Lactobacillus rhamnosus for improving oral health and application of lactobacillus rhamnosus

Abstract

The invention relates to an oral microbiome regulation and control technology, in particular to lactobacillus rhamnosus capable of improving oral health and application of the lactobacillus rhamnosus. The preservation number of the LRa37 strain is CGMCC No.34558, the LRa37 strain is preserved in China General Microbiological Culture Collection Center on May 15, 2025, and the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The LRa37 strain inhibits oral pathogenic bacteria through targeted inhibition of prevotella intermedia, streptococcus pasteurii and streptococcus parablood, improves oral micro-ecological balance and further improves the oral health state.

Description

Technical Field

[0001] The present application relates to oral microbiome regulation technology, and specifically to a Lactobacillus rhamnosus strain for improving oral health and its application. Background Art

[0002] The oral cavity is a complex microbial ecosystem, containing hundreds of bacterial species. Imbalances in certain specific bacterial communities are closely associated with oral diseases. Studies have shown that the occurrence of diseases such as periodontitis and dental caries is associated with the overgrowth of pathogenic bacteria and the imbalance of symbiotic flora.

[0003] Prevotella intermedia, a Gram-negative anaerobic bacterium, has been widely recognized as a key pathogen in periodontitis. It secretes proteolytic enzymes (such as gingipains) that damage periodontal tissues and promote inflammation. Furthermore, Prevotella intermedia can synergize with other pathogens in dental plaque, such as Porphyromonas gingivalis, to exacerbate periodontal destruction.

[0004] Streptococcus pasteurianus, a member of the oral Streptococcus genus, has been recently shown to be involved in the early formation of oral biofilms. This bacterium promotes the accumulation of dental plaque by producing exopolysaccharides (EPS) and provides a niche for other pathogens, such as Streptococcus mutans, thereby increasing the risk of dental caries.

[0005] Streptococcus parasanguinis is generally considered an oral commensal bacterium, but recent studies have found that under certain conditions (such as oral microecological imbalance), it may promote biofilm formation and engage in cross-trophy with pathogenic bacteria such as Prevotella intermedia, exacerbating periodontal inflammation.

[0006] Currently, the prevention and treatment of oral diseases primarily rely on mechanical cleaning (such as brushing and scaling) and antibiotic therapy. However, mechanical cleaning is difficult to completely eliminate pathogens within biofilms, while antibiotic use can lead to drug resistance and microecological imbalance. Therefore, there is an urgent need to develop precision intervention approaches based on microbiome regulation to restore oral microecological balance by targeting key pathogens (such as Prevotella intermedia and Streptococcus pasteurianus) and regulating commensal bacteria (such as Streptococcus parasanguinis). Summary of the Invention

[0007] The present application provides a strain of Lactobacillus rhamnosus, which is Lactobacillus rhamnosus LRa37 with a deposit number of CGMCC No. 34558. The LRa37 strain was deposited on May 15, 2025, at the General Microbiology Center of the China Culture Collection Administration, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The LRa37 strain inhibits oral pathogens by targeting Prevotella intermedia, Streptococcus pasteurianus, and Streptococcus parasanguinis, thereby improving the balance of oral microecology and thus oral health, and can also improve the balance of intestinal microecology.

[0008] The present application also provides a composition comprising live bacteria of Lactobacillus rhamnosus LRa37; dead bacteria of Lactobacillus rhamnosus LRa37; inactivated bacteria of Lactobacillus rhamnosus LRa37; one or more lysates of Lactobacillus rhamnosus LRa37; one or more metabolites of Lactobacillus rhamnosus LRa37; one or more analogs of Lactobacillus rhamnosus LRa37; one or more fragments of Lactobacillus rhamnosus LRa37; or a combination thereof.

[0009] The present application also provides a preparation containing Lactobacillus rhamnosus LRa37 as an active ingredient and auxiliary materials for forming the preparation.

