Use of a microbial composition for the preparation of a probiotic for the prevention and / or treatment of helicobacter pylori infection

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to the use of a microbial composition in preparing a postbiotic for preventing and / or treating Helicobacter pylori infection. The microbial composition comprises Lactobacillus plantarum RH03147 with a deposit number of CGMCC No. 25775, Lactobacillus plantarum HCS03-001 with a deposit number of CGMCC No. 16258, and Lactobacillus reuteri HCS02-001 with a deposit number of CGMCC No. 19746. The postbiotic prepared from the microbial composition provided by the present invention has a good anti-Helicobacter pylori effect and is relatively safe within a specific concentration range. Therefore, it can be used in the production of probiotic products or prepared into a medicine for clinical use.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a microbial composition in the preparation of a probiotic for preventing and / or treating Helicobacter pylori infection. BACKGROUND

[0002] A probiotic is a preparation of microorganisms, which has health benefits for the host, and / or components thereof, including non-living bacterial cells, bacterial metabolites and / or components after bacterial lysis. Among them, the metabolites mainly include exopolysaccharides, short-chain fatty acids, bacteriocins and organic acids, etc.; and the bacterial cell components mainly include lipoteichoic acid, teichoic acid, peptidoglycan, derived polypeptide and cell surface protein, etc.

[0003] The probiotic has the following health-promoting functions: enhancing the body's immunity, for example, butyrate in the probiotic can reduce intestinal inflammatory response; regulating intestinal flora, the probiotic contains antibacterial substances such as lactic acid and bacteriocins, which can promote intestinal microecological balance; regulating body metabolism, the probiotic plays a role in combating metabolic diseases such as obesity, hypertension and cardiovascular disease; and enhancing the epithelial barrier function, the probiotic can protect the gastrointestinal mucosa. For example, the probiotic derived from Lactobacillus paracasei can maintain the integrity of the intestinal barrier.

[0004] On the one hand, the probiotic has great application value in the food industry, and can be used as a food nutrition supplement or a biological preservative, etc. On the other hand, the probiotic has a positive effect on the treatment of certain diseases, and can be used to improve intestinal inflammation, relieve constipation, and also can be used to relieve allergies, and has great application potential in the medical field. Adding the probiotic to drugs and nutritional health products may become a new method and means for the auxiliary treatment of certain diseases.

[0005] Chinese patent application CN116763829A discloses a Lactobacillus plantarum LZ010 probiotic composition, a preparation method thereof and an application of the probiotic composition in inhibiting Helicobacter pylori, which comprises the following components in parts by weight: probiotic 30-40 parts, xylitol 10-25 parts, fructooligosaccharide 20-30 parts, and calcium carbonate 0.006-0.008 parts. The probiotic is composed of bacterial cells and metabolites of Lactobacillus plantarum LZ010, and the Lactobacillus plantarum LZ010 probiotic composition has obvious inhibitory effect on the growth of Helicobacter pylori and has the ability to resist Helicobacter pylori.

[0006] Chinese patent application CN117343874A discloses a preparation method of Paracaseicillus paracasei IOB413 probiotic extract product, the Paracaseicillus paracasei IOB413 probiotic extract product obtained according to the method, and the application of the Paracaseicillus paracasei IOB413 probiotic extract product in preparing a drug for resisting Helicobacter pylori infection. The Paracaseicillus paracasei IOB413 probiotic extract product has a significant effect of inhibiting the growth of Helicobacter pylori, relieving Helicobacter pylori infection, and improving bacterial flora disorder on mice infected with Helicobacter pylori.

[0007] Although there are some probiotics for resisting Helicobacter pylori infection in the art, the treatment of Helicobacter pylori infection still has an unmet need, and there is still a need for more probiotics that can effectively treat and / or prevent Helicobacter pylori infection in the art. SUMMARY

[0008] To achieve the above technical purposes, the present application provides the following technical solutions:

[0009] One of the purposes of the present application is to provide an application of a microbial composition in preparing a product for preventing and / or treating Helicobacter pylori infection, wherein the microbial composition comprises Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001, and Lactobacillus reuteri HCS02-001;

[0010] The Lactobacillus plantarum RH03147 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 25775;

[0011] The Lactobacillus plantarum HCS03-001 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 16258;

[0012] The Lactobacillus reuteri HCS02-001 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 19746.

[0013] In some examples, the product is a drug (a pharmaceutical composition).

[0014] In some examples, the product comprises a probiotic.

[0015] Another purpose of the present application is to provide a probiotic, wherein the raw material of the probiotic comprises a culture, a fermentation product, and / or a metabolic product of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001, and Lactobacillus reuteri HCS02-001;

[0016] The Lactiplantibacillus plantarum RH03147 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 25775;

[0017] The Lactiplantibacillus plantarum HCS03-001 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 16258;

[0018] The Lactobacillus rhamnosus HCS02-001 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 19746.

