RU2805957C1 - Liquid symbiotic and method for obtaining it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: liquid symbiotic containing at least 10⁸ CFU/ml of living microbial cells Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 39, Limosilactobacillus fermentum 90TC-4, Escherichia coli M-17 and products of their metabolism, including neurometabolites, is proposed. A method for producing symbiotics from producer strains cultivated on a hydrolyzate-casein nutrient medium containing casein hydrolysate, sodium chloride, peptone, fructose, agar-agar, and ascorbic acid, is proposed. Three generations of producer strains are separately cultivated. The resulting three generations of biomass producer strains are mixed.

EFFECT: method makes it possible to obtain a liquid symbiotic with a wide spectrum of physiological effects and a high level of neurometabolites.

2 cl, 8 tbl, 1 ex

Description

The group of inventions relates to the field of medical and food biotechnology and can be used for the preparation of probiotics in the form of therapeutic and prophylactic drugs, dietary supplements and food products using live probiotic strains of microorganisms as starter cultures.

The subject of the patent is the probiotic “BiCoel”, which is a symbiotic in composition and a psychobiotic in its functional effects.

Symbiotics-multiprobiotics are preparations or products containing several symbiotic strains of probiotic microorganisms of one or more species.

Probiotic microorganisms are live non-pathogenic, non-toxigenic microorganisms that enter the human intestine with food, have a beneficial effect on the human body and normalize the composition and biological activity of the microbiota of the digestive tract.

Most often, bacteria of the genera Bifidobacterium and Lactobacillus are considered as probiotic microorganisms. An extensive list of requirements is imposed on probiotic microorganisms [Guidelines for the control of biological and microbiological factors. System of pre-registration preclinical study of drug safety. Selection, testing and storage of production strains used in the production of probiotics: guidelines No. 4.2.2602-10. - M.: Rospotrebnadzor. - 2011. - 80 p.; Guidelines for sanitary and epidemiological assessment of the safety and functional potential of probiotic microorganisms used for food production: guidelines No. 2.3.2.2789-10 M.: Rospotrebnadzor. 2010. 103 p.], which correspond to only individual strains, and not all representatives of these species.

Bifidobacteria perform and regulate numerous body functions. In the process of vital activity, they form organic acids, which helps establish normal intestinal pH values, prevents the proliferation of pathogenic, putrefactive and gas-forming microflora, take part in the processes of enzymatic digestion of food, enhancing the hydrolysis of proteins, ferment carbohydrates, saponify fats, dissolve fiber, stimulate intestinal motility, promote normal evacuation of intestinal contents. In addition, they perform a vitamin-forming function, synthesize B vitamins, vitamin K, folic and nicotinic acids, promote the synthesis of essential amino acids, better absorption of calcium salts, vitamin D, which, in turn, has antianemic, antirachitic and antiallergic effects [Bondarenko V .M. Molecular-cellular mechanisms of the therapeutic action of probiotic drugs // Farmateka. - 2010. - No. 2. - P. 26-32.; Bukharin O.V. Bifidoflora in human associative symbiosis / Ekaterinburg: Ural Branch of the Russian Academy of Sciences, 2014. - 212 pp.]. An important function of bifidobacteria is their participation in the formation of immunological tolerance of the body. Bifidobacteria stimulate lymphoid tissue, the synthesis of immunoglobulins, increase the activity of lysozyme and help reduce the permeability of tissue barriers to toxic products of pathogenic and conditionally pathogenic microorganisms [Yankovsky D.S. Human microbial ecology: modern possibilities for its maintenance and restoration. - K.: Expert LTD, 2005. - 362 p.; Shenderov B.A. Medical microbial ecology and functional nutrition. T. 3. Probiotics and functional nutrition. - M.: Publishing house "GRANT", 2001. - 288 p.].

Lactobacilli - in the process of fermentation of carbohydrates form short-chain fatty acids (SCFA) - lactic, acetic, butyric, propionic, in the presence of which the development of opportunistic strains, which mostly have a proteolytic type of metabolism, is inhibited [Zorina, V.V. The influence of bacteria of the genus Lactobacillus on the migratory activity of macrophages // Journal. microbiology, epidemiology and immunobiology. - 2006. - No. 6. - P. 40-44]. Inhibition of proteolytic strains is accompanied by inhibition of putrefactive processes and suppression of the formation of ammonia, aromatic amines, sulfides, and endogenous carcinogens. Thanks to the production of fatty acids, the pH of the intestinal contents is regulated [Surzhik A.V. The influence of the probiotic culture Lactobacillus rhamnosus GG on the body’s immune response // Issues of modern pediatrics. - 2009. - No. 2. - P. 54-58.; Andreeva I.V. Potential possibilities of using probiotics in clinical practice // Clinical. microbiol. antimicrobial, chemotherapy. - 2006 - T. 8, No. 8. - P. 151-172.; Kavitha, S. Antibiosis of bacteriocins with domestic Lactobacilli isolated from prepared curd // Emir. J. Food Agric. - 2010. - Vol. 22, No. 5. - P. 398-400]. It has been shown that pronounced inhibition of growth, reproduction and adhesion of pathogenic and opportunistic microorganisms depends on the presence of the entire spectrum of acids produced by the normal flora of the gastrointestinal tract. The synergism of this combination ensures inhibition of not only bacteria, but also some types of yeast, while acid-resistant normal microflora is practically not affected. The antimicrobial effect of lactic and acetic acids has been well studied. They ensure the maintenance of the pH of the intestinal contents at the level of 4.0-5.8, thereby inhibiting the growth and reproduction of opportunistic and putrefactive microorganisms in the intestine [Ermolenko E.I. Antimicrobial effect of lactobacilli // Medicine - XXI century. - 2007. - No. 5. - P. 41-49.; J. Nissen-Meyer [et al.] A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides III. Bacteriol. - 1992 - Vol. 174, No. 17 - P. 5686 - 5692], and also penetrating the membrane, they release hydroxide ion into the neutral cytoplasm, which leads to the suppression of the vital functions of the cell. Thus, acetic acid at a pH above 4.5 exhibits a more pronounced inhibitory effect than lactic acid, and, conversely, at a pH below 4.0, stronger antimicrobial activity is observed in lactic acid [V.M. Bondarenko [et al.] Probiotics and mechanisms of their therapeutic action // Experiment. and clinical gastroenterology. - 2004. - No. 3. - P. 83-87.; S.V. Sidorenko Molecular basis of antibiotic resistance // Advances in biol. chemistry. - 2004. - T. 44. - P. 263-306]. A special place among antibacterial metabolites of lactobacilli is occupied by hydrogen peroxide, it inhibits the growth of a number of microorganisms - E. coli, staphylococci, pseudo -monon, and others. Starts Lactobacillus Fermentum, Lactobacillus Casei and Lactobacillus Acidoph ILUS, which make up the normal microflora of the intestines and human vaginal, actively produce lysozyme [ Pospelova V.V. Biological characteristics of some production and freshly isolated strains of lactobacilli // Med. aspects of micro. ecology. - 1992. - Issue. 6. - pp. 54-57.; Shenderov B.A. Medical and microbial ecology and functional nutrition: in 3 volumes. T. 3: Probiotics and functional nutrition / Moscow: GRANT, 2001. - 286 pp.].

