with respect to a number of pathogenic bacteria.

[0079] 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 p VMV105, B. subtilis TPI 9; B. subtilis pVMV 105. In a preferred embodiment, the probiotic strain of B. subtilis is B. subtilis VKM B-3536D and/or VKPM No. B-2335 (3).

[0080]As probiotic strains bacteria of the familyLactobacillaceaestrains can be usedL. reuteriDSM-17648 or ATCC-55730, SALR01, GMNL-0902, and strainsL. plantarum -VKPM B-11342 or SALP01.

[0081] The use of the said metabolite complex 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 the metabolite complex 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. In a preferred embodiment, the metabolite complex is immobilized on a sorbent. Due to this, the biologically active substances contained in the metabolite complex are released from the porous surface of the sorbent gradually during movement through the gastrointestinal tract. This allows maintaining the activity of the metabolites in the gastrointestinal tract for a long time.

[0082] The sorbent is one or more silicate minerals, including aluminosilicate minerals. In a preferred embodiment of the composition, the sorbent is smectite, bentonite, zeolite or a mixture thereof. The sorbent has a large active surface area, high adsorption capacity with respect to toxins, and selectivity of action with a minimum loss of essential microelements. The sorbent used is not destroyed in the gastrointestinal tract, is non-toxic, does not cause allergic reactions, and has a neutral taste. The sorbent helps maintain the integrity of the composition, preserve the biological activity of the substances included in the metabolite complex, and can also enhance the effect of the composition due to its own sorption properties.

[0083] In a preferred embodiment, all components of the claimed composition are dried to such a state that their moisture content is no more than 6% by weight. This makes it possible to create a dry composition suitable for long-term storage and convenient for transportation.

[0084] 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 urinary tract diseases in a wide range of patients. In addition, inactivated vegetative cells and spores of B. subtilis or inactivated cells of bacteria of the Lactobacillaceae family do not create competition for the intestinal microflora, which is unique for each patient, and accordingly, do not disturb its balance.

[0085] The claimed composition can be used for the manufacture of preparations that include additional components.

[0086] The claimed composition can be used for the treatment and prevention of intestinal dysbacteriosis of various origins: in chronic diseases of the digestive tract, after acute intestinal infections, during and after taking antibiotics, after chemotherapy, during long-term hormonal therapy, in conditions of chronic stress, with an irrational diet.

[0087] The claimed composition can be used to produce a food additive or a pharmaceutical preparation in the form of a powder, emulsion, tablet or capsule. The latter form is the most convenient for use. In this case, the capsule shell can be made of gelatin.

[0088] The method for producing the claimed composition includes the following steps. In step a), one or more probiotic strains of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family (Example 4) are submerged 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 for avoiding undesirable foaming, and on the other hand, allows for the most efficient use of the equipment for producing a culture suspension saturated with metabolites of B. subtilis bacteria and/or bacteria of the Lactobacillaceae family. Submerged cultivation is carried out at a temperature of 30-40°C with constant stirring. When cultivating B. subtilis bacteria with constant aeration, submerged cultivation is carried out until the pH of the culture suspension reaches 8.5-8.9 and/or until maturation reaches more than 90% . When cultivating bacteria of the Lactobacillaceae family, submerged cultivation is completed when the pH does not drop from the set value while the bacteria are in the exponential growth phase. When leaving the exponential growth zone, the pH drops.

[0089] At step b), the culture suspension is centrifuged in a flow centrifuge or ultrafiltered until a transparent supernatant is obtained, which is the culture liquid. In this case, the main amount of cells and spores are separated from the culture liquid, including metabolites.

