Lactobacillus reuteri strain for suppressing helicobacter pylori activity

Abstract

The invention relates to the field of biotechnology. The invention consists in a Lactobacillus reuteri strain SALR01 deposited in the All-Russian Collection of Microorganisms at the G.K. Skryabin Institute of Microbial Biochemistry and Physiology, an independent division of the Federal Research Centre "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences", under registration number VKM B-3734D. The claimed strain exhibits antagonistic activity against Helicobacter pylori. At the same time, the strain is not toxic to mammalian cells. This makes it possible to use the claimed strain to create probiotics and metabiotics.

Description

A strain of lactobacillus reuteri bacteria that suppresses the activity of helicobacter pylori AREA OF TECHNOLOGY

[0001] The present invention relates to the field of biotechnology and medicine, namely to the use of the Lactobacillus reuteri SALR01 strain, suitable for the creation of probiotics and metabiotics due to the antagonistic effect against Helicobacter pylori. LEVEL OF TECHNOLOGY

[0002] The activity of Helicobacter pylori bacteria (hereinafter H. pylori, or Hp) is one of the main causes of pathological processes leading to gastrointestinal diseases. However, eradication treatment of H. pylori-associated diseases is only a temporary measure, since a high risk of re-infection with H. pylori remains [1]. It has also become known that the majority of people infected with H. pylori do not have the above-mentioned diseases [2]. This led to the conclusion that the cause of Hp-associated diseases lies not in the presence of H. pylori in the patient, but in the increased activity of this pathogen, caused, among other things, by dysbacteriosis. Currently, great efforts are being directed at developing drugs that could help patients maintain the balance of gastrointestinal microbiota, thereby reducing the manifestation of diseases. Probiotics for the treatment of Hp-associated diseases such as gastritis, duodenitis, peptic ulcer disease, etc., Lactobacillus reuteri bacteria (hereinafter L. reuteri) and their metabolites are often used due to their repeatedly demonstrated anti-Helicobacter activity [3]. L. reuteri is a species of facultative anaerobic gram-positive non-spore-forming lactic acid bacteria that are part of the normal microflora of the colon of humans and some animals. L. reuteri is not a pathogenic microorganism [4].

[0003] Microorganisms of the Lactobacillaceae family secrete bacteriocins as metabolites [5]. 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 the distinctive bacteriocins of L. reuteri are the antimicrobial compounds reuterin and reutericyclin, which inhibit the growth of harmful gram-positive and gram-negative bacteria, as well as some types of mold, yeast, and protozoa [6]. L. reuteri secretes reuterin in quantities sufficient to inhibit the growth of harmful microorganisms in the gastrointestinal tract, but does not affect the rest of the microflora [7].

[0004] The present invention provides a new strain belonging to the species L. reuteri.

[0005] There are many known strains of the L. reuteri species: ATCC PTA 4660, ATCC PTA 4964, ATCC PTA 5289, ATCC 55730, DSM 17938, DSM 20016, DSM17648, NCIMB 30242, SD-RD83O-FR, YLR001. Patent RU 2 435 844 C2 (published: 10.12.2011; MIC: C12N1 / 20; A61K35 / 74; A61P1 / 06; A61K47 / 44; C12R1 / 225) describes a probiotic product for the treatment of colic in newborn infants, obtained using the L. reuteri strain DSM 17938 as an active ingredient. The resulting probiotic product, produced using L. reuteri, reduced daily crying time in infants by reducing excessive intestinal motility activation through IL-10 stimulation.

[0006] Currently, there is a need to expand the arsenal of L. reuteri strains with anti-Helicobacter activity. TERMS AND ABBREVIATIONS

[0007] Dysbacteriosis is a qualitative and / or quantitative change in the body's bacterial microflora, the displacement of various members of the microflora into uncharacteristic habitats, accompanied by metabolic and immune disturbances. Moreover, these disturbances do not disappear after the elimination of the unfavorable factor that caused the dysbacteriosis. Dysbacteriosis most often refers to a disruption of the intestinal microflora [8].