[0010] In some embodiments, the preparation uses a mixture of one or more of the live, inactivated and dead cells of Lactobacillus rhamnosus LRa37, one or more lysates of Lactobacillus rhamnosus LRa37, or one or more metabolites of Lactobacillus rhamnosus LRa37, one or more analogs of Lactobacillus rhamnosus LRa37, one or more fragments of Lactobacillus rhamnosus LRa37, or a combination thereof as an active ingredient.

[0011] In some embodiments, the concentration of the active ingredient is 0.0001% (w / w) to 99% (w / w).

[0012] In some embodiments, the preparation is an oral prebiotic preparation, an intestinal prebiotic preparation, a preparation for maintaining physiological balance, a preparation for regulating intestinal microbial flora, a food, a health product, or a dietary supplement.

[0013] The present application also provides a probiotic composition, which contains Lactobacillus rhamnosus LRa37 as an effective ingredient and one or more prebiotics.

[0014] The present application also provides the use of a strain of Lactobacillus rhamnosus in the preparation of a preparation. The Lactobacillus rhamnosus is Lactobacillus rhamnosus LRa37 with a deposit number of CGMCC No. 34558. The LRa37 strain was deposited on May 15, 2025 at the General Microbiology Center of the China Culture Collection Administration, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preparation is selected from at least one of the following: oral prebiotic preparations, intestinal prebiotic preparations, preparations for maintaining physiological balance, preparations for regulating intestinal microbial flora, foods, health products, and dietary supplements.

[0015] According to the technical solution provided in this application, the LRa37 strain inhibits oral pathogens by targeted inhibition of Prevotella intermedia, Streptococcus pasteurianus and Streptococcus parasanguinis, thereby improving the balance of oral microecology and thus improving oral health.

[0016] According to the technical solution provided in this application, Lactobacillus rhamnosus LRa37 can colonize the oral cavity and effectively alleviate weight loss and alveolar bone resorption caused by periodontitis. It can be applied to the oral cavity in a specific form or formulation. Lactobacillus rhamnosus LRa37 has the potential to be used as an oral probiotic formulation.

[0017] According to the technical solution provided in this application, Lactobacillus rhamnosus LRa37 can alleviate intestinal inflammation caused by periodontal pathogens and has the application prospect of being prepared into oral probiotic preparations and intestinal probiotic preparations.

[0018] According to the technical solution provided in the present application, Lactobacillus rhamnosus LRa37 can reduce inflammatory factors in the serum of rats with periodontal disease and has the application prospect of being prepared into a preparation for maintaining physiological balance.

[0019] According to the technical solution provided in this application, Lactobacillus rhamnosus LRa37 can target and inhibit Prevotella intermedia, Streptococcus pasteurianus and Streptococcus parasanguinis, affect the composition of intestinal flora through the oral-intestinal axis, alleviate intestinal flora imbalance caused by periodontitis, and promote the restoration of intestinal microbial status and composition to a healthy state. It has application prospects for preparing preparations for regulating intestinal microbial flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Images of the inhibition plates for Prevotella intermedia (A), Streptococcus pasteurianus (B), and Streptococcus parasanguinis (C) provided for the experimental examples.

[0021] Figure 2 The weight curves of rats in the NC, MC and LRa37 groups provided for experimental examples.

[0022] Figure 3Micro-CT scans of rats in the NC, MC, and LRa37 groups provided as experimental examples.

[0023] Figure 4 HE staining of periodontal tissues from rats in the NC, MC, and LRa37 groups, provided as experimental examples. In the figure, the arrow represents the cementoenamel junction, the box indicates the area of periodontal fibrolysis, AB represents the alveolar bone, ABC represents the alveolar ridge, FR represents the furcation, FM represents the first molar, and SM represents the second molar.

[0024] Figure 5 HE staining of colon tissue from rats in the NC, MC, and LRa37 groups, provided as experimental examples. Arrows indicate inflammatory infiltration, and boxes represent magnified areas.