Bacteriocins and bacteriocin-like substances are considered the most significant for the implementation of the antagonistic activity of lactobacilli [L.P. Blinkova [et al.] Bacteriocins and bacteriocin-like substances as biologically active agents // Probiotics, prebiotics, synbiotics and functional foods: fundamental and clinical aspects: materials of the 9th International. Slavyano-Balt. scientific forum "St. Petersburg GASTRO-2007", St. Petersburg, May 16-19, 2007. - St. Petersburg, 2007. - P. 22.; J.R. Tagg, A.S. Dajani, L.W. Wannamaker Bacteriocins of gram-positive bacteria // Bacteriol. Rev. - 1976. - Vol. 40, no. 3. - P. 722-756].

Bacteria of the families Lactobacillaceae and Bifidobacteriaceae synthesize bacteriocins - antibiotic substances of peptide nature that kill related species or strains, inhibit their growth, or have a wider spectrum of antibacterial action [Pokhilenko, V.D. Bacteriocins: their biological role and application trends // Electronic scientific journal “Researched in Russia” - P. 164-198]. For example, lactobacilli L. plantarum and L. fermentum produce bacteriocins and bacteriocin-like substances: lantibiotics (class I) - thermostable peptides with a molecular weight of less than 5 kDa, consisting of unusual, post-translationally modified amino acids (dehydroalanine, lanthionine, α-methyllanthionine); non-lantibiotics (class II) - thermostable peptides with a molecular weight of less than 10 kDa, which do not contain lanthionine and retain their activity over a wide pH range (3-9); high molecular weight (more than 30 kDa) thermolabile peptides (class III); cationic, amphiphilic, membrane-permeable antimicrobial peptides ranging in size from 2 to 6 kDa (lipo- and glycoproteins) class IV. Thus, L. plantarum 8 RA-3 is capable of producing the lantibiotic plantaricin. Some strains of B. bifidum and B. longum synthesize bacteriocin-like substances and bacteriocins of various classes.

Metabolites produced by bifidobacteria and lactobacilli have a stimulating effect on the indigenous microflora of the macroorganism, exhibit an antimicrobial effect against representatives of pathogenic and conditionally pathogenic microflora, as well as an immunotropic effect. In addition, the exometabolites of lacto- and bifidobacteria contain stimulants and inhibitors of the growth of bacterial cultures, as well as substances that affect the survival and antagonistic activity of microbial cells.

Probiotic strains are used for the preparation of various medicines, food products, incl. Dietary supplements for food, fermented milk products and biological products, both in the form of monocomponent probiotics and as part of multicomponent consortia of bacteria belonging to different species and even genera of microorganisms.

Psychobiotics are probiotic bacteria that have beneficial effects on human mental health.

Psychobiotics are live microorganisms that, when taken in adequate quantities, improve the health of patients with psychiatric problems.

According to the latest scientific data, the symbiotic microbiota of the human digestive tract is actively involved in the regulation of the development and functioning of the brain as a result of the interaction of low-molecular compounds of microbial origin with nerve cells. Thus, it is known that bacteria are capable of both recognizing and synthesizing neurotransmitters and neuroendocrine hormones. The interaction of the intestinal microbiota with the enteric nervous system and brain of mammals has been confirmed in many works [Stilling R.M., Bordenstein S.R., Dinan T.G., Cryan J.F. Friends with social benefits: host-microbe interactions as a driver of brain evolution and development? // Frontiers in Cellular and Infection Microbiology. - 2014. - DOI: 10.3389/fcimb.2014.00147; Oleskin A.B., Shenderov B.A., Rogovsky B.S. Sociality of microorganisms and relationships in the microbiota-host system: the role of neurotransmitters. Monograph. - Moscow: Moscow University Publishing House, 2020. - 286 p. - ISBN 978-5-19-011-450-8]. A fairly serious hypothesis has been put forward that without the microbiota a person would not be able to achieve the current level of cognitive abilities [Montiel-Castro A.J., Gonzalez-Cervantes R.M., Bravo-Ruiseco G., Pacheco-Lopez G. The microbiota-gut-brain axis: neurobehavioral correlates, health and sociality // Frontiers in Integrative Neuroscience. - 2013. October. - DOI: 10.3389/fnint.2013.00070]. A number of researchers consider microbiota as a risk factor for individuals genetically predisposed to ASD; It is believed that changes in the intestinal microbiota increase the risk of developing ASD by affecting the immune system and metabolism [De Angelis M., Francavilla R., Piccolo M., De Giacomo A., Gobbetti M. Autism spectrum disorders and intestinal microbiota. Gut Microbes. - 2015. - Vol. 6? N.3 - P. 207-213. https://doi.org/10.1080/19490976.2015.1035855; Blagonravova A.S., Zhilyaeva T.B., Kvashnina D.V. Disturbances of the intestinal microbiota in autism spectrum disorders: new horizons in the search for pathogenetic approaches to therapy. Part 1. Features of the intestinal microbiota in autism spectrum disorders // Journal of microbiology, epidemiology and immunobiology. - 2021 - v. 98, no. 1 - pp. 65-72 DOI: https://doi.org/10.36233/0372-9311-62]. The hypothesis about the role of the gut microbiota in the pathogenesis of autism is supported by studies showing that the manifestation of autism is often accompanied by complaints of gastrointestinal symptoms, which are preceded by the use of antibiotics: many children with autism spectrum disorder (ASD) are often treated with antibiotics during the first 3 years of life, which , presumably destabilizes their microbiota and opens the door for competitive potential pathogens to contribute to the severity of ASD [Niehus R., Lord C. Early medical history of children with autism spectrum disorders. J.Dev. Behav. Pediatr. 2006; 27(2): SI 20-7. https://doi.org/10.1097/00004703-200604002-00010; Willing B.P., Russell S.L., Finlay B.B. Shifting the balance: antibiotic effects on host-microbiota mutualism. Nat. Rev. Microbiol. 2011; 9(4): 233-43. https://doi.org/10.1038/nrmicro2536]. That is, it can be stated that the risk of neurodegenerative diseases should be associated with a deep imbalance of the microbiota-gut-brain axis. This imbalance is largely explained by the widespread use in modern society of antibiotics and other chemotherapeutic agents, hormonal drugs, xenobiotics, etc. [Shenderov B.A., Golubev V.L., Danilov A.B., Prishchepa A.V. Human intestinal microbiota and neurodegenerative diseases // Neurology. - 2016. - No. 1. - P. 7-13].

