Lactobacillus reuteri LR69 capable of enhancing gastric mucosal barrier and application, product and method thereof

Abstract

The invention discloses lactobacillus reuteri LR69 capable of enhancing a gastric mucosal barrier and application, a product and a method thereof, and belongs to the technical field of microorganisms. The invention provides a lactobacillus reuteri LR69, and the preservation number of the lactobacillus reuteri LR69 is CGMCC (China General Microbiological Culture Collection Center) No.33731. The lactobacillus reuteri LR69 is a lactobacillus reuteri LR69. The invention also provides application of the strain in preparation of food with an auxiliary protection function on gastric mucosa, and / or food with an intestinal flora regulating function, and / or a product for relieving gastritis, and / or food with an immunity enhancing function, and / or a bacteriostatic agent. The invention further discloses a food with an auxiliary protection function on gastric mucosa, a food with an intestinal flora regulating function, a product for relieving gastritis, a food with an immunity enhancing function, a bacteriostatic agent and an in-vitro bacteriostasis method based on the strain. The lactobacillus reuteri LR69 disclosed by the invention can effectively assist in protecting gastric mucosa, regulating intestinal flora, relieving gastritis, enhancing immunity and realizing broad-spectrum bacteriostasis.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Lactobacillus mucosae LR69 capable of enhancing the gastric mucosal barrier, and its application, product and method. Background Art

[0002] Helicobacter pylori ( Helicobacter pylori ) infection is one of the main causes of chronic gastritis, peptic ulcer and gastric cancer. At present, the eradication of Helicobacter pylori infection mainly relies on antibiotic treatment, but problems such as the increase in antibiotic resistance and the side effects brought by the treatment are becoming increasingly prominent. Therefore, finding safe and effective alternative or adjuvant treatment methods has become a research hotspot. In recent years, probiotics have received extensive attention due to their regulatory effects on the intestinal flora and potential benefits to host health. Lactobacillus mucosae is a lactic acid bacterium widely present in the intestines of humans and animals, and has a variety of probiotic functions, including inhibiting the growth of pathogenic bacteria, regulating immune responses and maintaining intestinal barrier function, etc. Research shows that Lactobacillus mucosae has a certain inhibitory effect on Helicobacter pylori infection, but its specific mechanism of action and application potential still need to be further studied. There has been no report on Lactobacillus mucosae that can inhibit Helicobacter pylori colonization, enhance the gastric mucosal barrier, and relieve Helicobacter pylori-induced gastritis based on regulating the intestinal flora.

[0003] Therefore, there is a need in the art to develop a new strain of Lactobacillus mucosae that simultaneously has the functions of enhancing the gastric mucosal barrier, regulating the intestinal flora, inhibiting the growth of Helicobacter pylori, and relieving Helicobacter pylori-induced gastritis. Summary of the Invention

[0004] For the purpose of solving the above-mentioned needs in the art, the present invention provides a Lactobacillus mucosae LR69 with the functions of inhibiting Helicobacter pylori, enhancing the gastric mucosal barrier, relieving Helicobacter pylori-induced gastritis, and regulating the intestinal flora, and its application, product and method.

[0005] The object of the present invention is achieved by the following technical solutions: A Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69, with a preservation number of CGMCC No. 33731.

[0006] The application of Lactobacillus mucosae LR69 with a preservation number of CGMCC No. 33731 in the preparation of a food with an auxiliary protective function for the gastric mucosa, and / or a food with a function of regulating the intestinal flora, and / or a product for relieving gastritis, and / or a food with an enhanced immune function, and / or an antibacterial agent.

[0007] The auxiliary protective function for the gastric mucosa includes: enhancing the gastric mucosal barrier and repairing gastric mucosal damage; Preferably, the alleviation of gastritis includes: inhibiting the growth of Helicobacter pylori, reducing the urease content, alleviating the inflammatory infiltration of gastric tissue, enhancing the gastric mucosal barrier, repairing gastric mucosal damage, inhibiting the adhesion and colonization of Helicobacter pylori, and regulating cytokine levels; Preferably, the enhancement of the gastric mucosal barrier includes: increasing the gene expression levels of the tight junction protein zonula occludens-1 and claudin in the gastric mucosa, and increasing the gene expression levels of mucin 1 and mucin 2 in the stomach; Preferably, the repair of gastric mucosal damage includes: increasing the contents of serum PGI and PGII; Preferably, the enhancement of immunity includes: regulating cytokine levels; Preferably, the regulation of cytokine levels includes: reducing the levels of IL-8, IL-6, IL-1β, and TNF-α; Preferably, the antibacterial spectrum of the bacteriostatic agent includes: Helicobacter pylori, Streptococcus mitis, Streptococcus paris, Streptococcus pasteurianus, Streptococcus sanguinis, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Klebsiella pneumoniae; Preferably, the regulation of the intestinal flora includes: increasing the relative abundances of Bacteroidales- Muribaculaceae、 Dubosiella Duboslella、 Lachnospiraceae -NK4A136-group and Akkermansia Akkermansia and decreasing the relative abundances of Helicobacter Helicobacter、 Bacteroides Bactrodales Turicibacter Tuicibacter and Helcococcus Mucispirillum ;

[0008] A food with auxiliary protection function for gastric mucosa, comprising: an active ingredient; the active ingredient includes: Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731.

[0009] A food for enhancing immune function, comprising: an active ingredient; the active ingredient includes: Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731.

[0010] A food for regulating intestinal flora, comprising: an active ingredient; the active ingredient includes: Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731.

[0011] A product for alleviating gastritis, comprising: an active ingredient; the active ingredient includes: Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731.

[0012] An antibacterial agent, comprising: an active ingredient; the active ingredient includes: Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731.

[0013] A method for antibacterial in vitro for non-therapeutic purposes, using Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731 for antibacterial.

[0014] The antibacterial spectrum of the antibacterial includes: Helicobacter pylori, Streptococcus mitis, Streptococcus paris, Streptococcus pasteurianus, Streptococcus sanguinis, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Klebsiella pneumoniae.

[0015] The beneficial effects of the present invention are as follows: A new strain was screened from adult fecal samples in the present invention. Through molecular identification, this strain is Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69. It has been confirmed by a large number of experiments in the present invention that the new strain LR69 has a high gastric juice tolerance ability, can enhance the gastric mucosal barrier, relieve gastric mucosal damage induced by Helicobacter pylori, relieve Helicobacter pylori-induced gastritis. This strain LR69 can effectively inhibit the growth of Helicobacter pylori, inhibit the urease activity of Helicobacter pylori, inhibit the secretion of pro-inflammatory factors IL-8, IL-6, IL-1β, TNF-α, and relieve Helicobacter pylori-induced gastritis in mice. At the same time, this strain LR69 also has a significant promoting effect on regulating the intestinal flora structure disorder caused by Helicobacter pylori, increasing the abundance of beneficial bacteria, and reducing the abundance of harmful bacteria, effectively regulating the intestinal flora.