[0025] Figure 6 The Simpson Index (A) and Chao1 Index (B) of the intestinal flora of rats in the NC, MC, and LRa37 groups are provided as experimental examples. In the figure, "a, b, ab" indicate significant differences.

[0026] Figure 7 The PCA analysis results of β-diversity of intestinal flora of rats in the NC group, MC group and LRa37 group provided as experimental examples.

[0027] Figure 8 Figure 1 shows the Bacteroidota (A), Proteobacteria (B), Firmicutes (C), and overall phylum-level statistical composition of the intestinal microbiota of rats in the NC, MC, and LRa37 groups (D). In the figure, "a, b, ab" indicate significant differences.

[0028] Figure 9 The genus-level composition of the intestinal microbiota of rats in the NC, MC, and LRa37 groups is shown in the figure. The genus-level composition of Escherichia Shigella (A), Bacteroides (B), Eubacterium (C), and Romboutsia (D) are shown. In the figure, "a, b, ab" indicate significant differences. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0030] Although Teughels, W., et al. " Journal of Oral Microbiology, 12(1), 1709372, DOI: 10.1080 / 20002297.2019.1709372" found that the metabolites secreted by Lactobacillus rhamnosus (such as lactic acid and bacteriocins) can interfere with the biofilm formation of pathogenic bacteria (such as Streptococcus mutans) and reduce dental plaque accumulation, not all Lactobacillus rhamnosus can produce these effects, nor can they have effects on all oral pathogens.

[0031] To this end, the present application still provides a strain of Lactobacillus rhamnosus, Lactobacillus rhamnosus LRa37, with the deposit number CGMCC No. 34558. The LRa37 strain was deposited with the China General Microbiological Culture Collection Center (CGMCC) on May 15, 2025, with the deposit number CGMCC No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The LRa37 strain inhibits oral pathogens by targeting Prevotella intermedia, Streptococcus pasteurianus, and Streptococcus parasanguinis, thereby improving the balance of the oral microecology and thus improving oral health.

[0032] In some embodiments, the Lactobacillus rhamnosus strain according to the present application may be an isolated bacterial strain.

[0033] The present application discloses Lactobacillus rhamnosus LRa37, including the following pure culture colonies of Lactobacillus rhamnosus deposited in the General Microbiological Center of China Culture Collection Administration Committee or their analogs, fragments, lysates, or combinations thereof.

[0034] The present application provides a composition comprising live bacteria of Lactobacillus rhamnosus LRa37; dead bacteria of Lactobacillus rhamnosus LRa37; inactivated bacteria of Lactobacillus rhamnosus LRa37; one or more lysates of Lactobacillus rhamnosus LRa37; one or more metabolites of Lactobacillus rhamnosus LRa37; one or more analogs of Lactobacillus rhamnosus LRa37; one or more fragments of Lactobacillus rhamnosus LRa37; or a combination thereof.

[0035] In the composition, Lactobacillus rhamnosus LRa37 can be provided as a dead or inactivated bacterium, a dead and a live bacterium, one or more lysates; one or more metabolites; one or more analogs, one or more fragments, or a combination thereof. The composition can promote and / or enhance serum SIgA secretion, thereby increasing its mucosal immune defense.

[0036] The present application also provides a preparation containing Lactobacillus rhamnosus LRa37 as an active ingredient and auxiliary materials for forming the preparation.

[0037] Some embodiments provide that the concentration of the inactivated bacteria or live bacteria is 10 3 to 10 16 In the range of colony forming units (CFU), for example, 10 5 -10 16 CFU range, for example, 10 6 -10 16 CFU range, for example, 10 7 -10 16 CFU range, for example, 10 8 -10 16 CFU range, for example, 10 9 -10 16 CFU range, for example, 10 10 -10 16 CFU range, for example, 10 11 -10 16 CFU range, for example, 10 12 -10 16 CFU range, for example, 10 13 -10 16 CFU range, for example, 10 7 -10 16 CFU range, for example, 10 8 -10 15 CFU range, for example, 10 9 -10 15 CFU range, for example, 10 10 -1015 CFU range, for example, 10 11 -10 15 CFU range, for example, 10 12 -10 15 CFU range.