[0090] In step c), the culture fluid obtained in step b) is concentrated by ultrafiltration. In a preferred embodiment, tangential ultrafiltration is used with a concentration degree of 10-15 times. The ultrafiltration process is completed when the filtration rate is reduced by 2-3 times. Any suitable filter membranes with a first cutoff threshold can be used for ultrafiltration. The first cutoff threshold can vary depending on the nature of the activity of the target product. In some embodiments, the first cutoff threshold is 100 kDa. As a result, a permeate is obtained that includes metabolites with a molecular weight of no more than the established cutoff threshold. Ultrafiltration makes it possible to remove low-molecular toxic substances that may end up in the final composition when using the entire culture fluid. In step d), the obtained metabolites are jointly dried on a sorbent until the metabolite content in the final composition is in the range of 0.1-7.0% by weight. During this process, the primary immobilization of active components on the silicate sorbent occurs. Preparation of the sorbent before immobilization of metabolites includes grinding, sifting, calcination, removal of magnetic impurities. The size of the particles used in the composition is in the range: 0.005-0.5 mm.

[0091]In some embodiments, the method further comprises step e), where vegetative cells and spores are inactivated.B. subtilisand/or inactivate cellsLactobacillaceae. Inactivation of vegetative cells and bacterial sporesB. subtilisand/or bacterial cellsLactobacillaceaeis carried out by incubating the sediment obtained in step b) at a temperature of 60-140°C for 5-120 minutes; cells of the probiotic strain of bacteria of the familyLactobacillaceaeinactivated by treating the sediment obtained at the stage of centrifugation of the culture suspension with heat treatment at 60-120°C for 20-40 minutes.

[0092] In some embodiments, the method includes step d), wherein the inactivated B. subtilis cells and spores and/or Lactobacillaceae cells are dried.

[0093] In one embodiment, all components of the claimed composition are dried together. In another embodiment, part of the composition is dried separately from each other, and then mixed together in dry form. In yet another embodiment, after mixing all components of the composition, regardless of their degree of moisture, final lyophilization drying is carried out. As a result of drying, a composition is obtained whose moisture content is no more than 6% by weight. Drying of individual components or the entire composition increases the shelf life of the finished composition, and also simplifies the storage and transportation of both individual components and the entire claimed composition.

[0094] The obtained metabolites can be lyophilized by standard methods known in the art. The lyophilized metabolites can be used to obtain the claimed composition in dry form. Lyophilization extends the shelf life of both the metabolites themselves and the claimed composition in dry form. In a preferred embodiment, the composition is obtained in dry form, for example, in the form of powder, capsules, tablets.

EXAMPLES OF IMPLEMENTATION OF THE INVENTION

Example 1. Study of the detoxifying activity of the claimed composition in case of intoxication with Clostridium septicum toxins

[0095] In this example, the antitoxic effect of the claimed composition, including a complex of B. subtilis metabolites immobilized on zeolite, was investigated. The complex of metabolites represented all metabolites obtained from the culture liquid of B. subtilis. In this case, the content of zeolite in the composition did not exceed 99.9% by weight.

[0096] The antitoxic effect was studied on white mice that were administered a complex of C. septicum ( Clostridium septicum ) toxins. Clostridium septicum is an anaerobic pathogen that is the main causative agent of gas gangrene in patients with immunosuppression, malignant intestinal neoplasms, or oncohematological diseases (Thomas et al., 2021). The development of pathology is caused by the action of a complex of specific toxins produced by these bacteria. The complex of known C. septicum toxins includes 4 biotoxins with different mechanisms of action (Bryant & Stevens, 2015).

[0097] The study used a complex of C. septicum type A biotoxins in quantities of 2 and 20 half-lethal doses (LD ₅₀ ). The toxins were administered to the experimental animals into the tail vein in a volume of 0.2 ml. The animals were observed for 4 days from the moment of toxin administration, with the number of live and dead animals recorded daily. The animals used were obtained from the Rappolovo nursery of the Russian Academy of Sciences (Leningrad Region). A total of 53 mice weighing 16-18 g were used.