[0016] The preservation information of Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69 of the present invention is as follows: Preservation number: CGMCC No. 33731; Classification and naming: Lactobacillus mucosae Limosilactobacillus reuteri ; Preservation date: March 6, 2025; Preservation unit: China General Microbiological Culture Collection Center; Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the Drawings

[0017] Figure 1 It is a rapid urease detection map of gastric tissues of mice in each group of the Helicobacter pylori-induced mouse gastritis model in part (2) of Experimental Example 6 of the present invention.

[0018] Figure 2 It is a H&E staining section map of gastric tissues of mice in each group of the Helicobacter pylori-induced gastritis model in part (3) of Experimental Example 6 of the present invention.

[0019] Figure 3 Bar chart showing the gene expression levels of tight junction protein zonula occludens-1 and claudin, as well as the gene expression levels of gastric mucin-1 and mucin-2 in the gastric mucosa of mice in each group of the Helicobacter pylori-induced mouse gastritis model in Part (4) of Experimental Example 6 of the present invention.

[0020] Figure 4 Bar chart showing the urease content in the serum samples of mice in the Helicobacter pylori-induced gastritis model in Part (5) of Experimental Example 6 of the present invention.

[0021] Figure 5 Bar chart showing the contents of PGI and PGII in the serum samples of mice in each group of the Helicobacter pylori-induced mouse gastritis model in Part (6) of Experimental Example 6 of the present invention.

[0022] Figure 6 Bar chart showing the levels of pro-inflammatory factors in mice in each group of the Helicobacter pylori-induced mouse gastritis model in Part (7) of Experimental Example 6 of the present invention.

[0023] Figure 7 Analysis diagrams of β-diversity and α-diversity of intestinal flora in mice in each group of the Helicobacter pylori-induced mouse gastritis model in Part (8) of Experimental Example 6 of the present invention; wherein, a is the analysis result diagram of β-diversity of intestinal flora in each group of mice; b is the bar chart of ACE index of intestinal flora in each group of mice, c is the bar chart of Chao1 index of intestinal flora in each group of mice, d is the bar chart of Simpson index of intestinal flora in each group of mice, and e is the bar chart of Shannon index of intestinal flora in each group of mice.

[0024] Figure 8 Analysis diagram of species diversity of intestinal flora at the phylum level in mice in each group of the Helicobacter pylori-induced mouse gastritis model in Part (8) of Experimental Example 6 of the present invention; wherein, a is the bar chart of relative abundance of species diversity at the phylum level of intestinal flora in each group of mice; b is the bar chart of relative abundance of Firmicutes, c is the bar chart of relative abundance of Bacteroidetes, and d is the bar chart of relative abundance of Proteobacteria.

[0025] Figure 9 Analysis diagram of species diversity of intestinal flora at the genus level in mice in each group of the Helicobacter pylori-induced mouse gastritis model in Part (8) of Experimental Example 6 of the present invention.

[0026] Figures 1 - 9 The meanings of each label are listed as follows: NC: blank control group; HP: model group; LR69: intervention group of Lactobacillus mucosae LR69.

[0027] Figure 7 The meanings of each label are listed as follows: a: represents β-diversity of intestinal flora analysis; b: represents ACE index; c: represents Chao1 index; d: represents Simpson index; e: represents Shannon index; Figure 8The meanings of the respective markers are listed as follows: a: represents the analysis of species diversity at the phylum level of the intestinal flora; b: represents the bar chart of Firmicutes at the phylum level for each group; c: represents the bar chart of Bacteroidetes at the phylum level for each group; e: represents the bar chart of Proteobacteria at the phylum level for each group; Detailed implementation mode

[0028] The following further specifically describes the detailed content of the present invention in combination with specific experimental examples, but does not limit the protection scope of the present invention hereby.

[0029] Source of biological material I. The Lactobacillus mucosae Roy used in Experimental Example 2 of the present invention is preserved by Wuhan Microkang Probiotics Research Institute Co., Ltd.; the applicant undertakes to make it available to the public for verifying the technical effects of the present invention within 20 years from the application date of the present invention.

[0030] II. Helicobacter pylori used in Experimental Example 2 and Experimental Example 6 of the present invention Helicobacter pylori SS1 is purchased from the Guangdong Microbial Preservation Center; the Lactobacillus casei rhamnosus LGG used in Experimental Example 2 is commercially available.

[0031] III. Streptococcus sanguinis ATCC10556, Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, Staphylococcus aureus ATCC25923 used in Experimental Example 5 of the present invention are purchased from the Guangdong Microbial Preservation Center; Klebsiella pneumoniae MT573143, this strain has been registered in NCBI, and its accession number is MT459365; Streptococcus mitis is Streptococcus mitis 5013, this strain has been registered in NCBI, and its accession number is MT512114; Streptococcus parasanguinis is Streptococcus parasanguinis 21178, this strain has been registered in NCBI, and its accession number is OM403351; Streptococcus pasteurianus is Streptococcus pasteurianus 12758, this strain has been registered in NCBI, and its accession number is MW445225; those skilled in the art can obtain the above-mentioned strains according to the NCBI registration information in combination with the conventional technical means in the field of molecular biology; the above-mentioned strains are all preserved in the applicant's laboratory, and the applicant undertakes to make them available to the public for verifying the technical effects of the present invention within 20 years from the application date of the present invention.

[0032] IV. The C57BL / 6 mice used in Experimental Example 6 of the present invention are purchased from the Animal Experiment Center of Huazhong Agricultural University.

[0033] Group 1 Example, Lactobacillus mucosae Roy LR69 of the present invention.

[0034] This group of examples provides a Lactobacillus mucosae Roy ( Limosilactobacillus reuteri)LR69. All the embodiments in this group have the following common features: The Lactobacillus mucosae LR69 with the functions of inhibiting the growth of Helicobacter pylori, enhancing the gastric mucosal barrier, and alleviating Helicobacter pylori-induced gastritis Limosilactobacillus reuteri )The preservation number of LR69 is CGMCC No. 33731.

[0035] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, reconstructing, selling, or offering to sell a strain of Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731, and / or, the act of combining a strain of Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731 with other probiotics, and / or, the antagonism of a strain of Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731 against various pathogenic bacteria, and / or, the preparation of bacteriostatic agents, and / or, the preparation of foods with enhanced immune function, and / or, the preparation of foods for regulating intestinal flora functions all fall within the protection scope of the present invention.

[0036] The other probiotics include, but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Torulaspora delbrueckii, Candida spp., Wickerhamomyces spp., Pichia spp., Saccharomyces boulardii, Trichosporonoides spp., Schwanniomyces spp., Rhodotorula rubra, Schizosaccharomyces pombe, Pichia kudriavzevii, Bacillus thuringiensis, Brevibacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotofixans, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium infantis (i.e., Bifidobacterium longum subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. lactis), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium thermophilum, and Bifidobacterium thermophilum acidophilus.

[0037] Those skilled in the art can, according to actual production needs, combine the conventional technical means or common knowledge in the production process of the pharmaceutical field (for example, "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Bacterial Agents", etc.), make conventional selections or adjustments to pharmaceutical excipients, and then prepare products of a strain of Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731 in different dosage forms, different storage conditions, and different shelf lives. This poses no technical obstacles for those skilled in the art and can be easily achieved.