[0019] In some examples, the culture includes a culture broth, a culture broth supernatant, a broken product, a concentrated product, a sterilized product, a gelatinized product, or a dried product, etc.

[0020] In some preferred examples, the culture is a culture broth or a dried product.

[0021] In some more preferred examples, the dried product is a powder after spray drying of the culture broth.

[0022] In some examples, the postbiotic includes a liquid postbiotic or a solid postbiotic.

[0023] In some examples, the postbiotic includes an inactivated bacterial postbiotic, a metabolite postbiotic, or a broken cell postbiotic.

[0024] In some preferred examples, the postbiotic is a metabolite postbiotic.

[0025] In some examples, the postbiotic further includes at least one physiologically acceptable excipient.

[0026] A third object of the present application is to provide a pharmaceutical composition comprising a culture, a fermentation product, or a metabolite of Lactiplantibacillus plantarum RH03147, Lactiplantibacillus plantarum HCS03-001, and Lactobacillus rhamnosus HCS02-001;

[0027] The Lactiplantibacillus plantarum RH03147 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 25775;

[0028] The Lactiplantibacillus plantarum HCS03-001 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 16258;

[0029] The Lactobacillus reuteri HCS02-001 is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 19746.

[0030] In some examples, the culture includes a culture broth, a culture broth supernatant, a fermentation broth, a fermentation broth supernatant, a broken product, a concentrated product, a sterilized product, a gelatinized product, or a dried product, etc.

[0031] In some preferred examples, the culture is a culture broth or a dried product.

[0032] In some more preferred examples, the dried product is a powder after spray drying of the culture broth.

[0033] The fourth object of the present application provides a preparation method of any of the aforementioned postbiotics, which comprises the following steps:

[0034] (1) inoculating Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001 and Lactobacillus reuteri HCS02-001 into a fermentation medium to perform primary fermentation, to obtain a primary fermentation product;

[0035] (2) inoculating the primary fermentation product into a fermentation medium to perform secondary fermentation, to obtain a secondary fermentation product;

[0036] (3) inactivating the secondary fermentation product, to obtain a postbiotic.

[0037] In some examples, the pH of the fermentation medium is 6.2-6.8.

[0038] In some examples, the fermentation medium in step (1) and step (2) comprises the following components:

[0039] yeast peptone 5.0-15.0 g / L, glucose 15.0-20.0 g / L, yeast extract 5.0-12.0 g / L, sodium acetate 3.0-5.0 g / L, citric acid 2.0-3.0 g / L, potassium dihydrogen phosphate 2.0-3.0 g / L, Tween 80 0.1-0.2 g / L, and magnesium sulfate 0.20-0.25 g / L.

[0040] The fifth object of the present application is to provide the postbiotic obtained by any of the aforementioned preparation methods, or the use of any of the aforementioned postbiotics in the preparation of a product for preventing and / or treating Helicobacter pylori infection.

[0041] In some examples, the product is a drug (pharmaceutical composition).

[0042] The application provides application of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001 and Lactobacillus rhamnosus HCS02-001 in preparation of a postbiotic. Research finds that the prepared postbiotic has a better anti-Helicobacter pylori effect, and has better safety in a specific concentration range, and therefore can be used in the production of probiotic products or prepared into a medicine for clinical use.

[0043] The Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001 and Lactobacillus rhamnosus HCS02-001 of the application are disclosed in CN116649578A, wherein the preservation number of the Lactobacillus plantarum RH03147 is CGMCC No.25775, the preservation number of the Lactobacillus plantarum HCS03-001 is CGMCC No.16258, and the preservation number of the Lactobacillus rhamnosus HCS02-001 is CGMCC No.19746. See CN116649578A for specific preservation information. BRIEF DESCRIPTION OF DRAWINGS

[0044] Fig. 1 Typical figures of fluorescence intensity of Helicobacter pylori in the gastrointestinal tract of zebrafish in different treatment groups are shown, wherein the scale is 100 µm.

[0045] Fig. 2 Fluorescence intensity of Helicobacter pylori in the gastrointestinal tract of zebrafish in different treatment groups is shown, wherein * represents comparison with the model control group, p <0.05; *** comparison with the model control group, p <0.001. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein and, unless otherwise specified, terms used in the singular will include the plural and vice versa.

[0047] The expressions "a" and "an" used herein include the plural reference unless the context clearly dictates otherwise.

[0048] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to examples. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturers are used. If the manufacturers of all reagents or instruments are not specified, they are all conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present application.

[0049] The materials and devices involved in the present application are shown in Table 1.