[0038] Some embodiments provide formulations that use a mixture of one or more of live, inactivated, and dead cells of Lactobacillus rhamnosus LRa37, one or more lysates of Lactobacillus rhamnosus LRa37, one or more metabolites of Lactobacillus rhamnosus LRa37, one or more analogs of Lactobacillus rhamnosus LRa37, one or more fragments of Lactobacillus rhamnosus LRa37, or a combination thereof as the active ingredient. The concentration of the active ingredient is 0.0001% (w / w) to 99% (w / w).

[0039] In the context of the present application, the Lactobacillus rhamnosus LRa37 defined herein can be provided in the composition of the present application in the form of a dead bacterial strain. The dead bacterial strain can be provided as a whole dead cell or as a lysate, metabolite, derivative, analog or extract obtained from the dead cell. Wherein "lysate" and the term "extract" specifically refer to a solution or suspension of microbial cells in an aqueous medium according to the present application, and contain, for example, macromolecules (such as DNA, RNA, proteins, peptides, lipids, carbohydrates, etc.) and cell debris. The lysate preferably includes cell walls or cell wall components, including binding receptors. Methods for producing lysates are well known to those skilled in the art, including, for example, using "French press" or enzymatic lysis, a ball mill with glass beads or iron beads. Cells can be broken by enzymatic, physical or chemical methods. Examples of enzymatic cell lysis can include individual enzymes and enzyme mixtures, such as proteases, proteinase K, lipases, glycosidases; chemical lysis can be induced by ionophores, detergents (such as SDS), acids or bases; physical methods can also be implemented by using, for example, high pressure, osmotic pressure, temperature changes or alternating hot and cold using French presses. Furthermore, it is of course possible to combine chemical, physical and enzymatic methods.

[0040] Some embodiments provide a formulation that is a topical, oral, gastric, or enteral formulation.

[0041] The formulations provided in some embodiments comprise at least a carrier or excipient that is acceptable in nutraceuticals, food science, or cosmetics. In some embodiments, the formulation can be provided in a solid form, a liquid form, a viscous form, an emulsion, or as a dry form.

[0042] Oral preparations can preferably be formulated as pastes, soft gelatin capsules, hard gelatin capsules, powders, talc, granules, beads, pastilles, effervescent tablets, lozenges, buccal tablets, chewable tablets, sublingual tablets, oils, liquids, solutions, tinctures, emulsions, juices, concentrates, syrups, sprays, mists, drinkable ampoules, gels, tablets, coated pills or as food or feed products or beverages.

[0043] The Lactobacillus rhamnosus LRa37 provided in the present application was found to have the ability to colonize the oral cavity and can be administered in the form of an oral colonizing microorganism or preparation.

[0044] As used herein, the terms "colonize the oral cavity" or "oral colonization" or other equivalent terms are given their standard and art-understood meaning of the presence and, preferably, proliferation of probiotic microorganisms in the oral cavity. Reference to proliferation means growth or reproduction of the microorganisms. Thus, the term encompasses the detectable presence or development of probiotic microorganisms in at least a portion of the oral cavity, i.e., at least a portion of the oral cavity is colonized with probiotic microorganisms.

[0045] Thus, for example, the present application relates to administering Lactobacillus rhamnosus LRa37 to a patient with an oral disease for a reasonable or appropriate period of time, and preferably allowing contact between Lactobacillus rhamnosus LRa37 and oral surfaces so that the microorganism can establish a detectable presence or colonize the oral cavity, so as to preferably allow Lactobacillus rhamnosus LRa37 to proliferate in the oral cavity. In other words, the presence of Lactobacillus rhamnosus LRa37 in the oral cavity is not transient, for example, the presence of the microorganism can still be detected in the oral cavity after the time window in which the microorganism is administered. This is different from, for example, a situation in which Lactobacillus rhamnosus LRa37 is administered as a standard coated tablet or capsule to be swallowed, such as a gelatin-coated capsule, in which case the microorganism is only temporarily present in the oral cavity during the passage of the tablet into the gastrointestinal tract, and the microorganism is not present in the oral cavity in an appropriate position to colonize the oral cavity.