[0038] Group 2 of Examples, Applications of the Lactobacillus mucosae LR69 of the Present Invention This group of examples provides the application of Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731 in the preparation of functional foods with an auxiliary protective function for gastric mucosa, and / or functional foods for regulating intestinal flora, and / or products for relieving gastritis, and / or functional foods for enhancing immune function, and / or bacteriostatic agents.

[0039] In a specific example, the auxiliary protective function for gastric mucosa includes: enhancing the gastric mucosa barrier and repairing gastric mucosa damage; Preferably, the relief of gastritis includes: inhibiting the growth of Helicobacter pylori, reducing the content of urease, relieving the inflammatory infiltration of gastric tissue, enhancing the gastric mucosa barrier, repairing gastric mucosa damage, inhibiting the adhesion and colonization of Helicobacter pylori, and regulating cytokine levels; Preferably, enhancing the gastric mucosa barrier includes: increasing the gene expression levels of tight junction protein zonula occludens-1 and claudin in the gastric mucosa, and increasing the gene expression levels of mucin 1 and mucin 2 in the stomach; Preferably, repairing gastric mucosa damage includes: increasing the contents of serum PGI and PGII; Preferably, enhancing immunity includes: regulating cytokine levels; Preferably, regulating cytokine levels includes: reducing the levels of IL-8, IL-6, IL-1β, and TNF-α; Preferably, the bacteriostatic spectrum of the bacteriostatic agent includes: Helicobacter pylori, Streptococcus mitis, Streptococcus paris, Streptococcus pasteurianus, Streptococcus sanguinis, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Klebsiella pneumoniae.

[0040] Preferably, regulating intestinal flora includes: increasing the relative abundances of Bacteroidales- Muribaculaceae、 Dubosiella Duboslella、 Lachnospiraceae -NK4A136-group and Akkermansia Akkermansia and reducing the relative abundances of Helicobacter Helicobacter、 Bacteroides Bactrodales , Turicibacter Tuicibacter and Helicophaga Mucispirillum relative abundances.

[0041] Group 3 of Examples, the Gastritis Relief Product of the Present Invention This group of examples provides a gastritis relief product. All examples in this group have the following common characteristics: the gastritis relief product includes: an active ingredient; the active ingredient includes: Lactobacillus mucosae ( Limosilactobacillus reuteri ), LR69 with the preservation number of CGMCC No. 33731.

[0042] In a further embodiment, the product for relieving Helicobacter pylori-induced gastritis is characterized by further comprising: excipients.

[0043] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0044] According to the content of the present invention, for different requirements in actual production applications, combined with conventional technical means in the field of preparation (such as "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Bacterial Agents", etc.), those skilled in the art can select and formulate the above excipients, and Lactobacillus mucosae LR69 with the preservation number CGMCC No. 33731 can be made into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0045] In a specific embodiment, the dosage form of the product for relieving Helicobacter pylori-induced gastritis is selected from one or more of: powders, tablets, liquids, capsules.

[0046] Group 4 Embodiments, Immune Function-Enhancing Food of the Present Invention This group of embodiments provides an immune function-enhancing food. All embodiments in this group have the following common features: The immune function-enhancing food includes: active ingredients; the active ingredients include: Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69 with the preservation number CGMCC No. 33731.

[0047] In a further embodiment, the immune function-enhancing food further comprises: excipients.

[0048] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0049] According to the content of the present invention, for different requirements in actual production applications, combined with the conventional technical means in the field of preparation technology (such as "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Inoculant Technology", etc.), those skilled in the art can select and formulate the above excipients, and prepare Lactobacillus mucosae LR69 with the preservation number CGMCC No. 33731 into different dosage forms, such as powder, tablet, oral liquid, gel, patch, spray, lotion, granule, etc.

[0050] In a specific embodiment, the dosage form of the food for enhancing immune function is selected from one or more of: powder, tablet, liquid, capsule.

[0051] Group 5 Examples, Food for Assisting in Protecting Gastric Mucosa According to the Present Invention This group of examples provides a food for assisting in protecting gastric mucosa. All examples in this group have the following common characteristics: The food for assisting in protecting gastric mucosa includes: active ingredients; the active ingredients include: Lactobacillus mucosae LR69 with the preservation number CGMCC No. 33731.

[0052] In a further embodiment, the food for assisting in protecting gastric mucosa further includes: excipients.

[0053] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0054] According to the content of the present invention, for different requirements in actual production applications, combined with the conventional technical means in the field of preparation technology (such as "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Inoculant Technology", etc.), those skilled in the art can select and formulate the above excipients, and prepare Lactobacillus mucosae LR69 with the preservation number CGMCC No. 33731 into different dosage forms, such as powder, tablet, oral liquid, gel, patch, spray, lotion, granule, etc.

[0055] In a specific embodiment, the dosage form of the functional food with an auxiliary protective function for gastric mucosa is selected from one or more of the following: powder, tablet, liquid, capsule.

[0056] Group 7 Examples, A Functional Food for Regulating Intestinal Flora of the Present Invention This group of examples provides a functional food for regulating intestinal flora. All examples in this group have the following common characteristics: The functional food for regulating intestinal flora includes: active ingredients; the active ingredients include: Lactobacillus mucosae LR69 with the preservation number CGMCC No. 33731.

[0057] In a further embodiment, the functional food for regulating intestinal flora further includes: excipients.

[0058] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0059] According to the content of the present invention, for different requirements in actual production applications, combined with conventional technical means in the field of preparation (for example, "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Bacterial Agents", etc.), those skilled in the art can select and formulate the above excipients, and make Lactobacillus mucosae LR69 with the preservation number CGMCC No. 33731 into different dosage forms, such as powder, tablet, oral liquid, gel, patch, spray, lotion, granule, etc.

[0060] In a specific embodiment, the dosage form of the functional food for regulating intestinal flora is selected from one or more of the following: powder, tablet, liquid, capsule.

[0061] Group 8 Examples, The Bacteriostatic Agent of the Present Invention This group of examples provides a bacteriostatic agent. All examples in this group have the following common characteristics: The bacteriostatic agent includes: active ingredients; the active ingredients include: Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69 with the preservation number CGMCC No. 33731.

[0062] In a further embodiment, the bacteriostatic agent further includes: excipients.

[0063] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

[0064] According to the content of the present invention, for different requirements in actual production applications, combined with conventional technical means in the field of preparation (for example, "Encyclopedia of Pharmaceutical Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Their Fermented Foods", "Research and Application of Microbial Inoculant Technology", etc.), those skilled in the art can select and formulate the above excipients, and make Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0065] In a specific embodiment, the dosage form of the bacteriostatic agent is selected from one or more of: powders, tablets, liquids, capsules.

[0066] Experimental Example Group 9, in vitro bacteriostatic method of the present invention This group of experimental examples provides a non-therapeutic in vitro bacteriostatic method. All experimental examples in this group have the following common characteristics: using Lactobacillus mucosae LR69 with the preservation number of CGMCC No. 33731 for bacteriostasis.