[0050] Table 1

[0051]

[0052] The experimental animals and bacteria involved in the present application are as follows:

[0053] The zebrafish are all bred in fish breeding water at 28℃ (water quality: 200 mg of instant sea salt is added to each 1 L of reverse osmosis water, the conductivity is 450-550 μS / cm, the pH is 6.5-8.5, and the hardness is 50-100 mg / L of CaCO3), which are bred and provided by the fish breeding center of the company, the experimental animal use license number is: SYXK (Zhe) 2022-0004, the breeding management meets the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number is: IACUC-2023-7775-01.

[0054] The wild type AB strain of zebrafish is bred by natural pair mating. Zebrafish at an age of 3 days post fertilization (3 dpf) are used to determine the maximum test concentration (MTC) of the sample anti-H. pylori efficacy determination and its efficacy evaluation.

[0055] Helicobacter pylori, Helicobacter pylori culture medium (7 mL of sterile defibrillated sheep blood and 1 bottle of Helicobacter pylori additive are added to each 100 mL), microaerophilic, 37℃, 150 rpm shaking culture.

[0056] Example 1 Preparation of lactic acid bacteria biopesticide

[0057] The biopesticide preparation steps described in this example are as follows:

[0058] (1) Take the plantarum RH03147, plantarum HCS03-001, and reuteri HCS02-001 strain cryopreservation tubes from the -80°C refrigerator, immediately place them in a 37°C water bath for 15-30 seconds, and then thaw the entire contents of the cryopreservation tube to recover the strain. Recovered strains are inoculated into different culture medium A at a 10% inoculation rate, incubated at 37°C for 17 hours to obtain seed liquid, and then inoculated into different culture medium A at a 5% inoculation rate, incubated at 37°C for 17 hours to obtain three strains of culture liquid.

[0059] (2) The plantarum RH03147, plantarum HCS03-001, and reuteri HCS02-001 culture liquids are inoculated into the same culture medium B at a ratio of 3.5:3.5:1, with an inoculation rate of 5%, and stirred at 35°C for 17 hours to obtain primary fermentation liquid.

[0060] (3) After step (2) is completed, the primary fermentation liquid is inoculated into culture medium B at a 5% inoculation rate, and stirred at 35°C for 48 hours to obtain secondary fermentation liquid.

[0061] (4) The secondary fermentation liquid is filtered to obtain colorless to light yellow transparent filtrate without impurities, and the filtrate is heated and concentrated by 2 times, and sterilized by cooking for 30 minutes to obtain metabiotic liquid.

[0062] 8% malt dextrin is added to the metabiotic liquid, mixed and stirred until completely dissolved, the environmental humidity is ≤50%, and the metabiotic powder is obtained by spray drying in a spray dryer with an inlet temperature of 180°C and an outlet temperature of 110°C.

[0063] (5) The preparation of culture medium A: yeast peptone 12.0 g / L, glucose 25.0 g / L, yeast extract 8.0 g / L, sodium acetate 8.0 g / L, citric acid monohydrate 3.0 g / L, potassium dihydrogen phosphate 3.0 g / L, Tween 80 0.2 g / L, magnesium sulfate 0.8 g / L, and the balance is purified water; according to the formula proportion, heat and dissolve, sterilize at 115°C for 30 minutes, and adjust the pH of the culture medium to 6.60 with 1 mol / L food-grade NaOH solution;

[0064] (6) The preparation of the culture medium B: yeast protein peptone 15.0 g / L, glucose 20.0 g / L, yeast extract 12.0 g / L, sodium acetate 5.0 g / L, citric acid 2.0 g / L, potassium dihydrogen phosphate 2.0 g / L, Tween 80 0.2 g / L, magnesium sulfate 0.6 g / L, manganese sulfate 0.25 g / L, and the balance is purified water; according to the formula proportion, heating and dissolving, sterilizing at 115°C for 30 min, adjusting the pH value of the medium to 6.80 with 1 mol / L food-grade NaOH solution.

[0065] Example 2 Determination of the maximum detection concentration (MTC) of the lactic acid bacterial bioinoculant

[0066] Randomly select 3 dpf wild type AB strain zebrafish in a 6-well plate, and treat 30 zebrafish in each well (experimental group) with lactic acid bacterial bioinoculant, and set up a normal control group, with a volume of 3 mL per well. After 48 h of treatment at 28°C, the MTC of the sample on normal zebrafish was determined.

[0067] The results are shown in Table 2, and the MTC of the lactic acid bacterial bioinoculant on normal zebrafish is 7.81 μL / mL.