Among the microorganisms that produce low-molecular compounds that can modify human behavioral reactions, various scientific studies include strains of Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus fermentum, etc. [Oleskin A.V., Shenderov B.A., Rogovsky B.S. Sociality of microorganisms and relationships in the microbiota-host system: the role of neurotransmitters. Monograph. - Moscow: Moscow University Publishing House, 2020. - 286 p. - ISBN 978-5-19-011-450-8]. Various strains of lactobacilli, bifidobacteria, and E. coli are capable of producing serotonin, tyramine, acetylcholine, agmatine, hydrogen sulfide, aspartate, gamma-aminobutyric acid (GABA), insulin, L-dioxyphenylalanine (DOPA). For example, L. plantarum and L. fermentum are capable of producing lactate (phenyllactate, hydroxyphenyllactate), SCFA (acetic, propionic, isobutyric, butyric (butyrate), isovaleric, valeric, isocaproic, caproic acids), formate, amino acids, ethanol, peroxide hydrogen, lysozyme, extracellular polysaccharides, etc. [Sitkin S.I., Tkachenko E.I., Vakhitov T.Ya. Metabolic dysbiosis of the intestine and its biomarkers // Experimental and clinical gastroenterology. - 2015. - No. 12 (124). - P. 6-29].

B. bifidum and B. longum synthesize lactate, SCFA (acetic, propionic acids), succinic acid, hydrogen peroxide, B vitamins, folate, lysozyme, tryptophan [Sitkin S.I., Tkachenko E.I., Vakhitov T.Ya. . Metabolic dysbiosis of the intestine and its biomarkers // Experimental and clinical gastroenterology. - 2015. - No. 12 (124). - P. 6-29].

Representatives of the E. coli species are capable of producing lactate, acetic acid (SCFA), succinic acid, ethanol, bacteriocins (colicin), vitamin K [Sitkin S.I., Tkachenko E.I., Vakhitov T.Ya. Metabolic dysbiosis of the intestine and its biomarkers // Experimental and clinical gastroenterology. - 2015. - No. 12 (124). - P. 6-29].

Neurotransmitters synthesized by microorganisms are involved in maintaining an active waking state and regulating the sleep-wake rhythm, stimulating hedonic behavior, executing voluntary movements, switching attention, regulating emotions, remembering, learning, cognitive activity, regulating appetite, pain sensitivity, alleviating anxiety and stress. [Oleskin A.V., Shenderov B.A., Rogovsky B.S. Sociality of microorganisms and relationships in the microbiota-host system: the role of neurotransmitters. Monograph. - Moscow: Moscow University Publishing House, 2020. - 286 p. - ISBN 978-5-19-011-450-8]. For example, it is known that bifidobacteria are capable of producing tryptophan, which is a precursor of serotonin, the deficiency of which subsequently causes a lack of dopamine and melatonin, resulting in the development of insomnia.

The production of biologically active metabolites is a strain characteristic of microorganisms, and therefore multicomponent probiotics have advantages over monocomponent ones. In multicomponent probiotics, each strain included in the composition has unique characteristics that determine the effectiveness of the product as a whole. For example, the possibility of taking a probiotic during antibiotic therapy is achieved by introducing into the consortium producers with chromosomal (non-transmissible) resistance to different groups of antimicrobial drugs so that there is always at least one strain resistant to a specific antibiotic. Taking into account the metabolic potential of the strains, it is possible to construct a probiotic with specified properties: having specific antagonistic activity against a certain group of microorganisms or having a set of specific metabolites that help compensate for their deficiency in specific pathologies. In addition, it is known that there are symbiotic relationships between lactic acid bacteria, in which lactobacilli can grow in associative environments poor in growth substances, mutually complementing each other’s needs. Also, when co-cultivating biocompatible strains, the goal of creating a consortium with an enhanced set of properties inherent in individual strains is achieved. The accumulation of lactic acid by some strains promotes the synthesis of probiotic substances by other strains, which enhances the antimicrobial activity of the consortium against pathogenic microflora.

The most common probiotics are those produced in dry, freeze-dried form, but a number of so-called liquid probiotics, which are suspensions in nature, are also known. Lyophilized probiotics have long shelf life (up to several years, ease of use and sale). But the greatest preservation of metabolites is ensured by the liquid form of probiotics, while during lyophilization most of the beneficial substances are destroyed. Bacteria in the liquid form of probiotics are in a physiologically active state and are able to actively colonize the intestines within 2 hours after entering the body, the structure of adhesins is not damaged, valuable bacterial metabolites are preserved in the nutritional base, in particular organic acids: acetic, lactic, vitamins B, C , K, which, when entering the intestine, change the properties of the environment in it, which has a beneficial effect on the development of its own microflora and inhibits pathogenic and opportunistic microorganisms [Bodarenko V.M., Shaposhnikova L.I. Clinical effect of liquid probiotic biocomplexes containing physiologically active cells of bifidobacteria and lactobacilli, 2007]. However, when developing liquid forms of probiotics, it is necessary to take into account their biocompatibility - the ability to coexist and function for a long time in a single nutrient substrate.

Essentially, liquid probiotics are both probiotics and metabiotics at the same time.

Metabiotics are metabolic products and structural components of probiotic microorganisms.

A more precise definition of this group was formulated by Professor B.A. Shenderov [Shenderov B.A. Microbial ecology of humans and its role in maintaining health // Metamorphoses. 2014. No. 5. pp. 72-80]: metabiotics are structural components of probiotic microorganisms and/or their metabolites, and/or signaling molecules with a certain (known) chemical structure that are capable of optimizing host-specific physiological functions, regulatory, metabolic and/or behavioral reactions associated with the activity of the indigenous microbiota of the host organism. Metabiotics begin to act in the macroorganism “here and now.” The therapeutic effect of metabiotics is due to a combination of several main actions: the ability to provide the homeostasis conditions necessary for normal interaction between the epithelium and microflora in the contact zone, as well as a direct effect on the physiological functions and biochemical reactions of the macroorganism, affecting the activity of cells and biofilms. At the same time, the body’s own microflora is stimulated.

Multi-strain probiotics containing lacto- and bifidobacteria are already known from the prior art, some of them are presented in the table (Table 1).