[0008] Living microorganisms are vegetative cells and spores, if the microorganisms are spore-forming, or cells of non-spore-forming microorganisms that retain the ability to carry out basic life processes.

[0009] 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 [9].

[0010] 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 symbiotic (indigenous) microflora of the host organism

[0010] . [UN] Microflora (microbiota) is the collection of various species of microorganisms that inhabit a particular environment. Microorganisms that live in symbiosis with their host are called normal, or symbiotic, microflora.

[0011] In this text, the term "microflora" refers to the microflora of the gastrointestinal tract.

[0012] Probiotics are a functional food ingredient in the form of non-pathogenic and non-toxicogenic live microorganisms that are beneficial to humans and, when systematically consumed in food in the form of preparations or as part of food products, provide a beneficial effect on the human body as a result of normalizing the composition and / or increasing the biological activity of normal intestinal microflora

[0012] .

[0013] Ultrafiltration is a membrane separation process, as well as fractionation and concentration of substances, achieved by filtering a liquid under the influence of a pressure difference before and after the membrane. The particle size of the separated particles (i.e., the size of particles that do not pass through the filter but remain in the concentrate) is approximately 0.001 - 0.05 µm (5 - 500 kDa).

[0014] GIT - gastrointestinal tract.

[0015] CFU - colony-forming unit.

[0016] PCR – polymerase chain reaction.

[0017] B. subtilis - Bacillus subtilis.

[0018] H. pylori - Helicobacter pylori.

[0019] L. reuteri - Lactobacillus reuteri. ESSENCE OF THE INVENTION

[0020] The objective of the present invention is to isolate a new strain of L. reuteri bacteria for the creation of probiotics and metabiotics for the treatment and / or prevention of Hp-associated diseases.

[0021] This problem is solved by the claimed invention by achieving such a technical result as the possibility of using the new strain of L. reuteri SALR01 to create probiotics and metabiotics for the treatment and / or prevention of Hp-associated diseases.

[0022] The stated technical result is achieved due to the antagonistic action of the L. reuteri SALR01 strain against H. pylori, as well as the adaptation of the L. strain. reuteri SALR01 for cultivation in laboratory and industrial nutrient media. DESCRIPTION OF DRAWINGS

[0023] The subject matter of the present application is described point by point and clearly stated in the claims. The above-mentioned objectives, features, and advantages of the invention are apparent from the following detailed description, taken in conjunction with the accompanying drawings, which show:

[0024] Fig. 1 shows the results of a study to evaluate the effectiveness of the use of the claimed composition in the treatment of Helicobacter pylori infection using a Mongolian gerbil model. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following detailed description of the invention includes numerous implementation details intended to provide a clear understanding of the present invention. However, one skilled in the art will readily appreciate 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 to avoid unnecessarily obscuring the features of the present invention.

[0026] Furthermore, it is clear from the foregoing description that the invention is not limited to the embodiment described. Numerous possible modifications, changes, variations, and substitutions, while preserving the spirit and form of the present invention, are apparent to those skilled in the art.

[0027] The declared L. reuteri strain SALR01 is deposited in the All-Russian Collection of Microorganisms of the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, a separate division of the Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences" under registration number VKM B-3734D.

[0028] The claimed strain was isolated from human intestinal contents. It was designated L. reuteri SALR01. It should be understood that the names "L. reuteri VKM B-3734D" and "L. reuteri SALR01" refer to the same strain.

[0029] L. reuteri strain SALR01 is a wild-type strain with unchanged properties. The strain is Gram-positive, non-motile, short, curved rods (2.0 - 5.0 x 0.7 - 1.0 µm) with rounded ends, located singly, in pairs, or in short chains. Spores are not formed; when deep-seeded on The semi-liquid nutrient medium is formed by crumbly colonies with a diameter of 1–2 mm. The optimal growth temperature is +37°C.