[0067] In a specific experimental example, the bacteria to be bacteriostatically inhibited include: Helicobacter pylori, Streptococcus mitis, Streptococcus paris, Streptococcus pasteurianus, Streptococcus sanguinis, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Klebsiella pneumoniae.

[0068] "Above" and "below" in this article both include the present number.

[0069] "Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69", "Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69", "Lactobacillus mucosae LR69", "LR69", "LR69 strain", "strain LR69" in this article all refer to: Lactobacillus mucosae ( Limosilactobacillus reuteri ) LR69 with the preservation number of CGMCC No. 33731.

[0070] The culture media and reagents used in the experimental examples of the present invention are as follows: MRS medium: Peptone 10 g / L, Beef extract powder 5 g / L, Yeast powder 5 g / L, Glucose 20 g / L, Sodium acetate anhydrous 5 g / L, Diammonium hydrogen citrate 2 g / L, Tween 80 1 g / L, K 2 HPO 4 2 g / L, MgSO 4 0.2 g / L, MnSO 4 0.05 g / L, pH = 6.80 ± 0.20, 121 °C, sterilized for 20 min.

[0071] LMRS medium: Based on MRS medium, add 0.05% (w / v) L-cysteine hydrochloride, pH = 6.80 ± 0.20, 121 °C, sterilized for 20 min.

[0072] BHI medium: Peptone 10 g / L, Dehydrated calf brain extract powder 12.5 g / L, Dehydrated beef heart extract powder 5 g / L, Sodium chloride 5 g / L, Glucose 2.0 g / L, Disodium hydrogen phosphate 2.5 g / L, pH = 7.40 ± 0.20, 121 °C, sterilized for 20 min.

[0073] BHI blood agar medium: Based on BHI medium, add 1.5% Columbia agar base and 5% defibrinated sheep blood.

[0074] Columbia agar base, BHI medium, Nutrient agar medium, TSA medium are all commercial media.

[0075] Mouse-derived urease (UE), Pepsinogen I (PGI), Pepsinogen II (PGII), IL-8, IL-6, IL-1β, TNF-α are all purchased from Wuhan Beinilai Biotechnology Co., Ltd.

[0076] Unless otherwise specified, all kinds of reagents and consumables used in the experimental examples and experimental examples of the present invention can be obtained commercially, and the relevant experimental steps are common operations in the art and have technical meanings that can be routinely understood by those skilled in the art.

[0077] The activation and preparation method of bacterial suspensions of the strains in the following experimental examples are as follows: Activated culture of Lactobacillus mucosae: The strain is taken out from the -80 °C refrigerator and inoculated into LMRS liquid medium at an inoculation amount of 2% (v / v), and cultured statically at 37 °C to obtain the first-generation Lactobacillus mucosae; then inoculated into LMRS liquid medium at an inoculation amount of 2% (v / v) and cultured statically at 37 °C for 16 h to obtain the Lactobacillus mucosae bacterial suspension for standby.

[0078] Preparation of Lactobacillus mucosae LR69 bacterial suspension: Lactobacillus mucosae LR69 was activated for 2 generations at an inoculation amount of 2%, and the cultured Lactobacillus mucosae bacterial solution was centrifuged at 10,000 r / min for 1 min. The supernatant was poured out, and the bacterial cells were washed twice with an equal volume of PBS and then resuspended with the same volume of PBS to obtain the Lactobacillus mucosae LR69 bacterial suspension for standby.

[0079] Culture of Helicobacter pylori SS1: Sydney strain of Helicobacter pylori H. pylori SS1 was inoculated onto a BHI blood agar plate at an inoculation amount of 1% and cultured in a triple-gas incubator at 37°C (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 72 - 96 h to obtain a Helicobacter pylori blood agar plate.

[0080] Preparation of Helicobacter pylori SS1 bacterial suspension: The cultured Helicobacter pylori Sydney strain SS1 bacterial cells were collected, washed twice with PBS buffer, resuspended in BHI medium, and the bacterial cell concentration was adjusted to 1×10 7 CFU / mL to obtain a Helicobacter pylori bacterial suspension.

[0081] Culture of Klebsiella pneumoniae: Klebsiella pneumoniae was activated on a Columbia blood agar plate medium and incubated in an incubator at 37°C for 24 h to obtain a Klebsiella pneumoniae blood agar plate.

[0082] Preparation of Klebsiella pneumoniae bacterial suspension: The Klebsiella pneumoniae bacterial cells were collected, washed twice with PBS buffer, and then resuspended in PBS buffer, and the concentration was adjusted to 10 8 CFU / mL for standby.

[0083] Culture of Streptococcus mitis: Streptococcus mitis was inoculated onto a BHI + defibrinated sheep blood agar plate medium at an inoculation amount of 1% and incubated in an incubator at 37°C for 24 h to obtain a Streptococcus mitis blood agar plate.

[0084] Preparation of Streptococcus mitis bacterial suspension: The cultured Streptococcus mitis bacterial cells of the second generation were collected, washed twice with PBS buffer, resuspended in PBS, and the bacterial cell concentration was adjusted to 1×10 8 CFU / mL to obtain a Streptococcus mitis bacterial suspension.

[0085] Culture of Streptococcus paris: Streptococcus paris was inoculated onto a TSA + defibrinated sheep blood agar plate medium at an inoculation amount of 1% and incubated in an incubator at 37°C for 24 h to obtain a Streptococcus paris blood agar plate.

[0086] Preparation of Streptococcus paris bacterial suspension: The cultured Streptococcus paris bacterial cells of the second generation were collected, washed twice with PBS buffer, resuspended in PBS, and the bacterial cell concentration was adjusted to 1×10 8 CFU / mL to obtain a Streptococcus paris bacterial suspension.

[0087] Culture of Streptococcus pasteurianus: Streptococcus pasteurianus was inoculated into BHI + defibrinated sheep blood agar plate medium at an inoculation amount of 1%, and cultured in a constant temperature incubator at 37°C for 24 h. A Streptococcus pasteurianus blood plate was obtained.

[0088] Preparation of Streptococcus pasteurianus bacterial suspension: The cultured Streptococcus pasteurianus cells of the second generation were collected, washed twice with PBS buffer, resuspended with PBS and adjusted to a cell concentration of 1×10 8 CFU / mL to obtain a Streptococcus pasteurianus bacterial suspension.

[0089] Culture of Streptococcus sanguinis: Streptococcus sanguinis was inoculated into Columbia blood agar plate medium at an inoculation amount of 1%, and cultured in a triple gas incubator at 37°C (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 24 h. A Streptococcus sanguinis blood plate was obtained.

[0090] Preparation of Streptococcus sanguinis bacterial suspension: The cultured Streptococcus sanguinis cells of the second generation were collected, washed twice with PBS buffer, resuspended with PBS and adjusted to a cell concentration of 1×10 8 CFU / mL to obtain a Streptococcus sanguinis bacterial suspension.

[0091] Culture of Escherichia coli: Escherichia coli was inoculated into LB liquid medium at an inoculation amount of 1%, and cultured in a constant temperature incubator at 37°C for 24 h. An Escherichia coli bacterial solution was obtained.