Probiotics are known that contain representatives of other types of microorganisms that are part of the normal microflora, such as Streptococcus spp., Enterococcus spp. etc., as well as microorganisms nonspecific to the human intestinal microbiota (for example, Bacillus cereus, Saccharomyces spp.), they are called self-eliminating antagonists. After ingestion, they pass through the gastrointestinal tract unchanged without colonization, but exhibit antagonism towards pathogenic and opportunistic microflora. The drugs are completely eliminated from the body within 2-4 days after stopping use [Novikov V.E. Pharmacological regulation of intestinal microbiocenosis // Reviews on wedge, pharmacocol. and lek. therapy. - 2009 - T. 7 - No. 2 - P. 51-57]. A list of some of these probiotics is presented in Table 2.

Preliminary studies found that children with ASD were characterized by the most frequent (p = 0.001) detection of intestinal dysbiosis in general and the detection of significant disorders (p = 0.001) in the form of grade 3-4 intestinal dysbiosis. Based on the results of a bacteriological study of the intestinal microbiota, it was found that children with ASD, compared with conditionally healthy children, were characterized by a significant decrease in the total bacterial mass of the intestinal microbiota (γ = 0.29, p-0.006); decrease in the representation of the main representatives of the phylometabolic core of the microbiota [Sitkin S.I., Tkachenko E.I., Vakhitov T.Ya. Phylometabolic core of the intestinal microbiota. Almanac of Clinical Medicine. 2015; 40: 12-34]: Lactobacillus spp (γ=0.47, p=0.001); Bifidobacterium spp. (γ=0.37, p=0.001); Bacteroides spp. (γ=0.34, p=0.002); Bacteroides thetaomicron (γ=0.33, p=0.021) Faecalibacterium (γ=0.40, p=0.001); Blautia spp. (γ=0.43, p=0.002); Eubacterium rectale (γ=0.44, p=0.001); Roseburia inulinivorans. (γ=0.41, p=0.001) and Ruminococcus spp. (γ=0.41, p=0.001). The obtained results of the analysis of the qualitative and quantitative composition of the intestinal microbiota according to polymerase chain reaction data with fluorescent detection of amplification results in real time are fully consistent with those from the analysis of the results of bacteriological research; as well as with the results of other researchers who found a significant decrease in microbial diversity in terms of the quantitative representation of individual representatives of the microbiota in ASD [Kang DW, Ilhan ZE, Isern NG, Hoyt DW, Howsmon DP, Shaffer M, et al. Differences in fecal microbial metabolites and microbiota of children with autism spectrum disorders. Anaerobe. Feb 2018; 49: 121-31. doi: 10.1016/j.anaerobe.2017.12.007.; Kang, D. W.; Park, J. G.; Ilhan, Z. E.; Wallstrom, G.; LaBaer, J.; Adams, J.W.; Krajmalnik-Brown, R. Reduced incidence of Prevotella and other fermenters in intestinal microflora of autistic children. PLoS ONE 2013, 8, e68322. doi: 10.1371/journal.pone.0068322].

The functional failure of the intestinal microbiota in ASD in relation to such short-chain fatty acids (SCFAs) as butyrate and propionate has been established. The results of metagenomic sequencing showed a significantly lower representation of butyrate producers {Coprococcus spp.), propionate and lactate producers (Fusicatenibacter spp., Sellimonas spp., Ruthenibacterium spp.) in patients with ASD. Differences were found in the microbial metabolism of fatty acids in children with ASD and healthy ones, and it was also found that Escherichia coli can act as a key resident in the regulation of the functional activity of fatty acid metabolism. In general, the data obtained indicate that in children with ASD there is a need to correct the disturbed microbiocenosis of the colon, and microorganisms of the genera Bifidobacterium spp., Lactobacillus spp. can act as probiotic producers. and Escherichia coli. Based on the data of functional profiling of microorganisms of the intestinal microbiota of healthy children and children with ASD, obtained by the authors, children with ASD show an increased content of lactic acid and glyoxylic acid and a decreased content of butyric, isobutyric, succinic, isovaleric and valeric acids, and a significant decrease in the concentration of pyruvic acid compared to healthy ones. In this regard, it is rational to include in the composition of the developed forms of symbiotics microorganisms that produce the smallest amount of lactic and glyoxylic acid (the latter acid is produced by all microorganisms in minimal quantities). Elevated levels of glyoxylic acid directly affect the production of large amounts of oxalate and, as a result, the occurrence of hyperoxaluria, which is often observed in children with autism spectrum disorders. The metabolism of oxalates is closely related to the production of pyruvic acid (in hyperoxaluria, the amount of the enzyme that converts glyoxylate into pyruvate is reduced), therefore, compositions of microorganisms that produce high concentrations of pyruvic, isobutyric, isovaleric and valeric acids are most preferable for inclusion in the composition of the symbiotic being developed.

Probiotics containing a coli component, Escherichia coli, are known from the prior art (Table 3).

To use probiotics as psychobiotics, preference should be given to the liquid form, since in addition to the microorganisms themselves, metabolites accumulate in the liquid base of probiotics, among which substances that affect the nervous system of the macroorganism (GABA, tryptophan, butyrates, folic acid, etc.) are of particular importance. , and during lyophilization most of the beneficial substances are destroyed. In addition, bacteria in the liquid form of probiotics are active immediately after administration; they do not need time to restore their physiological and metabolic functions, unlike dry probiotic preparations and dietary supplements. The production of biologically active metabolites is a strain characteristic of microorganisms, and therefore multicomponent probiotics have advantages over monocomponent ones. However, when developing liquid forms of multicomponent probiotics, it is necessary to take into account their biocompatibility - the ability to coexist and function for a long time in a single nutrient substrate.

Intraspecific antagonism of lactobacilli, bifidobacteria and Escherichia coli is a strain characteristic and is widespread. In this regard, studying the nature of the relationships between different types of probiotic microorganisms in vitro is one of the most important issues in the design of multi-strain probiotics. One of the necessary conditions that ensures the high quality of multicomponent probiotics and their good specific activity and preservation is the synergistic nature of the relationship (biocompatibility) of the strains of probiotic bacteria included in them.