[0098] The claimed composition was dissolved in a physiological solution at a concentration of 100 μg/ml and administered to animals in a volume of 0.3 ml, 24 hours before the introduction of the toxin. The criterion characterizing the effectiveness of the composition was an increase in the survival rate (%) of animals receiving the combination, in relation to the control animals that did not receive the composition. The percentage of surviving animals in the experimental and control groups was determined according to the tables of V.S. Genes (Genes, 1964). The results obtained are presented in Table 1.

Table 1. The effect of the claimed composition on the resistance of white mice to poisoning with type A toxin of C. septicum .

Group Substances introduced Number of animals Survival, X (X-tm÷X+tm), % Composition Composition + Toxins (2*) 20 60 (36-81) Composition Composition + Toxins (20*) 13 54 (25-81) Control Toxins (2*) 10 0 (0-31) Control Toxins (20*) 10 0 (0-31)

* - LD ₅₀ dose

[0099]The results obtained show that the toxin dose is 2 LD₅₀resulted in an average 100% lethality, however, the introduction of the claimed composition before the introduction of toxins increased the survival rate of mice to 60%. These data indicate that the claimed composition has an antitoxic effect. Moreover, this effect slightly depends on the infecting dose of the toxin complex: with a 10-fold increase in dose (up to 20 LD₅₀) the antitoxic effect decreased by only 6%.

[00100] Thus, the claimed composition has an antitoxic effect in case of intoxication with Clostridium septicum type A toxin.

Example 2. Study of the detoxifying activity of the claimed composition in case of intoxication with Clostridium novyi (oedematiens) toxins

[00101] In this example, the antitoxic effect of the claimed composition, including a complex of B. subtilis metabolites immobilized on zeolite, was investigated. The complex of metabolites represented all metabolites obtained from the culture liquid of B. subtilis. In this case, the content of zeolite in the composition did not exceed 99.9% by weight.

[00102]Antitoxic effect the stated composition were studied on 2 types of animals – white mice and guinea pigs. The animals were obtained from the Rappolovo nursery of the Russian Academy of Sciences (Leningrad Region). A total of 60 mice weighing 16-18 g and 60 guinea pigs weighing 200-250 g were used.

[00103] Animals were injected with type A toxin produced by Clostridium novyi (oedematiens) ( C. novyi ). This toxin is the most common causative agent of gas gangrene in humans and malignant edema in various animal species. The mortality rate for gas gangrene caused by C. novyi is at least 50% (Kapustin, 2019).

[00104] In this study, a complex of C. novyi type A biotoxins was used in quantities of 2 and 20 half-lethal doses (LD ₅₀ ). The toxins were administered to experimental animals into the tail vein in a volume of 0.2 ml (mice) or intraperitoneally in a volume of 0.5 ml (guinea pigs). The animals were observed for 4 days from the moment of toxin administration, with the number of live and dead animals recorded daily.

[00105] The claimed composition was dissolved in a physiological solution at a concentration of 100 μg/ml and administered to animals in a volume of 0.3 ml, 24 hours before the introduction of toxins. The criterion characterizing the effectiveness of the combination was an increase in the survival rate (%) of animals receiving the kmposition, in relation to the control animals that did not receive the composition. The percentage of surviving animals in the experimental and control groups was determined according to the tables of V.S. Genes (Genes, 1964). The results obtained are presented in Table 2.

Table 2. Effect of the claimed composition on the resistance of white mice and guinea pigs to poisoning with type A toxin C. novyi

Group Animal species Substances introduced Number of animals Survival, X (X-tm÷X+tm), % Composition Mice Composition + Toxins (2*) 20 95 (75-100)** Composition Mice Composition + Toxins (20*) 20 60 (36-81) Composition Guinea pigs Composition + Toxins (2*) 20 90 (56-100)** Composition Guinea pigs Composition + Toxins (20*) 20 50 (21-79) Control Mice Toxins (2*) 10 0 (0-31) Control Mice Toxins (20*) 10 0 (0-31) Control Guinea pigs Toxins (2*) 10 0 (0-31) Control Guinea pigs Toxins (20*) 10 0 (0-31)

* - LD ₅₀ dose

** - differences with the corresponding control are significant at p<0.05.

[00106] The obtained results show that a dose of toxins of 2 LD ₅₀ resulted in an average 100% lethality both in mice and in guinea pigs. At the same time, the introduction of the composition before the introduction of toxins increased the survival rate for this dose to 95% in mice and 90% in guinea pigs. These data indicate that the claimed composition has an antitoxic effect in case of intoxication with C. novyi type A toxin.