[0092] Preparation of Escherichia coli bacterial suspension: The Escherichia coli bacterial solution was washed twice with PBS buffer, the cells were collected, resuspended with PBS and adjusted to a cell concentration of 1×10 8 CFU / mL to obtain an Escherichia coli bacterial suspension.

[0093] Culture of Staphylococcus aureus: Staphylococcus aureus was inoculated into LB liquid medium at an inoculation amount of 1%, and cultured in a constant temperature incubator at 37°C for 24 h. A Staphylococcus aureus bacterial solution was obtained.

[0094] Preparation of Staphylococcus aureus bacterial suspension: The Staphylococcus aureus bacterial solution was washed twice with PBS buffer, the cells were collected, resuspended with PBS and adjusted to a cell concentration of 1×10 8 CFU / mL to obtain a Staphylococcus aureus bacterial suspension.

[0095] Culture of Salmonella enteritidis: Salmonella enteritidis was inoculated into LB liquid medium at an inoculation amount of 1%, and cultured in a constant temperature incubator at 37°C for 24 h. A Salmonella enteritidis bacterial solution was obtained.

[0096] Preparation of Salmonella enteritidis bacterial suspension: The Salmonella enteritidis bacterial solution was washed twice with PBS buffer, the cells were collected, resuspended with PBS and adjusted to a cell concentration of 1×10 8A Salmonella enteritidis bacterial suspension was obtained at CFU / mL.

[0097] Experimental Example 1: Isolation and Identification of Lactobacillus mucosae (1)Isolation and screening of Lactobacillus mucosae The collected adult feces were placed in a sterile sampling tube and transported in an ice box. Under sterile conditions, they were serially diluted with 0.85% saline. An appropriate dilution gradient was selected and spread on an LBS agar plate, and cultured at 37°C for 48 - 72 hours. Suspected single colonies were picked by observing their colony morphology with the naked eye, and were microscopically examined, preliminarily screened, and purified. After purification, they were cultured in MRS liquid medium at 37°C for 8 - 12 hours. After centrifugation to remove the supernatant, they were resuspended in a sterile 25% glycerol aqueous solution and stored in the strain library of Wuhan Microkang Probiotics Research Institute.

[0098] (2)Identification of Lactobacillus mucosae: The target strain was liquid-cultured for expansion, the bacterial cells were collected, genomic DNA was extracted, and the 16S rDNA fragment was amplified using the universal primers 27F and 1492R described in paragraph 58 of Chinese invention patent ZL202210478937.4. The PCR amplification product was detected by agarose gel electrophoresis, and the PCR product was sequenced. The PCR reaction system was as follows: 10×buffer 10 μL, 10 mM dNTP 2 μL, 1 μL each of the upstream and downstream primers, 2 μL of DNA template, 0.5 μL of Taq enzyme, and ddH 2 O 34 μL. Pre-denaturation at 95°C for 10 min; then 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and after completion, extension at 72°C for 5 min. The PCR product was detected by gel electrophoresis and then sent to Wuhan Kingcare Bioengineering Co., Ltd. for sequencing. The identified gene sequence was aligned in the NCBI database using the BLAST tool. According to the molecular biology identification results, the Latin name of the strain category was Limosilactobacillus reuteri , and it was determined that the isolated strain was Lactobacillus mucosae.

[0099] Experimental Example 2: Inhibition of Helicobacter pylori H. pylori SS1 Screening of Lactobacillus mucosae (1)Preparation of Lactobacillus mucosae bacterial liquid: Lactobacillus mucosae 01 - 05 was activated for 2 generations at an inoculation amount of 2%. The cultured Lactobacillus mucosae bacterial liquid was centrifuged at 10000 r / min for 1 min, the supernatant was poured out, the bacterial cells were washed twice with an equal volume of PBS, and then resuspended with the same volume of PBS to obtain the Lactobacillus mucosae LR69 bacterial suspension for standby.

[0100] (2)Helicobacter pylori H. pylori SS1Preparation of bacterial suspension: The activated Helicobacter pylori SS1 was rinsed with PBS buffer and then resuspended in BHI liquid medium, and the concentration was adjusted to 1×10 8 CFU / mL.

[0101] (3)Antagonism experiment: An appropriate amount of Helicobacter pylori was scraped from the plate and resuspended in 5 mL of BHI culture medium to prepare a bacterial suspension. Its concentration was adjusted to 1×10 7 CFU / mL with BHI. Fixed with a sterile punch on the plate, and 20 mL of BHI blood agar medium mixed with H. pylori SS1 was poured. After the medium solidified, the punch was carefully picked out with forceps to make a round hole, and 150 μL of probiotic bacterial liquid was injected into the hole. It was cultured for 72 h under microaerobic conditions at 37°C, and the size of the inhibition zone was measured. Each treatment was repeated 3 times.

[0102] Table 1. Antagonism experiment results of Lactobacillus mucosae on H. pylori SS1

[0103] Note: "abcde" indicates significant differences between strains, p <0.05;

[0104] Table 1 Antagonism experiment results of different Lactobacillus mucosae on Helicobacter pylori H. pylori SS1 The results of the antagonism experiment showed that different Lactobacillus mucosae had inhibitory effects on Helicobacter pylori. However, there were significant differences between strains (p<0.05). Among them, Lactobacillus mucosae LR69 had the strongest inhibitory ability on Helicobacter pylori, and the diameter of the inhibition zone was as high as 19.5 mm, significantly higher than that of the control strain Lactobacillus rhamnosus GG, indicating that Lactobacillus mucosae 05 had the potential to inhibit Helicobacter pylori. This strain was named Lactobacillus mucosae LR69 and deposited, and its deposit information is as follows: H. pylori SS1 Deposit number: CGMCC No. 33731; H. pylori SS1 Taxonomic name: Lactobacillus mucosae ; Deposit date: March 6, 2025; Limosilactobacillus reuteri ; Deposit unit: China General Microbiological Culture Collection Center; Deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0105] Experimental Example 3, Surface hydrophobicity of Lactobacillus mucosae LR69 ​Centrifuge the activated and cultured LR69 bacterial solution at 5000 rmp for 5 min to collect the bacterial cells. Wash the cells twice with an equal volume of PBS buffer, resuspend them in PBS buffer, and adjust the OD600 value of the bacterial suspension to around 1.0. At the same time, measure the absorbance value of the bacterial suspension at 600 nm (A 0 ). Take 12 mL of the above bacterial suspension and 4 mL of xylene in a test tube, mix well, let it stand at room temperature for 10 min, then vortex mix again, and let it stand at room temperature for 20 min for stratification. The transparent lower aqueous layer and the upper solvent layer containing cells are obtained. Take the aqueous phase and measure the absorbance value of the aqueous phase at 600 nm (A 1 ). Use the buffer as a blank control. The surface hydrophobicity is expressed as the percentage of bacteria adhering to the organic solvent: Surface hydrophobicity (%) = (1 - A 1 / A 0 ) × 100% Table 2. Results of the surface hydrophobicity of Lactobacillus mucosae LR69

[0106] The experimental results in Table 2 show that the surface hydrophobicity of LR69 is 94.98%. Some studies have shown that the stronger the surface hydrophobicity, the stronger the adhesion ability. This result indicates the possibility that Lactobacillus mucosae LR69 exhibits good adhesion ability.