[0046] For example, the Lactobacillus rhamnosus LRa37 is administered in a form or formulation that directly contacts the oral cavity (e.g., oral surfaces) for an appropriate amount of time and / or in an appropriate location to allow Lactobacillus rhamnosus LRa37 to colonize the oral cavity (or at least a portion of the oral cavity). Therefore, the Lactobacillus rhamnosus LRa37 of the present invention is generally not administered in the form of a coated tablet or protected capsule, or other forms or formulations designed to release its microbial contents in the gastrointestinal tract, such as the lower gastrointestinal tract, and contact the body surface.

[0047] In some embodiments, the formulation is in a chewable or suckable form or formulation, such as a chewing gum or chewable tablet or suckable tablet such as a lozenge, or some other form or formulation that will remain in the patient's oral cavity or mouth long enough to allow microbial colonization of the oral cavity (or at least a portion of the oral cavity) to eventually occur. Alternatively, a viscous syrup may be used, for example, that allows the oral cavity to be coated for an extended period of time.

[0048] In some embodiments, the preparation is an oral health food or an oral hygiene product. The oral health food is a lozenge, capsule, oral liquid, or chewing gum. The oral hygiene product is a mouthwash or toothpaste.

[0049] In some embodiments, the formulation further comprises a thickener, a filler, a binder, a disintegrant, a suspending agent, an adjuvant, a preservative, a stabilizer, a colorant, or a flavoring.

[0050] The preparations provided in some embodiments may contain one or more thickeners, and / or one or more sweeteners and / or one or more artificial sweeteners, wherein the thickener is preferably selected from cellulose ethers, polysaccharides, selected from the group comprising: xanthan gum, gelatin, highly dispersed silicon dioxide, starch, carrageenan, alginate, tragacanth gum, agar, gum arabic, pectin and polyvinyl ester, and the sweetener is selected from the group comprising: glucose, fructose, sucrose, glucose syrup, sorbitol, mannitol, xylitol, maltitol, steviol glycosides, saccharin, cyclamate, acesulfame K and / or aspartame.

[0051] Preferred formulations in the sense of the present application may include effervescent tablets, vitamin tablets, mineral tablets, trace element tablets, beverage powders, beverages, juices, milk drinks, yogurt, mineral water, non-carbonated water, filled gummies, chewable tablets, juices or syrups, coated pills and lozenges, and aerosols.

[0052] Furthermore, the formulations may comprise builders, enzymes, electrolytes, pH regulators, thickeners, prebiotics, optical brighteners, graying inhibitors, dye transfer inhibitors, foam regulators and / or colorants.

[0053] Astonishingly, the Lactobacillus rhamnosus LRa37 and its composition provided in this application not only have the ability to target and inhibit Prevotella intermedia, Streptococcus pasteurianus, and Streptococcus parasanguinis, improving microecological balance, but can also tolerate high concentrations of oral lysozyme and adhere to human gingival epithelial cells. The Lactobacillus rhamnosus LRa37 provided in this application can colonize the oral cavity, offering potential applications as a raw material for oral probiotics, oral cleaning preparations, and preparations that regulate the oral microecological environment.

[0054] According to another aspect of the present application, a probiotic composition comprising Lactobacillus rhamnosus LRa37 and at least one or more prebiotics is provided.

[0055] In the context of this application, a "prebiotic" is a non-digestible food ingredient that promotes the growth of specific microorganisms. A "synbiotic" is a composition comprising at least one probiotic and at least one prebiotic. Such compositions are understood to promote the growth of beneficial bacteria (e.g., probiotics). Thus, powerful synbiotics are based on the combination of specific strains of probiotics with carefully selected prebiotics. They can provide important health benefits to mammals.