Example 3. Study of the detoxifying activity of the claimed composition, including zeolite, in case of intoxication with the alpha-toxin of Clostridium perfringens type A

[00107] In this example, the antitoxic effect of the claimed composition, including a complex of B. subtilis metabolites immobilized on zeolite, was investigated. The complex of metabolites represented all metabolites obtained from the B. subtilis culture liquid. The zeolite content in the composition did not exceed 99.9% by weight.

[00108] The antitoxic effect of the claimed composition was studied on 2 animal species – white mice and guinea pigs. The animals were obtained from the Rappolovo nursery of the Russian Academy of Sciences (Leningrad Region). A total of 60 mice weighing 16-18 g and 60 guinea pigs weighing 200-250 g were used.

[00109] Animals were injected with alpha-toxin produced by Clostridium perfringens ( C. perfringens ). This toxin is the most common cause of post-traumatic gas gangrene, the symptoms of which develop within 24 hours. C. perfringens alpha-toxin also causes food poisoning in humans and animals, and necrotizing enteritis and mastitis in animals (Goossens et al., 2017).

[00110] The study used alpha-toxin of C. perfringens type A in quantities of 2 and 20 half-lethal doses (LD ₅₀ ). The toxin was administered to experimental animals into the tail vein in a volume of 0.2 ml (mice) or intraperitoneally in a volume of 0.5 ml (guinea pigs). The animals were observed for 4 days from the moment of toxin administration, with the number of live and dead animals noted daily.

[00111] The claimed composition was dissolved in a physiological solution at a concentration of 100 μg/ml and administered to animals in a volume of 0.3 ml, 24 hours before the introduction of the toxin. The criterion characterizing the effectiveness of the combination was an increase in the survival rate (%) of animals receiving the combination, in relation to the control animals that did not receive the composition. The percentage of surviving animals in the experimental and control groups was determined according to the tables of V.S. Genes (Genes, 1964). The results obtained are presented in Table 3.

Table 3. Effect of the declared composition on the resistance of white mice and guinea pigs to poisoning with C. perfringens alpha-toxin

Group Animal species Substances introduced Number of animals Survival, X (X-tm÷X+tm), % Composition Mice Composition + Toxins (2*) 20 100 (69-100)** Composition Mice Composition + Toxins (20*) 20 60 (36-81) Composition Guinea pigs Composition + Toxins (2*) 20 100 (69-100)** Composition Guinea pigs Composition + Toxins (20*) 20 50 (21-79) Control Mice Toxins (2*) 10 0 (0-31) Control Mice Toxins (20*) 10 0 (0-31) Control Guinea pigs Toxins (2*) 10 0 (0-31) Control Guinea pigs Toxins (20*) 10 0 (0-31)

* - LD ₅₀ dose

** - differences with the corresponding control are significant at p<0.05.

[00112] The obtained results show that the alpha-toxin dose of 2 LD ₅₀ resulted in an average 100% lethality in both mice and guinea pigs. At the same time, the introduction of the composition before the introduction of the toxin increased the survival rate for this dose to 100% in both mice and guinea pigs. These data indicate that the claimed composition has an antitoxic effect in case of intoxication with C. perfringens alpha-toxin.