[0107] Experimental Example 4. Tolerance of Lactobacillus mucosae LR69 to artificial gastric juice (1) Preparation of reagents: Preparation of artificial gastric juice: Prepare 0.5% normal saline, add 0.3% pepsin, adjust the pH to 2.5 with dilute hydrochloric acid 1M HCl, filter and sterilize with a 0.22 μm microporous filter membrane for standby after complete dissolution; (2) Preparation of experimental strains: According to the preparation method of the Lactobacillus mucosae LR69 bacterial suspension, obtain the Lactobacillus mucosae LR69 bacterial suspension, and adjust the viable cell count of the bacterial solution to 10 8 CFU / mL for the experiment according to the results of the previous viable cell count.

[0108] (3) Experiment on tolerance to artificial gastric juice: Take 0.1 mL of the bacterial suspension and add it to 0.9 mL of artificial gastric juice (pH = 2.5) respectively, and incubate at 37 °C for 3 h; perform gradient dilution on the treatment solution at 0 h and 3 h respectively, and spread it on the LMRS plate for viable cell count. Calculate the survival rate of the strain in artificial gastric juice and bile salts respectively according to the following calculation formula:

[0109] Among them, N1 represents the number of viable bacteria in the treated strain system, (CFU / mL); N0 represents the initial number of viable bacteria in the strain system, that is, the number of viable bacteria measured at 0 h (CFU / mL).

[0110] Table 3. Tolerance results of Lactobacillus mucosae LR69 in artificial gastric juice (pH = 2.5)

[0111] According to the experimental results in Table 3, the survival rate of Lactobacillus mucosae LR69 after 3 h of treatment in artificial gastric juice (pH = 2.5) is 75.47%, indicating good tolerance and suggesting that Lactobacillus mucosae LR69 can survive in the low-acid gastric juice environment.

[0112] Experimental Example 5. Inhibition of the growth of pathogenic bacteria by Lactobacillus mucosae LR69 Fix with a sterile puncher on the plate, pour 20 mL of LB agar medium mixed with 1% Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus respectively, pour 20 mL of BHI blood agar medium mixed with 1% Streptococcus mitis and Streptococcus pasteurianus respectively for the medium, pour 20 mL of nutrient agar medium mixed with 1% Klebsiella pneumoniae, pour 20 mL of TSA blood agar medium mixed with 1% Streptococcus paris, and pour 20 mL of Columbia blood agar medium mixed with Streptococcus sanguinis. After the medium solidifies, carefully pick out the puncher with forceps to make round holes, inject 150 μL of the fermentation supernatant of Lactobacillus mucosae LR69 into the holes, and culture for 24 h under microaerobic conditions at 37 °C. Measure the size of the inhibition zone, and repeat each treatment 3 times.

[0113] Table 4. Experimental results of the antagonism between Lactobacillus mucosae LR69 and different pathogenic bacteria

[0114] Note: "abcde" indicates significant differences between strains, p <0.05;

[0115] The experimental results in Table 4 show that Lactobacillus mucosae LR69 involved in this invention patent exhibits significant antagonistic effects against pathogenic bacteria such as Streptococcus mitis, Streptococcus paris, Streptococcus pasteurianus, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Salmonella enteritidis; among them, the inhibition zone diameters against Streptococcus paris, Streptococcus pasteurianus, Klebsiella pneumoniae, Escherichia coli, and Salmonella enteritidis are all greater than 20 mm. It shows that Lactobacillus mucosae has broad-spectrum antibacterial properties.

[0116] Experimental Example 6. Verification of the effect of Lactobacillus mucosae LR69 on relieving Helicobacter pylori-induced gastritis in mice (1) Establishment of Helicobacter pylori-induced murine gastritis model A total of 30 C57BL / 6 female mice (18 - 20 g) were housed in a controlled environment (12-hour light cycle, 22 ± 2 °C) with normal access to food and water. The experiment began after 7 days of adaptation in the experimental room. NC group (blank control group, 10 mice): normal diet, 200 μL of normal saline, intragastric administration once a day throughout the period after the adaptation period; HP group (model group, 10 mice): normal diet, 200 μL of normal saline, intragastric administration once a day throughout the period after the adaptation period; starting from the 14th, 16th, 18th, 20th, and 22nd days, 200 μL of HPSS1 live cells at 1 × 10 10 cfu / mL were intragastrically administered; LR69 group (Lactobacillus mucosae LR69 intervention group, 10 mice): normal diet, 200 μL of probiotic LR69 live cells (8 × 10 9 cfu / mL) resuspended in normal saline, intragastric administration once a day throughout the period after the adaptation period, and on the 14th, 16th, 18th, 20th, 22nd, 24th, and 26th days, 200 μL of HPSS1 live cells at 1 × 10 10 CFU / mL were intragastrically administered. After the infection ended, two randomly selected mice from the HP group were dissected to determine the colonization of Helicobacter pylori in the stomach; the remaining groups were not otherwise treated and were normally raised for 7 days, and then fasted. Blood was collected from the eye before euthanasia, and after euthanasia, the stomach was taken. The stomach was dissected and opened along the greater curvature. After washing with cold sterile normal saline, the stomach was divided into three longitudinal strips, fixed with 10% formaldehyde and frozen in an -80 °C refrigerator. One strip was used for histological staining (H&E). One strip was used for urease indicator detection. One strip was used for real-time fluorescence quantitative PCR to measure the gastric tissue mucosal barrier.

[0117] (2) Determination of urease activity in gastric tissue of Helicobacter pylori-induced gastritis mice After the mice were euthanized, the stomachs were taken. The stomachs were dissected and opened along the greater curvature. After washing with cold sterile normal saline, the stomachs were divided into three longitudinal strips. One piece was cut and placed in a rapid urease reagent, and the gastric glandular tissue was incubated in an oven at 37 °C for 6 h. A change in color to red indicated successful infection.

[0118] Table 5. Results of urease determination in gastric tissue of Lactobacillus mucosae LR69-treated Helicobacter pylori-induced gastritis mice

[0119]

[0120] According to Table 5 and Figure 1The results showed that after rapid urease determination of the gastric tissues of the mice in the model group, all turned red, and the positive rate of urease determination was 100%. This indicated that Helicobacter pylori was successfully colonized in the gastric tissues of the mice. The LR69 intervention group showed that only 2 mice turned red, and the positive rate of urease determination was 20%, suggesting that the intervention of Lactobacillus mucosae LR69 could inhibit the colonization of Helicobacter pylori.

[0121] (3)Effect of Lactobacillus mucosae LR69 on gastric tissue inflammatory infiltration in Helicobacter pylori-induced gastritis mice (H&E) After the mice were sacrificed, the stomachs were taken. The stomachs were dissected and opened along the greater curvature. After washing with cold sterile saline, the stomachs were divided into three longitudinal strips. One part was fixed in 10% formaldehyde solution for inflammatory infiltration and immunohistochemical analysis of the gastric tissues of the mice.