In preliminary studies, the biocompatibility of 9 industrial probiotic strains was determined by co-cultivation on solid nutrient media [Glushanova N.A. Experimental substantiation of new approaches to the correction of intestinal microbiocenosis: Abstract of thesis. dis. ... doc. honey. Sci. - Moscow, 2005. - 56 p.]. In the process of creating probiotics “LB-complex PLUS” “LB-complex L” using a unified methodological approach, we proved the biocompatibility of the strains Lactiplantibacillus plantarum 8 RA 3, Limosilactobacillus fermentum 39, Limosilactobacillus fermentum 90 TC-4, and bifidobacteria Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379 [Tochilina, A.G. Biochemical and molecular genetic identification of bacteria of the genus Lactobacillus: specialty 03.00.04, 03.00.07: abstract of the dissertation for the degree of candidate of biological sciences / Tochilina Anna Georgievna. - Nizhny Novgorod, 2009. - 25 p.; Belova I.V., Tochilina A.G., Solovyova I.V., Novikova N.A., Efimov E.I., Ivanova T.P., Zhirnov V.A. The use of zeolites in the composition of immobilized multiprobiotics // Medical almanac. - 2014. - No. 2(32). - P. 74-77].

Strains Lactiplantibacillus plantarum 38 and Bifidobacterium bifidum LVA-3 were also considered promising. Using the same method, the type of relationship between E. coli M-17, Lactiplantibacillus plantarum 38, Bifidobacterium bifidum LVA-3 was studied both among themselves and with representatives of the consortium Lactiplantibacillus plantarum 8 RA-3, Limosilactobacillus fermentum 39, Limosilactobacillus fermentum 90 TC-4 , and bifidobacteria Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379.

The research results are presented in Table 4.

As can be seen from Table 4, strains Lactiplantibacillus plantarum 8 RA-3 and Limosilactobacillus fermentum 39 showed pronounced antagonism to strain Lactiplantibacillus plantarum 38 (suppressed its growth), which, in turn, demonstrated pronounced antagonism to strains Bifidobacterium bifidum 1 and E. coli M-17. The Bifidobacterium bifidum strain LBA-3 was suppressed by the Lactiplantibacillus plantarum 8 RA-3 strain and the Escherichia coli strain M-17. The conducted studies identified a possible consortium of producer strains for creating a liquid form of a multi-strain probiotic - these are Lactiplantibacillus plantarum 8 RA-3, Limosilactobacillus fermentum 39, Limosilactobacillus fermentum 90 TC-4, Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 3 79, Escherichia coli M- 17.

Metabolomic profiling of all consortium strains was carried out using high-performance liquid chromatography-mass spectrometry HPLC-MS. The results are presented in Table 5.

Probiotic compositions are prepared by methods traditional to the art, which are selected depending on the type and form of the finished preparation.

Thus, there is a known method for preparing the drug “Bifikol, lyophilisate for the preparation of a suspension for oral administration” in dry freeze-dried form, the starter cultures of which are B. bifidum 1, E. coli M-17 (FS 42-3268-99, ND LS 002258-190618, RU LS 002158), including separate cultivation of strains of bifidobacteria and Escherichia coli on different nutrient media. For the cultivation of bifidobacteria in accordance with the production regulations, the KD-5 nutrient medium of the following composition is used:

- pancreatic casein hydrolyzate with a final concentration of amine nitrogen 150±10_mg/% - 350 ml/l

- yeast autolysate - 650 ml/l

- NaCl - 5.0 g/l

- lactose - 10.0 g/l

- L-cysteine hydrochloride - 0.1 g/l

- microbiological agar - 0.75 g/l

To scale the biomass, three generations are cultivated: I generation - 48 hours, II generation - 48 hours, III generation in the reactor - 18 hours.

For cultivation of E. coli M-17 - casein broth:

- Casein hydrolyzate with an amine nitrogen content of 220-270 mg/% - 1 l

- Peptone 0.5-0.6%

- pH 7.6-7.7

To scale the biomass, three generations are cultivated: I generation - 18 hours, II generation - 18 hours, III generation in the reactor - 7 hours.

At the end of cultivation, the third generations of starter cultures are mixed in accordance with the production regulations in the ratio of 4 parts bifidobacteria and 1 part E. coli (4:1). Next, the SLM protective environment is added. The biomass is bottled and freeze-dried.

The disadvantages of this probiotic are, firstly, the lyophilized form, during which most of the beneficial metabolites are destroyed. In addition, bacteria in dry form need time to restore their physiological and metabolic functions. Secondly, the use of lactose in the medium formulation in an amount of 10.0 g/l makes it impossible to use the drug prepared using this method in people with lactase deficiency. Thirdly, the use of only strain B. bifidum 1, 1 strain of Escherichia coli and the absence of lactobacilli does not solve the problem of creating a psychobiotic, since this drug does not contain active producers of GABA, butyric, succinic, isovaleric and valeric acids, and does not make it possible to use this probiotic against the background of the use of any antibacterial drugs. Its capabilities are limited by the antibiotic resistance of two producing strains.

As a prototype, we are considering a method for preparing a therapeutic and prophylactic drug from live strains of microorganisms lactobacilli and bifidobacteria “LB-complex PLUS” (RF patent No. 2517734, 05/27/2014). The method includes co-cultivation of strains B. bifidum 791, B. longum 379 starting from the first generation and separate cultivation of B. bifidum 1; joint cultivation of lactobacilli L.plantarum 8RA-3, L.fermentum 39 starting from the first generation and separate cultivation of L.fermentum 90-TC-4. After 24 hours of cultivation, the first generation biomass is mixed with fresh nutrient medium and continued to be cultivated for 48 hours. At the end of cultivation, the resulting biomass is mixed in a ratio of 2:1:2:1. The method involves the use of two hydrolyzate-casein media GKS-L for lactobacilli and GKS-B for bifidobacteria, prepared on the basis of one intermediate product - casein hydrolyzate - with an amine nitrogen level of 450-500 mg%. The media differ from each other: the level of amine nitrogen is 160-170 mg% and 180-200 mg%, the agar content is 0.75±0.1 g and 1.0±0.1 g, the pH level is 7.8-8.0 and 8.5-8.6 respectively. Fructose is used as the carbohydrate component of both media in an amount of 10.0±0.1 g/l. The finished drug is packaged in vials taking into account the required daily dose. The method makes it possible within 72 hours to obtain a ready-to-use preparation with a high content of living microbial cells that affects the lacto- and bifidocomponents of the human microflora.

The disadvantage of this method is the inability to change the ratio of the number of individual strains in the finished product depending on their metabolic activity. The two-stage accelerated cultivation method does not allow the metabolic potential of the selected producer strains to be fully realized. The probiotic “LB-complex PLUS”, obtained by the method considered as a prototype, does not contain E. coli, as a result of which it does not contain a sufficient amount of neurometabolites (GABA, butyric, succinic, valeric acids, etc.) necessary for its use by patients with cognitive impairments, including autism spectrum disorders (ASD).

These shortcomings are eliminated by the proposed technical solution.

Problem solved: creation of a liquid multicomponent bifido-, lacto- and coli-containing symbiotic with a high level of neurometabolites produced by seven strains included in the consortium, and a method for its preparation for the correction of intestinal microbiota and cognitive impairment, including autism spectrum disorders.