Example 4. Study of the detoxifying activity of the claimed composition, including bentonite or smectite, in case of intoxication with the alpha-toxin of Clostridium perfringens

[00113] In this example, the effects of combining other sorbents - bentonite or smectite with a complex of B. subtilis metabolites on the resistance of white mice to poisoning with C. perfringens alpha-toxin were tested. The complex of metabolites was a fraction of substances with a molecular weight of 5-100 kDa, obtained from the culture liquid of B. subtilis. The content of the sorbent in the composition did not exceed 99.9% by weight.

[00114] The studies were carried out in a manner similar to that described in Example 3. The effect of the compositions including bentonite or smectite was similar to the effect of the composition with zeolite.

Table 4. Effect of a combination of bentonite and a complex of B. subtilis metabolites (composition) on the resistance of white mice to poisoning with C. perfringens alpha-toxin

Group Animal species Substances introduced Number of animals Survival, X (X-tm÷X+tm), % Composition Mice Composition + Toxins (2*) 20 100 (69-100)** Composition Mice Composition + Toxins (20*) 20 60 (36-81) Control Mice Toxins (2*) 10 0 (0-31) Control Mice Toxins (20*) 10 0 (0-31)

* - LD ₅₀ dose

** - differences with the corresponding control are significant at p<0.05.

Table 5. Effect of a combination of smectite and B. subtilis metabolites (composition) on the resistance of white mice to C. perfringens alpha-toxin poisoning

Group Animal species Substances introduced Number of animals Survival, X (X-tm÷X+tm), % Composition Mice Composition + Toxins (2*) 20 100 (69-100)** Composition Mice Composition + Toxins (20*) 20 55 (28-82) Control Mice Toxins (2*) 10 0 (0-31) Control Mice Toxins (20*) 10 0 (0-31)

* - LD ₅₀ dose

** - differences with the corresponding control are significant at p<0.05.

[00115] Administration of the compositions prior to administration of the toxin increased the survival rate of mice for a toxin dose of 2 LD ₅₀ to 100%. This effect was observed in both the bentonite and smectite compositions. These data indicate the antitoxic effect of these compositions.

[00116] Thus, examples 1-4 reliably demonstrate the detoxifying activity of different variants of the claimed composition.

Example 5. Obtaining a complex including inactivated cells and metabolites of bacteria of the Lactobacillaceae family immobilized on zeolite, using L. plantarum as an example

[00117] The L. plantarum strain VKPM B-11342 was used to obtain the metabolite complex. The culture was grown at a temperature of 37±1°C for 24 h. The pH of the nutrient medium was adjusted to 6.8–7.0 using alkali before the start of cultivation. Cultivation was carried out in a BioR-01 fermenter in a nutrient medium volume of 30 l. The total time of biomass accumulation in the reactor was 18 h. The completion of the cultivation process was considered to be the absence of a drop in pH from the specified value.

[00118] After completion of cultivation, the culture was cooled to 10°C. The cell titer in the culture was 6×10 ⁹ CFU/ml. Then, concentration was performed on an ultrafiltration unit without the stage of separating the culture fluid from the cells by centrifugation, since a mixture of metabolite concentrate from the culture fluid and cells is required to prepare the composition. Completion of the concentration process was considered to be a decrease in the efficiency of the cassette operation by at least 5 times in terms of the centrate yield. A concentrated cell suspension was obtained.

[00119] The obtained cells were inactivated by heating at 80°C for 40 minutes. The sorbent - zeolite - was mixed with distilled water. Inactivated cells were added to the resulting mixture. CFU analysis before lyophilization showed the absence of viable cells. Lyophilization of the mixture of sorbent and inactivated cells was carried out on a TG-50 unit with standard lyophilization parameters. The output was a complex of metabolites immobilized on the sorbent, with a moisture content of 2%.