[0122] The inflammatory infiltration of the gastric tissues of the mice was as Figure 2 shown. In the gastric mucosa of the mice infected with Helicobacter pylori, gastric mucosal lesions, inflammatory cell infiltration, necrosis and exfoliation of red blood cells and mucosal surface cells all occurred. While in the LR69 intervention group, red blood cells and a small amount of cell exfoliation appeared in the gastric mucosa, and the damage symptoms were significantly lighter than those in the HP group, indicating that Lactobacillus mucosae LR69 could relieve the mucosal inflammatory damage of the gastric tissues of the mice.

[0123] (4)Determination of gastric mucosal barrier function in Helicobacter pylori-induced gastritis mice After the mice were sacrificed, the stomachs were taken. The stomachs were dissected and opened along the greater curvature. After washing with cold sterile saline, the stomachs were divided into three longitudinal strips. One part was used for real-time fluorescence quantitative PCR technology to detect the gene expression of the tight junction proteins zonula occludens-1 and occludin in the gastric mucosa, and to detect the gene expression of the mucins mucin-1 and mucin-2 in the stomach.

[0124] The gene expression of the tight junction proteins zonula occludens-1 and occludin in the gastric mucosa can reflect the tight junction degree of the gastric mucosa, and the gene expression of the mucins mucin-1 and mucin-2 in the stomach reflects the function of the gastric mucosa to secrete mucins. The results were as Figure 3 shown. Compared with the NC group, after Helicobacter pylori infection, the gene expression levels of the tight junction proteins zonula occludens-1 and occludin in the gastric mucosa of the mice decreased significantly ( p <0.01). After intervention with Lactobacillus mucosae LR69, the gene expression levels of the tight junction proteins zonula occludens-1 and occludin in the gastric mucosa of the mice increased significantly ( p <0.01). Compared with the NC group, after Helicobacter pylori infection, the gene expression levels of the mucins MUC-1 and MUC-2 in the stomach of the mice decreased significantly ( p <0.01). After intervention with Lactobacillus mucosae LR69, the gene expression levels of the mucins mucin-1 and mucin-2 in the stomach of the mice increased significantly ( p(<0.01). These results indicate that Lactobacillus mucosae LR69 can significantly enhance the gastric mucosal barrier in mice and alleviate gastric mucosal injury in mice.

[0125] (5) Determination of the content of urease (UE) in the serum of mice with Helicobacter pylori-induced gastritis The grouping and modeling of mice were the same as those in the first part of this experimental example. After the experiment, before sacrificing the mice, blood was collected from the eyes of the mice. The plasma was centrifuged at 3000 rpm for 15 min, and the upper serum was collected. The content of urease (UE) in the serum of mice was determined according to the method of the ELISA kit.

[0126] The experimental results are as Figure 4 shown. Compared with the NC group, after Helicobacter pylori infection, the urease content in the serum of mice in the HP group increased significantly ( p <0.05), and the urease content was 9.34 ± 0.82 pg / mL; the urease content in the serum of mice intervened with Lactobacillus mucosae LR69 was 6.85 ± 1.90 pg / mL. This result is consistent with the apparent result of urease in the gastric tissue in Experimental Example 6, indicating that Lactobacillus mucosae LR69 can reduce the urease content secreted by Helicobacter pylori in the gastric tissue, indirectly reflecting that the load of Helicobacter pylori in the gastric tissue can be reduced after the intervention of Lactobacillus mucosae LR69.

[0127] (6) Determination of the contents of PGI and PGII in the serum of mice with Helicobacter pylori-induced gastritis The grouping and modeling of mice were the same as those in the first part of this experimental example. After the experiment, before sacrificing the mice, blood was collected from the eyes of the mice. The plasma was centrifuged at 3000 rpm for 15 min, and the upper serum was collected. The contents of PGI and PGII in the serum of mice were determined according to the method of the ELISA kit.

[0128] Serum PG can reflect the strength of gastric mucosal secretion function and further reflect the state of the gastric mucosa. PGI is secreted by the chief cells and neck mucous cells of the gastric fundic glands, reflecting the function of glandular cells secreting gastric acid. PGII is produced by the glands in the gastric antrum and cardiac chambers that secrete mucus. The experimental results are as Figure 5 shown. The contents of PGI and PGII in the NC group were 31.68 ± 6.19 pg / mL and 21.68 ± 3.74 pg / mL, respectively. Compared with the NC group, after Helicobacter pylori infection, the contents of PGI and PGII in the HP group decreased significantly ( p <0.05), being 18.39 ± 2.87 pg / mL and 12.39 ± 1.44 pg / mL, respectively, indicating a decrease in the secretion function of the gastric mucosa. After intervention with Lactobacillus mucosae LR69, the contents of PGI and PGII in the LR69 group recovered to 27.91 ± 3.69 pg / mL and 20.15 ± 3.61 pg / mL, respectively (p (<0.05), indicating that Lactobacillus mucosae LR69 can alleviate the impaired gastric mucosal secretion function in mice.

[0129] Combined with the results of part (4) of this experimental example, it reflects that Lactobacillus mucosae LR69 can significantly enhance the gastric mucosal barrier in mice and repair the gastric mucosal damage induced by Helicobacter pylori in mice.

[0130] (7) Determination of inflammatory factors in the serum of mice with Helicobacter pylori-induced gastritis The grouping and modeling of mice were the same as those in part (1) of this experimental example. After the experiment ended and before the mice were sacrificed, blood was collected from the eyes of the mice. The plasma was centrifuged at 3000 rpm for 15 min, and the upper serum was collected. The contents of IL-8, IL-1β, IL-6, and TNF-α in the serum of the mice were determined according to the method of the ELISA kit.

[0131] The results are shown in Figure 6 , compared with the control group, the contents of pro-inflammatory factors IL-8, IL-1β, IL-6, and TNF-α in the model group were significantly increased ( p <0.01), compared with the model group, the contents of pro-inflammatory factors IL-8, IL-1β, IL-6, and TNF-α in the Lactobacillus mucosae LR69 group were significantly decreased ( p <0.01). It shows that Lactobacillus mucosae LR69 can alleviate Helicobacter pylori-induced gastritis in mice.

[0132] (8) 16S rRNA sequencing of the intestinal flora of mice with Helicobacter pylori-induced gastritis The grouping and modeling of mice were the same as those in part (1) of this experimental example. After the experiment ended, the mice were sacrificed and dissected, and the cecal contents were collected. The 16S rRNA of the cecal contents of the mice was sequenced and analyzed. The results showed ( Figure 7 ) that Helicobacter pylori caused disorders in the intestinal flora of mice. Compared with the normal group, the ACE index, Chao1 index, Simpson index, and Shannon index in the model group were significantly decreased (p<0.05). The ACE index, Chao1 index, and Shannon index in the Lactobacillus mucosae LR69 intervention group were significantly increased (p<0.01). The above results indicate that supplementing Lactobacillus mucosae LR69 can improve the richness and community diversity of the intestinal microbiota.