The technical result of the proposed technical solutions is the creation of a liquid symbiotic from seven living strains of bifidobacteria, lactobacilli and E. coli M-17, which contains an increased level of neurometabolites compared to the "LB-complex PLUS" such as GABA, butyrate, pyruvic acid, succinic acid, isobutyric, isovaleric and valeric acids with reduced levels of lactic and glyoxylic acids, which makes it possible to use it to normalize the intestinal microbiota and correct cognitive impairment, including autism spectrum disorders.

The specified technical result is achieved by a liquid symbiotic, including biomass of strains Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 39, Limosilactobacillus fermentum 90TC-4, Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379, and Escherichia coli M-17 with content of CFU/ml not less than 10 ⁸ in the following percentages: Lactiplantibacillus plantarum 8RA-3 - 14-16 vol. %, Limosilactobacillus fermentum 39 - 14-16 vol. %, L. fermentum 90-TC-4 - 15-17 vol. %, Bifidobacterium bifidum 1 - 15-17 vol. %, B. bifidum 791 - 16-18 vol. %, B. longum 379 - 13-15 vol. %, Escherichia coli M-17 - 6-8 vol. % and metabolic products of all producer strains.

The specified technical result is also achieved by the fact that the method of obtaining it, including the use of producer strains Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 90-TC-4, Limosilactobacillus fermentum 39, Bifidobacterium bifidum 1, Bifidobacterium bifidum 791 and Bifidobacterium longum 379, their cultivation on hydrolyzate-casein nutrient medium containing casein hydrolyzate, sodium chloride, peptone, fructose, agar-agar, ascorbic acid as a nutrient base, consists of using Escherichia coli M-17 as an additional producer strain, separately cultivating three generations of strains - producers on a hydrolyzate-casein nutrient medium, the use of MALDI TOF mass spectrometry after cultivation of the first generation, which makes it possible to confirm the purity of the strains and ensure, when using the method, the exact compliance of the manufactured drug with the declared one, mixing the resulting third generations of biomass producer strains Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 90-TC-4, Limosilactobacillus fermentum 39, Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Escherichia coli M-17 in percentages: Lactiplantibacillus plantarum 8RA-3 - 14-16 vol. %, Limosilactobacillus fermentum 39 - 14-16 vol. %, L. fermentum 90-TC-4 - 15-17 vol. %, Bifidobacterium bifidum 1 - 15-17 vol. %, B. bifidum 791 - 16-18 vol. %, B. longum 379 - 13-15 vol. %, Escherichia coli M-17 - 6-8 vol. % and packaging taking into account the daily dose required for patients.

The method is carried out as follows:

At the first stage, the semi-product is prepared for future use as follows: drinking water (GOST 2874-82) is heated to 48±1°C, casein (edible acid casein according to GOST 4960-74) is added in an amount of 60-70 g per liter, stirred, and the pH is adjusted up to 7.8-8.2 units with a 20% NaOH solution, place in a thermostat on a rotary shaker for mixing at a speed of 20-30 rpm at a temperature of 48±1°C for 24±1 hour. Then turn off the rocker and leave it to settle for another 24±1 hour; after keeping it in the thermostat, drain the supernatant liquid through a paper filter and set the pH to 8.2-8.5.

Two nutrient media are prepared from the resulting intermediate product: GKS and GKS-M according to the following recipes.

To prepare GCS, the whole hydrolyzate is diluted with distilled water to an amine nitrogen level of 220-230 mg%. To the diluted hydrolyzate add 5 g of sodium chloride, 2 g of peptone, 10 g of fructose, 0.25 g of ascorbic acid and 0.75 g of previously prepared standard agar-agar per 1 liter. Adjust pH to 8.1-8.3 with 20% NaOH solution.

GKS-M medium is prepared in the same way as GKS, but agar-agar is not added. Ready-made GKS and GKS-M media are sterilized using the same method at 0.5 atm for 30 minutes.

Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 90-TC-4, L. fermentum 39, Bifidobacterium bifidum 1, B. Bifidum 791, B. longum 379 and additionally Escherichia coli M-17 are used as producer strains.

Cultivation of producing strains of lactobacilli and bifidobacteria is carried out as follows.

I generation: 1 ml of GKS-M medium is added to ampoules with dry strains, then the contents of each ampoule are transferred to a separate bottle with 10.5 ml of GKS-M. Then the producing strains are cultivated for 24±1 hour at a temperature of 37±1°C. After cultivation, MALDI TOF mass spectrometry is performed to confirm the purity of the strain and the compliance of the grown strain with that stated in the formulation.

II generation: the obtained biomasses of the I generation, each separately, are mixed with the GCS nutrient medium; 10 ml of the I generation are added to 110 ml of the medium, and continue to be cultivated for 24±1 hour at a temperature of 37±1°C. After cultivation, the concentration of bacterial cells is monitored.

III generation: the obtained biomass of the II generation of each strain is separately mixed with the GCS nutrient medium; 100 ml of the II generation are added to 1000 ml of GCS, thermostated for 48 ± 1 hour at a temperature of 37 ± 1 ° C. After cultivation, the concentration of bacterial cells is monitored.

The cultivation of E. coli M17 strains is carried out in the same way as the cultivation of producer strains of lactobacilli and bifidobacteria, with the difference that the incubation duration is 18 ± 1 hour for all generations.

At the end of cultivation, the resulting biomass of all seven producer strains is mixed in the following percentage ratios: Lactiplantibacillus plantarum 8RA-3 - 14-16 vol. %, Limosilactobacillus fermentum 39 - 14-16 vol. %, L. fermentum 90-TC-4 - 15-17 vol. %, Bifidobacterium bifidum 1 - 15-17 vol. %, B. bifidum 791 - 16-18 vol. %, B. longum 379 - 13-15 vol. %, Escherichia coli M-17 - 6-8 vol. %.

A mixture of biomass starter cultures of lactobacilli, bifidobacteria and E. coli is poured into sterile vials, taking into account the daily dose of the probiotic. Then the bottles are labeled and packed into boxes.

The symbiotic can be obtained by the reactor method.

The liquid symbiotic obtained by the claimed method is called “BiCoel”.

We conducted a study of the obtained probiotics using the proposed method (option 1), and methods that differ from the proposed one in other percentages of mixed generation III producer strains (option 2 and option 3) (Table 5).

As can be seen from Table 6, the symbiotic in option 2 was characterized by undesirably high levels of lactic and glyoxylic acids and insufficient levels of GABA, butyrate, valeric and succinic acids. Based on the number of living microbial cells on the 20th day of storage, a decrease in the number of individual strains of lactobacilli and bifidobacteria and inhibition of the growth of E. coli M-17 to 10 ⁵ CFU/ml were noted (Table 7).