[0030] L. reuteri strain SALR01 can grow at 37±2°C in an anerobic atmosphere on modified MRS nutrient medium for lactobacilli and a pH of 6.8-6.9. This strain exhibits the following biochemical characteristics: facultative anaerobe, obligate heterofermentative. It ferments glucose, galactose, maltose, sucrose, arabinose, and ribose. It does not ferment xylose, trealose, mannose, or cellobiose.

[0031] As part of the claimed invention, the L. reuteri SALR01 strain has been adapted for cultivation in laboratory and industrial nutrient media. In one embodiment, the L. reuteri SALR01 strain is cultivated on a solid nutrient medium. MRS lactobacillus medium supplemented with 15 g / L of agar-agar can be used. Cultivation of the strain on the solid nutrient medium is carried out by streaking.

[0032] In another embodiment, the L. reuteri SALR01 strain is cultivated in a liquid nutrient medium. The liquid nutrient medium for culturing the L. reuteri SALR01 strain may have an initial pH of 6.8-6.9. The liquid nutrient medium comprises peptone, yeast extract, sucrose, meat-peptone broth, ammonium citrate, magnesium sulfate, calcium chloride, manganese sulfate, potassium salts of orthophosphoric acid, sodium acetate, sodium hydroxide, and water.

[0033] In one embodiment, the nutrient medium may additionally include growth factors and / or special additives. In a preferred embodiment, the nutrient medium does not include additional components.

[0034] The method of storing the L. reuteri SALR01 strain is by periodic subcultures with storage of the culture in a refrigerator at 4–6°C, or in a lyophilized state.

[0035] The method for producing inactivated cells of the L. reuteri SALR01 strain includes the following steps. The L. reuteri SALR01 strain is cultured in a liquid nutrient medium. After cultivation, the culture is cooled to 10°C, then the culture suspension is centrifuged in a continuous-flow centrifuge. The resulting cells are inactivated by heating at 80°C for 40 minutes.

[0036] The L. reuteri SALR01 strain can be used to produce probiotics and / or metabiotics that inhibit H. pylori activity. However, the applicability of this strain is not limited to the treatment of Helicobacter pylori but can also be used to suppress other pathogenic and / or opportunistic microorganisms. EXAMPLES OF IMPLEMENTATION OF THE INVENTION

[0037] Example 1. Genetic analysis of L. reuteri SALR01.

[0038] To identify the declared strain, DNA was isolated from a L. reuteri SALR01

[0013] cell culture. A fragment of the gyrB gene was then amplified by PCR on a GeneAmp PCR System 2700 (Applied Biosystems, USA) using the universal primers UplF and UP2R. The nucleotide sequence of the PCR product was determined using the ABI PRISM® BigDye™ Terminator v. 3.1 reagent kit with the Up IS primer, followed by analysis of the reaction products on an Applied Biosystems 3730 DNA Analyzer automated sequencer. The nucleotide sequence of the gyrB gene fragment of the declared L. reuteri strain SALR01 was compared with the nucleotide sequences of similar fragments of the gyrB gene of the strains Lactobacillus reuteri DSM 20016, Limosilactobacillus reuteri strain reuteri (CP045049), Limosilactobacillus reuteri SD-RD83O-FR (CP080621), Limosilactobacillus reuteri YLR001 (CP065540).Phylogenetic analysis of nucleotide sequences of gyrB gene fragments was performed using the BLAST program (http: / / www.ncbi.nlm.nih.gov). The phylogenetic position of the strains and the percentage of similarity were determined using the TaxonDC 1.3.1

[0014] program. Phylogenetic analysis showed a high similarity of the declared L. reuteri strain SALR01 with all the above-mentioned strains: 99.95% homology for Lactobacillus reuteri DSM 20016, 100% for Limosilactobacillus reuteri strain reuteri (CP045049), Limosilactobacillus reuteri SD-RD83O-FR (CP080621), Limosilactobacillus reuteri YLR001 (CP065540) (Table 1).