[0056] Prebiotics are chemical products that induce the growth and / or activity of commensal microorganisms (e.g., bacteria and fungi) that contribute to the health of the host. Prebiotics are non-digestible carbohydrates that pass through the upper gastrointestinal tract undigested and stimulate the growth and / or activity of beneficial bacteria that colonize the intestinal or skin microbiome.

[0057] Some oligosaccharides used as prebiotics are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose, pectin, galacto-oligosaccharides (GOS) or human milk oligosaccharides (HMO). In addition, disaccharides such as lactulose or some monosaccharides such as lactose or tagatose can also be used as prebiotics.

[0058] In one embodiment of the present application, at least one prebiotic compound may be included in the composition of the present application. In a very broad concept, prebiotics are all compounds that can be metabolized by probiotics.

[0059] Preferably, prebiotics are indigestible or difficult to digest for mammals. Therefore, after being taken in by mammals, indigestible prebiotics can pass through the small intestine and enter the large intestine to stimulate the growth of probiotics in this compartment. Therefore, prebiotics can be used as a food source for probiotics. It is believed that prebiotics (many of which are indigestible carbohydrates) promote the growth of probiotics. Prebiotics are naturally present in, for example, cabbage, onion, whole grains, banana, garlic, honey, leek, artichoke, fortified food and beverage and dietary supplements. Prebiotics are well known in the art, and when used for the present application, there is no particular limitation on prebiotics themselves.

[0060] In one embodiment, the at least one prebiotic product in the composition is selected from the following compounds and compositions: non-digestible carbohydrates, β-glucans, manno-oligosaccharides, inulin, fructooligosaccharides, human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), lactulose, lactofructooligosaccharides, galactotriose, fructooligosaccharides (FOS), cellobiose, cellodextrins, cyclodextrins, maltitol, lactitol, glycosilsucrose, betaine, vitamin E or a variant thereof (wherein the variant is selected from α, β, γ, δ tocopherol, tocotrienols and tocopherols). Optionally, manno-oligosaccharides and / or inulin may be preferred. HMOs may include lacto-N-tetraose, lacto-N-fucopentaose, lacto-N-triose, 3'-sialyllactose, lacto-N-neofucopentaose, sialic acid, L-fucose, 2-fucosyllactose, 6'-sialyllactose, lacto-N-neotetraose, and 3-fucosyllactose.

[0061] Prebiotics may also be used in the topical compositions of the present application.

[0062] In one embodiment, at least one of the following prebiotic compounds is used in the topical composition of the present application: lactose, β-glucan, manno-oligosaccharide, inulin, fructooligosaccharide, galacto-oligosaccharide (GOS), lactulose, lactofructooligosaccharide, galactotriose, fructooligosaccharide (FOS), cellobiose, cellodextrin, cyclodextrin, maltitol, lactitol, glucosylsucrose, betaine, vitamin E or a variant thereof (wherein the variant is selected from α, β, γ, δ tocopherol, tocotrienol and tocopherol), lacto-N-tetraose, lacto-N-fucosylpentaose, lacto-N-triose, 3'-sialyllactose, lacto-N-neofucopentaose, sialic acid, 2-fucosyllactose, 6'-sialyllactose, lacto-N-neotetraose and 3-fucosyllactose. Optionally, lactose and / or manno-oligosaccharide and / or inulin may be preferred.

[0063] D- and L-fucose strengthen the natural defenses of the skin or mucous membranes, stimulate epidermal immune defenses and / or prevent and / or treat skin autoimmune diseases. In one embodiment of the present application, the composition comprises D- or L-fucose.

[0064] In one embodiment of the present application, the composition further comprises L-fucose at a concentration of 0 to 0.3 g / kg in the composition.

[0065] In the context of the present application, acceptable additives and / or excipients for pharmaceutical or food applications include auxiliary substances known to those skilled in the art for the preparation of solid, semi-solid or liquid forms, for example, diluents, solvents (including water, glycerol, ethanol), solubilizers, acidulants, thickeners, sweeteners, flavoring agents, colorants, sweeteners, lubricants, surfactants, preservatives, pH stability buffers and mixtures thereof.