The symbiotic according to option 3 was distinguished by an even higher concentration of lactic and glyoxylic acids and a low level of GABA and butyrate, and against the background of a sufficient level of lacto- and bifidobacteria, inhibition of E. coli - M-17 to 10 ⁵ CFU/ml (Table 6.7).

In this regard, symbiotics prepared according to options two and three cannot be recommended for use in patients with ASD, since patients with ASD have an increased content of lactic and glyoxylic acids, the increased content of the latter directly affects the production of large amounts of oxalates, resulting in the development hyperoxaluria, against the background of decreased levels of neurotransmitters (GABA, butyrate, etc.). In terms of the number of living microbial cells of the producing strains, the second and third options do not meet the goal of obtaining a multi-strain probiotic with the number of each member of the consortium at least 10 ⁸ CFU/ml.

As can be seen from tables 6 and 7, the drug according to option 1 is optimal in terms of the amount of metabolites and living microbial cells, and if the values go beyond the intervals according to option 1, we do not receive a drug with the declared properties.

The claimed method makes it possible to obtain a liquid symbiotic for use in the correction of intestinal microbiota and cognitive impairment, including in ASD. Three-stage scaling of biomass makes it possible to increase the level of metabolite yield, and monitoring the compliance of producer strains with those declared at the first stage of cultivation contributes to the high quality of the finished product. The claimed liquid symbiotic is a hypoallergenic, lactose-free multicomponent probiotic (symbiotic, psychobiotic) with a high content of living microbial cells of all seven starter cultures per unit volume with a sufficient concentration of neurometabolites (GABA, SCFA, tryptophan, butyrate, succinic acid, etc.).

An example of obtaining the liquid symbiotic “BiCoel” using the proposed method

At the first stage, the intermediate product was prepared for future use as follows: drinking water (GOST 2874-82) was heated to 48±1°C, casein (edible acid casein according to GOST 4960-74) was added in an amount of 60-70 g per liter, stirred, and the pH was adjusted up to 7.8-8.2 units with a 20% NaOH solution, placed in a thermostat in a 5 liter bottle on a rotary shaker for mixing at a speed of 20-30 rpm at a temperature of 48±1°C for 24±1 hour. Then the shaker was turned off and left to settle for another 24±1 hour; after keeping in the thermostat, the supernatant liquid was drained through a paper filter and the pH was set to 8.2-8.5. The finished hydrolyzate contained 490-520 mg% amine nitrogen. The hydrolyzate was stored for future use under chloroform 1% by volume at a temperature of 4±1°C.

From the resulting intermediate product, two nutrient media were prepared: GKS and GKS-M according to the following recipes.

To prepare GCS, the whole hydrolyzate was diluted with distilled water to an amine nitrogen value of 220-230 mg% (on average, based on 1.8 liters of water per 1 liter of intermediate product). 5 g of sodium chloride, 2 g of peptone, 10 g of fructose, 0.25 g of ascorbic acid and 0.75 g of previously prepared standard agar-agar were added to the diluted hydrolyzate per 1 liter. The pH was adjusted to 8.1-8.3 with a 20% NaOH solution.

The GKS-M medium was distinguished by the absence of agar-agar in its composition. The prepared GKS and GKS-M media were sterilized using the same method at 0.5 atm for 30 minutes.

The use of continuous stirring during the hydrolysis process reduced the hydrolysis time compared to the prototype by 24 hours and provided an intermediate product with a higher level of amine nitrogen of 490-520 mg% compared to the prototype 450-500 mg%, which increased the level of amine nitrogen, providing a high biomass yield of the producer strain, up to 220-230 mg% compared to the prototype 160-200 mg%.

The use of this composition of media prepared according to the presented method, in addition to maintaining a high level of biomass yield, also contributed to the accumulation of valuable bacterial metabolites in the product, such as GABA SCFA, tryptophan necessary for patients with ASD (Table 8). Ascorbic acid has been used as a growth promoter for bifidobacteria and lactobacilli.

Peptone was a mixture of poly- and oligopeptides, amino acids, salts and trace elements, respectively, and was a source of nutrients.

Agar increased the viscosity of the medium, which ensured uniform growth of microorganisms throughout the entire thickness of the medium and uniform consumption of growth factors and nutrients.

Fructose is a carbohydrate - an energy substrate and a source of carbon.

Sodium chloride maintained optimal osmotic pressure in the cell.

Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 90-TC-4, L. fermentum 39, Bifidobacterium bifidum 1, B. Bifidum 791, B. longum 379 and additionally Escherichia coli M-l 7 were used as producer strains.

Cultivation of producer strains of lactobacilli and bifidobacteria was carried out as follows.

Generation I: 1 ml of GKS-M medium was added to ampoules with dry strains, then the contents of each ampoule were transferred to a separate bottle with 10.5 ml of GKS-M. Then the producer strains were cultivated for 24±1 hour at a temperature of 37±1°C. After cultivation, 1.5 ml of the first generation of each culture was selected and MALDI TOF mass spectrometry was performed to confirm the purity of the strain and the compliance of the grown strain with that stated in the recipe. MALDI TOF mass spectrometry allowed us to obtain a unique individual spectrum of each strain. A limitation of the method was the possibility of studying the strain only in liquid non-agar media, therefore, to obtain the first generation of strains, the agar-free liquid medium GKS-M was used.

II generation: the obtained biomasses of the I generation, each separately, were mixed with the GCS nutrient medium; 10 ml of the I generation were added to 110 ml of the medium, and continued to be cultivated for 24±1 hour at a temperature of 37±1°C. After cultivation, 10 ml of the second generation of each strain were selected to control the concentration of bacterial cells in 1 ml using a densitometer. Concentration of bacterial cells (cells/ml) - 10 ¹⁰ -10 ¹¹ .

III generation: the obtained biomass of the II generation of each strain was separately mixed with the GCS nutrient medium; 100 ml of the II generation were added to 1000 ml of GCS, thermostated for 48 ± 1 hour at a temperature of 37 ± 1 ° C. At the end of cultivation, 10 ml of the third generation of each strain were selected to control the concentration of bacterial cells in 1 ml using a densitometer. Concentration of bacterial cells (cells/ml) - 10 ¹⁰ -10 ¹² .

As a result, we obtained 1.09 liters of the third generation of three strains of lactobacilli and three strains of bifidobacteria.

Cultivation of E. coli MP strains was carried out according to the method described above, first on GKS-M medium, then on GKS medium in the same ratios of generation I, II and III. The duration of incubation was changed; it was 18±1 hour for all generations at a temperature of 37±1°C.