[0039]

[0040] Example 2. Obtaining inactivated cells of the L. reuteri SALR01 strain, dried on maltodextrin.

[0041] To obtain inactivated cells extracted from the culture fluid, the L. reuteri strain SALR01 was used. The culture was grown at a temperature of 36 ± 1 °C for 24 hours. Before cultivation, the pH of the nutrient medium was adjusted to 6.8 - 7.0 with alkali. Cultivation was carried out in an ANKUM 210 fermenter in a nutrient medium volume of 10 liters. The total biomass accumulation time in the reactor was 18 hours. The completion of the cultivation process was considered to be the absence of a drop in pH from the set value.

[0042] After completion of cultivation, the culture was cooled to 10°C. The cell titer in the culture was 6x10 9 CFU / ml. The culture fluid was then separated using a GF105B flow-through ultracentrifuge. A pellet was obtained after ultracentrifugation. The titer in the pellet was 2.5x10. 10CFU / 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. A physiologically acceptable carrier, maltodextrin, was mixed with distilled water. Inactivated cells were added to the resulting mixture. A CFU analysis before lyophilization revealed the absence of viable cells. Lyophilization of the carrier and inactivated cell mixture was performed on a TG-50 system using standard lyophilization parameters. The resulting inactivated cells were extracted from the culture fluid and dried on maltodextrin with a moisture content of 2%.

[0043] Example 3. Evaluation of the anti-Helicobacter effect of compositions obtained using the L. reuteri strain SALR01 on a Mongolian gerbil model.

[0044] Mongolian gerbils are an effective model for studying H. pylori pathogenesis, reflecting many of the features of gastric inflammation caused by H. pylori in humans. Animals were infected with an intragastric suspension of H. pylori bacteria twice (every other day). After the double infection, the animals were observed for 11 weeks. By the 12th week after infection, the animals developed gastritis and ulcerative lesions of the gastric tissue. Starting from the 12th week, administration of the test substances began according to the schedule (Table 2). The test substance was a mixture of inactivated L. reuteri SALR01 cells and a physiologically acceptable carrier.

[0045] Starch at 1% wt % was used as a physiologically acceptable carrier. Standard human H. pylori eradication therapy, including the proton pump inhibitor omeprazole and the antibiotics clarithromycin and amoxicillin, served as positive controls. The carrier alone served as a negative control. The effects of this substance in combination with the positive control drugs were also studied.

[0046]

[0047] The vehicle, positive control drugs, and the test substance were administered to gerbils intragastrically, since this method is analogous to the oral route of administration in humans. At the end of the experiment, the animals were sacrificed for histological examination of the stomach and duodenum tissues. The stomach condition was assessed based on the following parameters: area of ​​inflammation; macroscopic changes in the mucosa; morphological changes (degree of inflammation; inflammatory activity; atrophy of the gastric glands; metaplasia; colonization of the mucosa with H. pylori). For morphological assessment of the severity of pathology, visual analogue scales proposed in the Sydney system were used. In this case, a point assessment system was used: 0 - normal; 1 - mild; 2 - moderate; 3 - severe.

[0048] Macroscopic examination of stomach and duodenal tissues in all experimental groups revealed no visible pathological changes. The results of microscopic histological examinations are presented in Table 3.

[0049]

[0050] The median, upper and lower quartiles (Me (Qi; Q3)) are shown in white cells, the sum of points in the group is shown in gray cells; a - significant difference from the intact group of mice, (p < 0.05); b - significant difference from the negative control group (p < 0.05). In the intact group of mice that were not infected and treated, the structure of the stomach wall was normal. In the negative control group, all animals showed morphological changes in the gastric mucosa characteristic of catarrhal gastritis of varying severity: from mild to moderate, with a tendency to chronicity of the process. The development of infection in animals of the negative control group led to the formation of predominantly pangastritis (inflammation area 3, average score 15, Table 3). In this group, a significant increase in the severity of pathological changes, the degree and activity of inflammation, atrophy of the gastric glands and dissemination of the gastric mucosa with H.pylori compared to the intact group (p<0.05). In the positive control group, there was a significant decrease in indicators for all studied criteria compared to the negative control group.