[0133] The 16S rRNA of the cecal contents of the mice was sequenced and the species diversity was analyzed. The results showed ( Figure 8 ) that at the phylum level, compared with the normal group, the Firmicutes in the model group decreased, and the Proteobacteria and Bacteroidetes increased significantly ( p <0.01, p(<0.01). After supplementing Lactobacillus reuteri LR69, the abundance of Firmicutes increased, while the abundances of Proteobacteria and Bacteroidetes decreased, indicating that supplementing Lactobacillus reuteri LR69 can regulate the intestinal flora structure and alleviate the intestinal flora imbalance caused by Helicobacter pylori infection.

[0134] The changes in the relative abundances of species at the genus level in the cecal contents of mice among groups were analyzed, and the results showed that ( Figure 9 ), in the Lactobacillus reuteri LR69 treatment group, the relative abundances of Bacteroidales- Muribaculaceae、 Dubosiella Duboslella、 Lachnospiraceae -NK4A136-group and Akkermansia Akkermansia were significantly increased compared with the model group ( P <0.05). In the model group (HP), the abundances of Helicobacter Helicobacter、 Bacteroides Bactrodales , Turicibacter Tuicibacter and Helicophaga Mucispirillum were significantly increased, but the intervention of LR69 could reduce their relative abundances. Muribaculaceae Produce short-chain fatty acids through endogenous (mucinoglycans) and exogenous polysaccharides (dietary fiber). The increased relative abundance is related to the enhancement of intestinal barrier function and the reduction of inflammatory response. It is shown that by supplementing Lactobacillus reuteri LR69, its abundance can be significantly increased. Dubosiella Duboslella The increased relative abundance is related to the increase in the production of short-chain fatty acids (SCFAs), which helps to improve metabolic health. Lachnospiraceae -NK4A136-group The increased relative abundance is related to anti-inflammatory effects and immunomodulatory functions. Akkermansia Akkermansia The increased relative abundance is related to the improvement of intestinal barrier function, the reduction of metabolic disorders, and the enhancement of immune response. The results show that after supplementing Lactobacillus reuteri LR69, the abundance of beneficial flora can be increased, thereby achieving the effects of enhancing intestinal barrier function, reducing intestinal inflammation, and regulating immune response. Helicobacter Helicobacter genus is the main genus of Helicobacter pylori. Supplementing Lactobacillus reuteri LR69 significantly reduces its abundance, indicating that Lactobacillus reuteri LR69 can reduce the load of Helicobacter pylori in vivo. Bacteroides Bactrodales The increased relative abundance may lead to the down-regulation of the expression of tight junction proteins in intestinal epithelial cells, thereby disrupting intestinal barrier function and increasing intestinal permeability. Bacteroides Bacteroidales may promote the release of pro-inflammatory factors (such as IL-6, TNF-α) by activating the TLR4 / NF-κB signaling pathway, leading to chronic inflammation. Turicibacter The increased relative abundance is related to the abnormal activation of Th17 cells and may lead to the occurrence of autoimmune diseases. Turicibacter TuricibacterMay produce secondary bile acids and other metabolites, which have toxic effects on the host liver and intestine. Lachnospiraceae Mucispirillum May regulate the host immune response, leading to overactivation or inhibition of the immune system, thus triggering chronic inflammation or immune deficiency. The results show that after supplementing with Limosilactobacillus reuteri LR69, the abundance of beneficial flora can be reduced and the inflammatory response can be decreased, thus achieving the effect of regulating the intestinal flora.

Claims

1. A Lactobacillus reuteri ( Limosilactobacillus reuteri ) LR69, characterized in that, Its deposit number is CGMCC No.33731.

2. Use of Lactobacillus reuteri LR69 with a preservation number of CGMCC No. 33731 in the preparation of foods with auxiliary protective functions for gastric mucosa, and / or foods with functional functions for regulating intestinal flora, and / or products for relieving gastritis, and / or foods for enhancing immune functions, and / or antibacterial agents.

3. The use of Lactobacillus reuteri LR69 with a deposit number of CGMCC No. 33731 according to claim 2 in the preparation of food with auxiliary protection function for gastric mucosa, and / or food with function of regulating intestinal flora, and / or product for relieving gastritis, and / or food for enhancing immune function, and / or antibacterial agent, characterized in that: The auxiliary protective function of gastric mucosa includes: strengthening gastric mucosal barrier and repairing gastric mucosal damage; And / or, the relief of gastritis includes: inhibiting the growth of Helicobacter pylori, reducing urease content, alleviating gastric tissue inflammatory infiltration, enhancing gastric mucosal barrier, repairing gastric mucosal damage, inhibiting Helicobacter pylori adhesion and colonization, and regulating cytokine levels; And / or, the enhancing the gastric mucosal barrier comprises: increasing the gene expression of gastric mucosal tight junction proteins zonula occludens 1 and occludin, and increasing the gene expression of gastric mucin 1 and mucin 2; And / or, the repairing of gastric mucosal damage includes: increasing the levels of serum PGI and PGII; And / or, the immunity enhancement comprises: regulating cytokine levels; And / or, the regulating cytokine levels comprises: reducing the levels of IL-8, IL-6, IL-1β, and TNF-α; And / or, the antibacterial spectrum of the antibacterial agent includes: Helicobacter pylori, Streptococcus mitis, Streptococcus Parisi, Streptococcus pasteurianus, Streptococcus sanguinis, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Klebsiella pneumoniae; And / or, the regulating intestinal flora comprises: increasing the number of Bacteroidetes- Muribaculaceae, Duboisia Duboslella, Lachnospiraceae -NK4A136-group and Akkermansia Akkermansia Relative abundance, reduced Helicobacter Helicobacter Bacteroides Bactrodales Zurichobacter Tuicibacter and Myxospirillum Mucispirillum Relative abundance.

4. A food having auxiliary protective function for gastric mucosa, comprising: Active ingredient; characterized in that the active ingredient includes: Lactobacillus reuteri LR69 with a preservation number of CGMCC No.33731.

5. A food for enhancing immune function, comprising: Active ingredient; characterized in that the active ingredient includes: Lactobacillus reuteri LR69 with a preservation number of CGMCC No.33731.

6. A functional food for regulating intestinal flora, comprising: Active ingredient; characterized in that the active ingredient includes: Lactobacillus reuteri LR69 with a preservation number of CGMCC No.33731.

7. A product for relieving gastritis, comprising: Active ingredient; characterized in that the active ingredient includes: Lactobacillus reuteri LR69 with a preservation number of CGMCC No.33731.

8. An antibacterial agent comprising: Active ingredient; characterized in that the active ingredient includes: Lactobacillus reuteri LR69 with a preservation number of CGMCC No.33731.

9. A non-therapeutic in vitro antibacterial method, characterized in that: Lactobacillus reuteri LR69 with a preservation number of CGMCC No.33731 was used for bacteriostasis.

10. The in vitro antibacterial method for non-therapeutic destinations according to claim 9, characterized in that: The antibacterial spectrum includes: Helicobacter pylori, Streptococcus mitis, Streptococcus Parisi, Streptococcus pasteurianus, Streptococcus sanguinis, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Klebsiella pneumoniae.

Images