[0066] In various embodiments of the present application, the composition of the present application containing Lactobacillus rhamnosus LRa37 can be an oral probiotic preparation, an intestinal probiotic preparation, a preparation for maintaining physiological balance, a preparation for regulating intestinal microbial flora, a food, a health product, or a dietary supplement.

[0067] For example, the inventors of the present application found through testing that the rhamnosus Lactobacillus LRa37 provided in the present application inhibits oral pathogens by targeting Prevotella intermedia, Streptococcus pasteurianus and Streptococcus parasanguinis, thereby regulating the balance of oral microecology and improving oral health.

[0068] For example, the inventors of this application have discovered through testing that the Lactobacillus rhamnosus LRa37 provided herein can colonize the oral cavity and effectively alleviate weight loss and alveolar bone resorption caused by periodontitis. This can be applied to the oral cavity in a specific form or formulation. Lactobacillus rhamnosus LRa37 has the potential to be used as an oral probiotic formulation.

[0069] For example, the inventors of the present application found through testing that the Lactobacillus rhamnosus LRa37 provided in the present application can alleviate intestinal inflammation caused by periodontal pathogens, and has application prospects in preparing oral probiotic preparations and intestinal probiotic preparations.

[0070] For example, the inventors of the present application found through testing that the Lactobacillus rhamnosus LRa37 provided in the present application can reduce inflammatory factors in the serum of rats with periodontal disease and has application prospects in preparing preparations for maintaining physiological balance.

[0071] For example, the inventors of the present application discovered through testing that the rhamnosus Lactobacillus LRa37 provided in the present application can target and inhibit Prevotella intermedia, Streptococcus pasteurianus and Streptococcus parasanguinis, affect the composition of the intestinal flora through the oral-intestinal axis, alleviate the intestinal flora imbalance caused by periodontitis, and promote the restoration of the intestinal microbial status and composition to a healthy state, and has application prospects for preparing preparations for regulating intestinal microbial flora.

[0072] In order to help understand the screening and identification of Lactobacillus rhamnosus LRa37 and its easy colonization of the oral cavity, improvement of oral microecology, relief of inflammation, prevention and improvement of serum inflammation, and the oral-gut axis affecting the composition of the intestinal flora, and relief of intestinal flora imbalance caused by periodontitis, etc., the following will be explained through a detailed experimental part, but it does not constitute a limitation to the implementation methods of this application.

[0073] 1. Strain isolation and identification

[0074] The collected oral samples were placed in sterile sampling tubes and transported in ice boxes. They were diluted with 0.85% saline gradients under sterile conditions, and the appropriate dilution gradient was selected for smearing on LBS agar plates and cultured at 37°C for 48-72 hours. Suspected single colonies were picked by visual observation of their colony morphology, and they were observed under a microscope and preliminarily screened and purified. After purification, they were cultured at 37°C for 12-16 hours using MRS liquid anaerobic tubes, centrifuged to remove the supernatant, and resuspended in a sterile 25% glycerol aqueous solution and stored in the strain bank of Wuhan Weikang Probiotics Research Institute. The screened target strain was liquid-cultured, diluted and coated, and its colony morphology was observed. An appropriate amount of bacterial liquid was taken for identification. The screened target strain was liquid-cultured, the bacteria were collected, the genomic DNA was extracted, and PCR amplification reaction was performed. The content and purity of the PCR amplification product were tested. After passing the test, it was sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. According to the sequencing results, the BLAST tool in the NCBI database was used for homology comparison, and the obtained strain was identified as rhamnosus casei. The strain was named rhamnosus casei LRa37 and sent for preservation.