[0051] Figure 1 shows the range diagrams of the total severity of pathological symptoms for the studied substances. Box plot 1 represents the negative control. Box plot 2 represents the positive control. The substance (L. reuteri + vehicle) represents box plot 3. The combination of the substance and positive control represents box plot 4. The ordinate axis shows the height of the diagrams corresponding to the total severity of pathological symptoms, expressed as points on the visual analogue scale. The mean and median values ​​for the experiments are shown, with seven replicates in each experiment.

[0052] Monotherapy with the substance (L. reuteri + vehicle) resulted in a significant reduction in most of the studied parameters: inflammation area and activity, morphological changes in the stomach, and the degree of H. pylori colonization of the gastric mucosa compared with the negative control. However, no significant differences were observed between the positive control group and the monotherapy group. Monotherapy with the substance was inferior to the positive control only in terms of reduction in the degree of inflammation.

[0053] Combination therapy with the test substance and positive control drugs outperformed the positive control in several respects. Thus, adding the test substance to the positive control drugs more effectively reduced inflammatory activity, the severity of gastric gland atrophy, and the degree of H. pylori colonization of the gastric mucosa (Table 3).

[0054] The obtained data show that a composition including L. reuteri can reduce the manifestations of H. pylori infection in monotherapy, and also increases the effectiveness of treatment with standard H. pylori eradication therapy drugs.

[0055] Example 4. Evaluation of the effectiveness of using a composition based on inactivated cells of the L. reuteri SALR01 strain in therapy in patients with chronic gastritis or functional dyspepsia associated with H. pylori infection.

[0056] In this example, an open-label, randomized, prospective, controlled study of the efficacy and safety of the claimed composition was conducted in patients with functional dyspepsia and / or chronic gastritis associated with H. pylori infection. The composition used in the example included inactivated cells of the probiotic strain L. reuteri SALR01, a complex of metabolites of the probiotic strain B. subtilis VKM B-3536D, and a physiologically acceptable carrier containing zinc citrate trihydrate and fructooligosaccharides. The study included 40 patients of both sexes, aged 18 to 65 years inclusive, with a confirmed diagnosis of functional dyspepsia or chronic gastritis and a positive test for the presence of H. pylori DNA in feces using real-time PCR. A separate requirement for patients was the absence of a history of previously administered eradication therapy less than a year before screening.

[0057] All patients were equally divided into experimental group 1 and control group 2. Patients from experimental group 1 received the stated composition twice daily in the morning and evening after meals for 14 days in addition to standard three-component first-line eradication therapy (esomeprazole 20 mg orally twice daily in the morning and evening after meals, amoxicillin 1000 mg twice daily in the morning and evening after meals, clarithromycin 500 mg twice daily in the morning and evening after meals). For the next 14 days after the end of therapy, patients in this group received the stated composition twice daily in the morning and evening after meals as a monotherapy. Patients from control group 2 received eradication therapy for 14 days, including esomeprazole 20 mg orally 2 times a day in the morning and evening after meals, amoxicillin 1000 mg orally 2 times a day in the morning and evening after meals, clarithromycin 500 mg orally 2 times a day in the morning and evening after meals.The severity of symptoms characteristic of chronic gastritis or functional dyspepsia was assessed using questionnaires that all patients completed. filled out before the start of the experiment and 28 days after the start of the experiment (Table 4).