[0068] Table 2

[0069]

[0070] Example 3 Evaluation of the efficacy of lactic acid bacterial bioinoculant against Helicobacter pylori

[0071] Randomly select 3 dpf wild type AB strain zebrafish in a 6-well plate, and treat 30 zebrafish in each well with lactic acid bacterial bioinoculant, and set up a positive control group with a concentration of 125 μg / mL of clarithromycin, and set up a model control group, with a volume of 3 mL per well. After 48 h of treatment at 28°C, 5×10 7 CFU / mL of Helicobacter pylori were added to each group, as shown in Table 3. The model control group had a high fluorescence intensity of Helicobacter pylori in the gastrointestinal tract, and the Helicobacter pylori infection model of zebrafish was successfully established. After 24 h of treatment at 28°C, 10 zebrafish were randomly taken from each group and photographed under a fluorescence microscope, and the pictures were saved. The data were collected using NIS-Elements D3.20 advanced image processing software, and the fluorescence intensity of Helicobacter pylori in the gastrointestinal tract of zebrafish was analyzed. The statistical results of this index were used to evaluate the efficacy of the sample against Helicobacter pylori. The statistical processing results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, p <0.05 indicates that the difference is statistically significant.

[0072] Positive control: clarithromycin, white powder, batch number E1529013, Shanghai Aladdin Biochem Technology Co., Ltd. Prepared into 12.5 mg / mL stock solution with DMSO, stored at -20℃.

[0073] The results showed that the lactobacillus bio-stimulant had the efficacy of resisting H. pylori (Table 3, Figs. 1-2 ).

[0074] Table 3

[0075]

[0076] Compared with the model control group, p <0.05, *** p <0.001.

[0077] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. Use of a postbiotic in the preparation of a medicament for preventing and / or treating Helicobacter pylori infection, characterized in that: The raw materials of the postbiotics are composed of culture solutions of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001 and Lactobacillus reuteri HCS02-001; The deposit number of the plant lactobacillus RH03147 is CGMCC No. 25775; The deposit number of the Lactobacillus plantarum HCS03-001 is CGMCC No. 16258; The deposit number of the Lactobacillus reuteri HCS02-001 is CGMCC No. 19746. The preparation method of the postbiotics is as follows: (1) Take the frozen tubes of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001, and Lactobacillus reuteri HCS02-001 stored in a -80℃ refrigerator, immediately place them in a 37℃ water bath for 15-30 seconds, and wait for the frozen tubes to completely melt before resuscitating the strains. Inoculate the revived strains into different culture media A at a 10% inoculation rate, and culture them in a 37℃ incubator for 17 hours to obtain seed liquid. Inoculate the seed liquid into different culture media A at a 5% inoculation rate, and culture them in a 37℃ incubator for 17 hours to obtain the culture liquid of the three strains. (2) The culture fluids of Lactobacillus plantarum RH03147, Lactobacillus plantarum HCS03-001, and Lactobacillus reuteri HCS02-001 were inoculated into the same culture medium B at a ratio of 3.5:3.5:1, with an inoculum size of 5%, and cultured at 35°C with stirring for 17 h to obtain a primary fermentation fluid; (3) Immediately after step (2), the primary fermentation broth was transferred to medium B again at a 5% inoculum volume and cultured at 35°C with stirring for 48 h to obtain the secondary fermentation broth; (4) After filtering the secondary fermentation broth, a colorless to light yellow, transparent, impurity-free filtrate was obtained. The filtrate was heated and concentrated twice, and then sterilized by steaming for 30 min to obtain a postbiotic liquid. 8% maltodextrin was added to the postbiotic liquid, and the mixture was mixed and stirred until completely dissolved. The ambient humidity was ≤50%, and the mixture was placed in a spray dryer for spray drying at an inlet air temperature of 180°C and an outlet air temperature of 110°C to obtain a postbiotic powder. (5) The preparation of the culture medium A is as follows: 12.0 g / L yeast peptone, 25.0 g / L glucose, 8.0 g / L yeast extract, 8.0 g / L sodium acetate, 3.0 g / L citric acid monohydrate, 3.0 g / L potassium dihydrogen phosphate, 0.2 g / L Tween 80, 0.8 g / L magnesium sulfate, and the balance is purified water; weigh according to the formula ratio, heat to dissolve, sterilize at 115°C for 30 min, and adjust the pH value of the culture medium to 6.60 with 1 mol / L food-grade NaOH solution; (6) The preparation of the culture medium B is as follows: yeast peptone 15.0 g / L, glucose 20.0 g / L, yeast extract 12.0 g / L, sodium acetate 5.0 g / L, citric acid monohydrate 2.0 g / L, potassium dihydrogen phosphate 2.0 g / L, Tween 80 0.2 g / L, magnesium sulfate 0.6 g / L, manganese sulfate 0.25 g / L, and the balance is purified water; weigh according to the formula ratio, heat to dissolve, sterilize at 115°C for 30 min, and adjust the pH value of the culture medium to 6.80 with 1 mol / L food-grade NaOH solution.

2. The use according to claim 1, characterized in that The postbiotics are metabolite postbiotics.

3. The use according to claim 1, characterized in that The postbiotics further include at least one physiologically acceptable excipient.

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