At the end of cultivation, the resulting biomass of all seven producer strains was mixed in the following percentages: Lactiplantibacillus plantarum 8RA-3 - 15 vol. %, Limosilactobacillus fermentum 39 - 15 vol. %, L. fermentum 90-TC-4 - 16 vol. %, Bifidobacterium bifidum 1-16 vol. %, B. bifidum 791 - 17 vol. %, B. longum 379 - 14 vol. %, Escherichia coli M-17 - 7 vol. % taking into account the metabolic potential of the producing strains.

Thus, a lactose-free multicomponent probiotic (symbiotic, psychobiotic, metabiotic) was obtained with a high content of living microbial cells of all seven biocompatible producer strains per unit volume with an increased level of neurometabolites such as GABA, butyrate, pyruvic acid compared to the “LB-complex PLUS” , succinic, isobutyric, isovaleric and valeric acids with reduced levels of lactic and glyoxylic acids (Table 8).

A mixture of biomass starter cultures of lactobacilli, bifidobacteria and E. coli was poured into sterile bottles of 2.5, 5.0 ml, taking into account the daily dose of the probiotic.

It was hermetically sealed with a rubber stopper and an aluminum cap. Then the bottles were labeled and packed into boxes of 9 pieces (the minimum course of treatment is 27 days, that is, three boxes per course of treatment). The drug was stored at a temperature of +6±2°C with the preservation of the specific activity of the symbiotic at least 10 ⁸ CFU/ml.

Liquid symbiotic can be used orally in 1 or 2 doses, 1 bottle per day before meals with water, compote, fruit drink, juice, etc. with a temperature not exceeding 30°C. Shake the bottle with the drug thoroughly before use.

Indications for use:

- as a probiotic component of diet therapy for any diseases complicated by intestinal dysbiosis, as a means of normalizing microflora: during long-term treatment with antibiotics, chemotherapy and hormonal drugs; for allergic diseases (allergodermatoses, eczema, etc.), for chronic gastrointestinal diseases, acute intestinal infections of bacterial and viral etiology (dysentery, coli-enteritis, salmonellosis, acute intestinal infections of unknown etiology, oral and enterovirus infections, as well as after suffering a new coronavirus infection, etc.), food toxic infections; for the correction of cognitive impairments, including autism spectrum disorders (ASD) in order to improve socialization, minimize unwanted behavior, increase learning potential, the effectiveness of psychological and pedagogical correction, etc.

Can be used for lactase deficiency and diabetes mellitus, as well as against the background of antibacterial therapy, taking into account the pharmacokinetics and pharmacodynamics of the antibiotic/chemo drug.

Preclinical studies of the liquid symbiotic "BiCoel", prepared according to the method presented in the application, were carried out in the central research laboratory of the Federal State Budgetary Educational Institution of Higher Education "PIMU" of the Ministry of Health of Russia on an animal model of "cognitive disorders" and on a model of "intestinal dysbiosis".

Clinical testing of the symbiotic "BiCoel", prepared according to the method presented in the application, was carried out in the following state budgetary healthcare institutions of the city of Nizhny Novgorod: State Budgetary Healthcare Institution "City Children's Clinical Hospital No. 1", Institute of Pediatrics of the University Clinic of the Federal State Budgetary Educational Institution of Higher Education "PIMU" of the Ministry of Health of Russia (2nd pediatric department), Institute of Traumatology and Orthopedics of the University Clinic of the Federal State Budgetary Educational Institution of Higher Education "PIMU" of the Ministry of Health of Russia (2nd pediatric burn department).

Preclinical and clinical trials have confirmed that the claimed multicomponent probiotic is a harmless and effective agent that normalizes intestinal microflora and corrects cognitive impairment, including autism spectrum disorder.

Claims (2)

1. Liquid symbiotic containing strains Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 90-TC-4, Limosilactobacillus fermentum 39, Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379, characterized in that it additionally contains Escherichia coli strain M-17 with the number of living microbial cells of each producer strain is at least 10 ⁸ CFU/ml in the ratios: Lactiplantibacillus plantarum 8RA-3 - 14-16 vol. %, Limosilactobacillus fermentum 39 - 14-16 vol. %, L. fermentum 90-TC-4 - 15-17 vol. %, Bifidobacterium bifidum 1 - 15-17 vol. %, B. bifidum 791 - 16-18 vol. %, B.longum 379 - 13-15 vol. %, Escherichia coli M-17 - 6-8 vol. % and their metabolic products, including neurometabolites. 2. A method for producing a liquid symbiotic, which consists in using the producer strains Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 90-TC-4, Limosilactobacillus fermentum 39, Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379, which are cultivated in hydro lickable - casein nutrient medium containing casein hydrolyzate, sodium chloride, peptone, fructose, agar-agar, ascorbic acid as a nutrient base, the resulting biomass of producer strains is mixed, packaged taking into account the daily dose required for patients, characterized in that it is used as additional producer strain Escherichia coli strain M-17; biomass of producer strains are separately cultivated on a hydrolyzate-casein nutrient medium, and the first generation of producer strains of lactobacilli and bifidobacteria is obtained from a dry mother culture by adding it to a hydrolyzate-casein nutrient medium without agar in a ratio of 1:11, followed by cultivation at a temperature of 37 ± 1 °C for 24±1 hours, then confirm the purity of the strains and the compliance of the grown strains with the stated in the recipe by MALDI TOF mass spectrometry, the obtained biomass of the first generation, each separately, is mixed with a hydrolyzate-casein nutrient medium in a ratio of 1:11 and cultivated at at a temperature of 37±1°C for 24±1 hours, the resulting biomass of the second generation of each strain is separately mixed with a hydrolyzate-casein nutrient medium in a ratio of 1:10 and thermostated at a temperature of 37±1°C for 48±1 hours, strains E .coli Ml7 is cultivated similarly to the producing strains of lactobacilli and bifidobacteria for 18±1 hours for all generations; the resulting generations of biomass producer strains Lactiplantibacillus plantarum 8RA-3, Limosilactobacillus fermentum 90-TC-4, Limosilactobacillus fermentum 39, Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Escherichia coli M-17 are mixed in percentage ratios: Lact iplantibacillus plantarum 8RA- 3 - 14-16 rev. %, Limosilactobacillus fermentum 39 - 14-16 vol. %, L. fermentum 90-TC-4 - 15-17 vol. %, Bifidobacterium bifidum 1 - 15-17 vol.%, B. bifidum 791 - 16-18 vol. %, B.longum 379 - 13-15 vol. %, Escherichia coli M-17 - 6-8 vol. %.