[0058] Table 4. Dynamics of gastrointestinal complaints in patients of the main and control groups

[0059] A statistical comparison of the treatment effects in the experimental and control groups showed that the use of the claimed composition significantly alleviated symptoms such as excessive gas, abdominal rumbling, and a tendency toward diarrhea. Moreover, in the experimental group, the frequency of these symptoms decreased after the course of therapy compared to the start of therapy. Meanwhile, in the control group, these symptoms became more common after therapy than at the beginning. This indicates that standard eradication therapy disrupts gastrointestinal function. This is most likely due to dysbacteriosis caused by broad-spectrum antibiotics. Moreover, the use of the claimed composition together with standard eradication therapy drugs reduced the frequency of almost all symptoms after therapy. This may be due to relief or prevention of dysbacteriosis, thanks to the beneficial effect of the stated composition on representatives of the normal intestinal microflora under conditions of taking antibiotics.

[0060] Example 5. Study of the effect of the L. reuteri strain SALR01 on the inhibition of the growth of pathogenic and opportunistic microflora.

[0061] In this example, the ability of the L. reuteri SALR01 strain to inhibit the growth of indicator cultures was studied. The study was conducted using an adapted standard disk method for determining the susceptibility of microorganisms to antibiotics. The indicator cultures used in this example represented pathogenic and opportunistic microorganisms in humans from various initial locations (intestine, oral cavity, skin, urogenital tract). To implement the method, indicator cultures were placed on the surface of agarized Luria-Bertani nutrient medium in a Petri dish at a rate of 10 7CFU. After solidification, the Petri dishes were dried for 15 minutes at 37°C. Then, 10 µl of the L. reuteri SALR01 strain culture, grown in liquid nutrient medium, were applied to the surface of the nutrient medium containing the indicator cultures in the form of droplets 5±1 mm in diameter.

[0062] Petri dishes with the applied solutions were left at room temperature until the drops were completely absorbed and then incubated at 37°C for 24–48 hours. Results were determined by determining the presence of growth inhibition zones for indicator cultures around the area where the L. reuteri SALR01 culture was applied. The appearance of zones free of indicator cultures around the area where the culture was applied indicates antagonistic activity of L. reuteri SALR01 against the indicator cultures listed in Table 5.

[0063] Table 5 shows that the L. reuteri SALR01 strain is capable of inhibiting the growth of a number of pathogenic and opportunistic bacteria in various locations. These include the opportunistic strain Enterococcus faecium, clinical isolates of the pathogen Staphylococcus aureus, two specific pathogens of the female urogenital tract, Streptococcus agalactiae, which are among the most dangerous pathogens of neonatal infections, and the pathogenic strains of the oral cavity, Streptococcus mutans and Streptococcus gordonii, which are involved in the pathogenesis of caries and periodontitis. These data demonstrate that the L. reuteri SALR01 strain has a diverse spectrum of antagonistic activity, ensuring its effectiveness in the prevention of various infectious diseases.

[0064] Note: * no growth retardation ** presence of a zone of inhibition of the growth of the indicator culture (bactericidal action) *** incomplete suppression of indicator culture growth (bacteriostatic effect)

[0065] Example 6. Study of the harmlessness, toxicity, allergenicity and toxigenic properties of the L. reuteri strain SALR01.

[0066] To determine its safety, a 0.5 ml dose of L. reuteri SALR01 was administered orally to 10 female white mice weighing 14-16 g. Observation of the animals over 5 days revealed that administration of the L. reuteri SALR01 lactic acid bacteria strain did not cause death, signs of intoxication, or a decrease in group body weight compared to baseline. Thus, the results demonstrate that the studied lactic acid bacteria strain is nonpathogenic and harmless to laboratory animals.

[0067] To determine toxicogenicity, a culture of L. reuteri SALR01 bacteria was administered subcutaneously to rats (10 animals of the same sex, weighing 250-300 g) at a dose of 0.5 ml. Sterile saline served as a control, also administered at a dose of 0.5 ml. The animals were observed for 14 days. No deaths were observed during the observation period. Thus, the results showed that the studied lactic acid bacteria strain L. reuteri SALR01 does not exhibit toxicogenic properties.