Example 6. Obtaining a complex including inactivated cells and metabolites of bacteria of the Lactobacillaceae family immobilized on zeolite, using L. reuteri as an example

[00120] The L. reuteri SALR01 strain was used to obtain a complex of metabolites and inactivated cells. The culture was grown at a temperature of 36±1°C for 24 h. Before the start of the cultivation, the pH of the nutrient medium was adjusted to 6.8–7.0 using alkali. The cultivation was carried out in an ANKUM 210 fermenter in a nutrient medium volume of 10 l. The total time of biomass accumulation in the reactor was 18 h. The completion of the cultivation process was considered to be the absence of a drop in pH from the specified value.

[00121] After completion of cultivation, the culture was cooled to 10°C. The cell titer in the culture was 6×10 ⁹ CFU/ml. The culture fluid was then separated in a GF105B flow ultracentrifuge. A sediment was obtained after the ultracentrifugation process. The titer in the sediment was 2.5°10 ¹⁰ CFU/ml. The sediment was mixed with sterile distilled water to obtain a cell suspension. The cells were inactivated by heating at 80°C for 40 minutes. The sorbent - zeolite - was mixed with distilled water. Inactivated cells were added to the resulting mixture. CFU analysis before lyophilization showed the absence of viable cells. Lyophilization of the mixture of sorbent and inactivated cells was carried out in a TG-50 unit with standard lyophilization parameters. The output was a complex of inactivated cells and metabolites immobilized on a sorbent with a moisture content of 2%.

Example 7. Obtaining B. subtilis metabolites with a molecular weight of 5-100 kDa

[00122] 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.

[00123] The B. subtilis VKM B-3536D culture suspension was centrifuged in a Sharpless AS-26 flow supercentrifuge. The resulting culture fluid was ultrafiltered in a tangential ultrafiltration unit in two stages. An AP-3-100 filter membrane was used in the first stage. This yielded concentrate No. 1 with a cutoff threshold of 100 kDa, including a high-molecular fraction of metabolites weighing more than 100 kDa. The permeate obtained after the first ultrafiltration stage was again subjected to tangential ultrafiltration using an AP-3-5 filter membrane. This yielded concentrate No. 2 with a cutoff threshold of 5 kDa. The resulting concentrate No. 2 constituted approximately 10% of the initial volume of the culture fluid. The resulting concentrate No. 2 included metabolites of B. subtilis VKM B-3536D with a molecular weight of 5-100 kDa.

Example 8. Evaluation of the immunomodulatory properties of B. subtilis metabolites with a molecular weight of 5-100 kDa

[00124] In this example, the immunomodulatory properties of metabolites with a molecular weight of 5-100 kDa of the probiotic strain B. subtilis VKM B-3536D were studied. For this purpose, lyophilized metabolites of 5-100 kDa with the addition of maltodextrin as a carrier, but not a sorbent, were added to a culture of human monocytes THP-1 cells. In this example, only the immunomodulatory properties of the metabolites of B. subtilis VKM B-3536D are considered, and not their effect on the body in combination with the maltodextrin carrier. 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 (RT-PCR), based on the expression of mRNA marker genes encoding interleukins and other factors: IL1b, IL6, IL10, 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 replicates 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 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 cytokine, high concentrations of which are also associated with an anti-inflammatory effect.

[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.

Example 9. Study of the effect of B. subtilis metabolites on the inhibition of the growth of pathogenic and opportunistic microflora

[00128] In this example, the ability of metabolites of the B. subtilis strain VKM B-3536D with a molecular weight of 5-100 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 concentrates No. 1 and No. 2, obtained earlier, 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. Then the Petri dishes 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 the indicator cultures around the area of application of the cultures, around the area of application of the concentrates indicates the antagonistic effect of the metabolites of B. subtilis VKM B-3536 in relation to the indicator cultures indicated in Table 6. The size of the zones free of indicator cultures indicates the effectiveness of the antagonistic effect of the metabolites of B. subtilis VKM B-3536.