[0068] To determine toxicity, heat-inactivated culture of L. reuteri SALR01 bacteria was administered to white rats (10 animals of the same sex weighing 250–300 g) intraperitoneally in a volume of 0.5 ml. The animals were observed for 7 days. During the entire observation period, there were no deaths of the experimental animals and no signs of intoxication; the group body weight of the animals did not decrease compared to the initial one. In this regard, we concluded that the studied strain of lactic acid bacteria L. reuteri SALR01 at a dose of 10 10 killed cells are not toxic.

[0069] To determine allergenicity, a single application of 0.02 ml of L. reuteri SALR01 bacterial culture was made to the lateral body surface of rats of one sex (10 animals weighing 250-300 g). The reaction was monitored visually after 24 hours. During the observation period, no allergic swelling or erythema was observed at the site of bacterial application, indicating the absence of allergenicity.

[0070] To determine its irritant effect on the skin, a single application of 0.1 ml of L. reuteri SALR01 bacteria to the skin of 10 rats weighing 180-200 g was performed. Skin reactions were recorded at the end of exposure, 1 hour later, and 16 hours later. No signs of irritation, such as erythema or swelling, were observed at the site of application of the test sample.

[0071] In another model, the irritant effect of the studied L. reuteri SALR01 strain on the conjunctiva was determined by applying a single dose of 0.02 ml of the bacterial culture to the conjunctival sac of rats (10 animals weighing 180-200 g). The condition of the ocular mucosa was monitored daily for 14 days. No signs of irritation—such as swelling, hyperemia, or other changes in the animals' ocular mucosa—were observed throughout the entire observation period.

[0072] Example 7. Study of the anti-Helicobacter coaggregation activity in vitro of the L. reuteri strain SALR01.

[0073] The study of the ability to co-aggregate between L. reuteri SALR01 and H. pylori was carried out using an adapted method of the co-aggregation test in artificial gastric juice

[0015] .

[0074] Clinical isolates obtained from patients with gastric pathology H. pylori-362 and H. pylori-420 were used. Passage of H. pylori strains was carried out on a blood nutrient medium (5-7% horse blood, Columbia agar base, 1% IsoVitalex (Becton Dickinson, USA)). Incubation of crops was carried out under microaerophilic conditions at an oxygen content of about 5%, using an anaerostat of the GasPaklOO system (BBL CampyPak System, Becton Dickinson, USA). To create microaerophilic conditions in the anaerostat, an atmospheric generation system for in vitro diagnostics CampyGen 2.5 L (Oxoid LTD, UK) was used. After loading The anaerostats were placed in a thermostat at 37°C, the optimal temperature for H. pylori growth. The incubation period for H. pylori strains, which produced a minimal number of coccoid forms, was 3-4 days.

[0075] Lyophilized inactivated substances of L. reuteri DSM17648 and L. plantarum SALP01 strains were used as positive and negative controls, respectively. Test samples included substances of L. reuteri SALR01 inactivated by different methods and dried using different technologies. Lyophilized samples were resuspended in PBS buffer and incubated for 2 hours at room temperature to rehydrate. Cells were washed and resuspended in PBS buffer to obtain a solution with ODeoo = 4.0.

[0076] The test was then conducted according to the specified method. None of the tested strains or samples showed autoaggregation, indicating high-quality sample preparation. The tests yielded identical results with both H. pylori-362 and H. pylori-420 strains used. The summarized results of three independent experiments are shown in Table 6.

[0077] Notes: T°C – temperature inactivation; H2O2 - inactivation with hydrogen peroxide; L - lyophilization; P - spray drying; * — the sample was evaluated in one experiment.

[0078] Samples of the negative and positive control strains yielded the expected results, confirming that the experiment was conducted correctly. Samples of the L. reuteri SALR01 strain yielded results comparable to those of the positive control. This confirms that dried, inactivated cells of the L. reuteri SALR01 strain exhibit anti-Helicobacter pylori coaggregation activity.

[0079] These application materials present a preferred disclosure of the implementation of the claimed technical solution, which should not be used as limiting other, particular embodiments of its implementation that do not go beyond the scope of the requested scope of legal protection and are obvious to specialists in the relevant field of technology. Sources of information: 1. Zimmerman Ya. S. Unresolved and controversial issues of modern gastroenterology. - M.: MEDpress-inform, 2013. - 224 p. 2. Algood Н.М., Cover T.L. Helicobacter pylori persistence: an overview of interactions between H. pylori and host immune defenses. Clin Microbiol Rev, 2006, 19(4): 597-613. 3. Kandler O., Stetter K., Kohl R. Lactobacillus reuteri sp. nov. a new species of heterofermentative lactobacilli. ZBL. Bakt. Hyg. Abt. Orig., 1980, Cl: 264-9. 4. Dore M.P., Cuccu M., Pes G.M. et al. Lactobacillus reuteri in the treatment of Helicobacter pylori infection. Intern Emerg Med 9, 2014, 649-654. 5. Cotter P., Ross R., Hill C. Bacteriocins — a viable alternative to antibiotics? / / Nat Rev Microbiol, 2012, 11, 95-105. 6. Talarico T.L., Dobrogosz W.J. Chemical characterization of an antimicrobial substance produced by Lactobacillus reuteri. / / Antimicrob Agents Chemother 33, 1989. 7. Casas IA, Dobrogosz WJ Validation of the Probiotic Concept: Lactobacillus reuteri Confers Broad-spectrum Protection against Disease in Humans and Animals. / / Microbial Ecology in Health and Disease, 2000, 12:4, 247-285. 8. Khursa PV, Mesnikova IL, Miksha Ya S. Intestinal microflora: role in maintaining health and developing pathology, correction possibilities: teaching aid. Minsk: BSMU, 2017, 36 p. 9. Tarantula V.Z. and others. Dictionary of biotechnological terms. Moscow: INITs Rospatent, 2005, 126 p. 10. Sheyderov B. A., Tkachenko E. I., Lazebnik L. B., Ardatskaya M. D., Sinitsa A. V., Zakharchenko M. M. Metabiotics - a new technology for the prevention and treatment of diseases associated with microecological disturbances in the human body. / / Experimental and clinical gastroenterology, 2018, 151 (3), 83-92. 11. Firsov N.N. Microbiology: dictionary of terms. Moscow: Bustard, 2026, 256 p. 12. GOST R 52349-2005 Food products. Functional food products. Terms and definitions (with Amendment No. 1) / / GOST R dated May 31, 2005 No. 52349-2005. 13. Ausubel FM et al. Current Protocols in Molecular Biology. Wiley, New York (1994). 14. Tarlachkov SV, Starodumova IP TaxonDC: calculating the similarity value of the 16S rRNA gene sequences of prokaryotes or ITS regions of fungi. / / Journal of Bioinformatics and Genomics, 2017, 3(5). 15. Holz, C., Busjahn, A., Mehling, H., Arya, S., Boehner, M., Habibi, H., & Lang, C. Significant Reduction in Helicobacter pylori Load in Humans with Non-viability Lactobacillus reuteri DSM17648: A Pilot Study. / / Probiotics and Antimicrobial Proteins, 2015, 7(2), 91-100.

Claims

CLAUSES OF THE INVENTION 1. The strain Lactobacillus reuteri SALR01, deposited in the All-Russian Collection of Microorganisms of the G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, a separate division of the Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences" under registration number VKM B-3734D, has an antagonistic effect against Helicobacter pylori.

2. Use of the L. reuteri strain SALR01 for the production of probiotics and / or metabiotics.

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