Table 6. Growth retardation of indicator cultures exposed to B. subtilis metabolites

Indicator culture Growth inhibition zone outside the concentrate droplet Concentrate No. 1 Concentrate No. 2 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* Klebsiella oxytoca cl.is. - - Pseudomonas spp. cl.is. - -

* – bacteriostatic action

"-" - no growth retardation was observed

[00129] It is evident from Table 6 that both high-molecular metabolites weighing more than 100 kDa (concentrate No. 1) and metabolites weighing 5-100 kDa (concentrate 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 cause a delay in the growth of E. coli , while target metabolites weighing 5-100 kDa do not. Given that E. coli is a representative of the normal intestinal microflora, the absence of a suppressive effect of concentrate 2 on this culture indicates the selectivity of the action of metabolites weighing 5-100 kDa.

[00130] It is worth noting that the culture suspension of the B. subtilis strain VKM B-3536D, including all metabolites, also caused a significant delay in the growth of E. coli . Such data were obtained in a study of the antagonistic activity of the B. subtilis strain VKM B-3536D using the perpendicular streak method.

[00131] The study revealed a pronounced selective ability of the metabolites of the B. subtilis strain with a molecular weight of 5-100 kDa to suppress the growth of pathogenic and opportunistic microorganisms. 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).

[00132] The culture fluid sample was divided into sub-samples after mixing and stored frozen at –18°C until analysis. The samples were then divided into aliquots, in which mass concentrations were measured. Screening was also performed using high-performance liquid chromatography-mass spectrometry with high resolution (HR-HPLC-MS). As a result of screening using HR-HPLC-MS, the compounds presented in Table 7 were identified. The difference in theoretical mass from practical mass is 0.7 ppm, thus, reliable identification can be stated.

Table 7. 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 Fengitsin A C16 – LCD C ₇₂ H ₁₁₀ N ₁₂ O ₂₀ 1463,803 732,405 11.55 0,000 Fengitsin A C17 – LCD C ₇₃ H ₁₁₂ N ₁₂ O ₂₀ 1477,819 739,413 12.74 0,000 Fengitsin n B C16 – ZhK C ₇₄ H ₁₁₄ N ₁₂ O ₂₀ 1491,835 749.42 13.11 0,000 Fengitsin B C17 – ZhK C ₇₅ H ₁₁₆ N ₁₂ O ₂₀ 1505,85 753,428 13.62 0,000 Fengitsin B C18 – ZhK C ₇₆ H ₁₁₈ N ₁₂ O ₂₀ 1519,866 760,436 15.12 0,000

RT – Retention time, FA – fatty acid

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Claims (28)

1. A metabiotic composition for the treatment and/or prevention of diseases associated with a disruption of the composition of the gastrointestinal tract microbiota and/or intoxication of the body, comprising a complex of metabolites obtained from the culture fluid of at least one probiotic strain of Bacillus subtilis bacteria and/or bacteria of the Lactobacillaceae family, as well as an aluminosilicate sorbent, wherein the ratio of components, wt. %: - a complex of metabolites obtained from the culture fluid of at least one probiotic strain of Bacillus subtilis bacteria and/or bacteria of the Lactobacillaceae family – 0.1-7.0; - aluminosilicate sorbent – 93.0-99.9. 2. A metabiotic composition according to claim 1, comprising metabolites of the probiotic strain of bacteria Bacillus subtilis VKM B-3536D and/or strain VKPM No. B-2335. 3. The metabiotic composition according to claim 1, additionally comprising inactivated cells and spores of Bacillus subtilis bacteria. 4. The metabiotic composition according to claim 1, additionally comprising inactivated cells of bacteria of the Lactobacillaceae family. 5. The metabiotic composition according to claim 1, comprising metabolites of bacteria of at least one probiotic strain from the following series: Lactobacillus reuteri GMNL-0902, Lactobacillus reuteri DSM-17648, Lactobacillus reuteri ATCC-55730, Lactobacillus reuteri SALR01, Lactobacillus plantarum VKPM B-11342, Lactobacillus plantarum SALP01. 6. A metabiotic composition according to claim 1, characterized in that the complex of metabolites is immobilized on an aluminosilicate sorbent. 7. The metabiotic composition according to claim 1, characterized in that the aluminosilicate sorbent includes an aluminosilicate mineral selected from the group: bentonite, smectite, zeolite. 8. A metabiotic composition according to claim 1, characterized by a moisture content of no more than 6% by weight. 9. A method for producing a composition according to item 1, comprising the following steps: a) at least one probiotic strain of Bacillus subtilis bacteria and/or bacteria of the Lactobacillaceae family is grown in a liquid nutrient medium to obtain a culture suspension; b) centrifuging the culture suspension in a flow centrifuge to obtain a culture liquid and sediment, including vegetative cells and spores of the probiotic strain of Bacillus subtilis and/or cells of the probiotic strain of bacteria of the Lactobacillaceae family; c) ultrafiltration of the culture fluid is carried out to obtain a concentrate; d) dry the resulting concentrate and mix it with an aluminosilicate sorbent or dry the concentrate on an aluminosilicate sorbent. 10. The method according to claim 9, wherein the cultivation of Bacillus subtilis bacteria and/or bacteria of the Lactobacillaceae family is carried out using the deep method. 11. The method according to claim 9, wherein the cultivation of Bacillus subtilis bacteria and/or strains of bacteria of the Lactobacillaceae family is carried out in a fermenter with a filling factor of 60-70% with stirring, wherein Bacillus subtilis is aerated. 12. The method according to claim 9, wherein the cultivation of Bacillus subtilis and/or Lactobacillaceae family bacteria is carried out at a temperature of 30-40°C. 13. The method according to item 9, in which the concentrate is dried on an aluminosilicate sorbent to such a state that its moisture content is no more than 6% by weight. 14. The method according to item 9, wherein the concentrate is dried on an aluminosilicate sorbent by lyophilization. 15. The method of claim 9, further comprising mixing the concentrate with inactivated vegetative cells and spores of the probiotic strain of bacteria Bacillus subtilis and/or inactivated cells of the probiotic strain of bacteria of the Lactobacillaceae family. 16. The method according to claim 15, wherein the vegetative cells and spores of the probiotic strain of Bacillus subtilis bacteria are inactivated by incubating the sediment obtained at the stage of centrifuging the culture suspension at a temperature of 60-140°C for 5-120 minutes. 17. The method according to claim 15, wherein the cells of the probiotic strain of bacteria of the Lactobacillaceae family are inactivated by incubating the sediment obtained at the stage of centrifuging the culture suspension at 60-120°C for 20-40 minutes. 18. The method according to claim 16 or 17, wherein after inactivation, the vegetative cells and spores of the probiotic strain of bacteria Bacillus subtilis and/or the cells of the probiotic strain of bacteria of the Lactobacillaceae family are dried to a moisture content of no more than 6% by weight. 19. The method according to claim 9, wherein the probiotic strain used is Bacillus subtilis VKM B-3536D, and/or Bacillus subtilis VKPM No. B-2335, and/or bacteria of the Lactobacillaceae family from the following series: Lactobacillus reuteri GMNL-0902, Lactobacillus reuteri DSM-17648, Lactobacillus reuteri ATCC-55730, Lactobacillus reuteri SALR01, Lactobacillus plantarum VKPM B-11342, Lactobacillus plantarum SALP01. 20. The method according to claim 19, wherein the inactivated cells and spores are dried by lyophilization on a sorbent until the cell concentrate content in the final composition is in the range of 0.1-7.0% by weight. 21. The method according to item 9, wherein the aluminosilicate sorbent is an aluminosilicate mineral selected from the group: bentonite, smectite, zeolite. 22. The method according to item 9, in which particles of aluminosilicate sorbent with a size of 0.005-0.5 mm are used.