[0075] 2. Strain preservation

[0076] The strain provided in this application is named Lactobacillus rhamnosus LRa37, and was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on May 15, 2025, with the deposit number CGMCC No. 34558, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0077] 3. Lactobacillus rhamnosus LRa37 targeted inhibition of Prevotella intermedia, Streptococcus pasteurianus and Streptococcus parasanguinis

[0078] Preparation of Porphyromonas gingivalis suspension: Porphyromonas gingivalis was inoculated into TSA solid plates containing 5% defibrinated sheep blood and cultured anaerobically at 37°C for 72 hours.

[0079] Preparation of Streptococcus mutans suspension: Streptococcus mutans was inoculated into BHI liquid culture medium at a 2% inoculum size and cultured at 37°C and 250 rpm / min in a shaking incubator for 24 h.

[0080] Preparation of Prevotella intermedia suspension: Prevotella intermedia was inoculated into BHI liquid medium at a 2% inoculum size and cultured at 37°C for 24 h.

[0081] Preparation of Streptococcus pasteurianus suspension: Streptococcus pasteurianus was inoculated into BHI liquid medium at a 2% inoculum size and cultured at 37°C for 24 h.

[0082] Preparation of Streptococcus parasanguinis suspension: Streptococcus parasanguinis was inoculated into BHI liquid culture medium at a 2% inoculum size and cultured at 37°C for 24 h.

[0083] Using the Oxford cup method, the suspension of Prevotella intermedia, Streptococcus pasteurianus and Streptococcus parasanguinis were mixed into BHI medium containing 5% defibrinated sheep blood. The number of viable pathogenic bacteria in the system was 10 6 CFU / mL order of magnitude, after taking out the Oxford cup, add 150 μL of rhamnosus Lactobacillus LRa37 bacterial solution, with a concentration of 5×10 9 CFU / mL, cultured overnight at 37°C, and the diameter of the inhibition zone was measured.

[0084] Table 1 Inhibitory ability of Lactobacillus rhamnosus LRa37 against pathogens

[0085] indicator strains Diameter of inhibition zone (mm) Streptococcus mutans 23.50±0.50 Porphyromonas gingivalis 25.33±0.29 Prevotella intermedia 16.17±0.29 Streptococcus pasteurianus 19.83±0.58 Streptococcus parasanguinis 17.67±0.58

[0086] As shown in Table 1, Lactobacillus rhamnosus LRa37 not only has an inhibitory effect on common oral pathogens Porphyromonas gingivalis and Streptococcus mutans, but can also target and inhibit Prevotella intermedia, Streptococcus pasteurianus and Streptococcus parasanguinis, and has application prospects in inhibiting oral pathogens, regulating symbiotic flora, and restoring the balance of oral microecology.

[0087] 4. Coaggregation ability test of Lactobacillus rhamnosus LRa37 and Streptococcus mutans

[0088] Lactobacillus rhamnosus LRa37 was inoculated into MRS liquid medium at a 2% inoculum volume, cultured at 37°C for 24 hours, and the culture solution was set aside. Streptococcus mutans was inoculated into BHI medium at a 1% inoculum volume, cultured at 37°C for 24 hours, and the culture solution was set aside. The cultured Lactobacillus rhamnosus LRa37 and Streptococcus mutans were centrifuged separately, 65.1 cells were collected, washed twice with PBS buffer, and then resuspended with PBS. The OD600 value of the Lactobacillus rhamnosus LRa37 bacterial solution was adjusted to 0.6 and the OD600 value of the Streptococcus mutans bacterial solution was 0.4. The mixture was fully shaken and the initial OD600 values Ax and Ay were measured respectively. 20 mL of each of Lactobacillus rhamnosus LRa37 and Streptococcus mutans were added to a 50 mL centrifuge tube and mixed. After vortexing for 20 seconds, the mixture was incubated at 37°C. After incubation for 2h, 5h, 21h, and 24h, 2.5 mL of the upper liquid was carefully aspirated, and the absorbance (A) of the bacterial suspension at 600 nm was measured to calculate the coagulation force between the strain and Streptococcus mutans. Coagulation force = [(Ax + Ay) / 2 - A(x + y)] / [Ax + Ay / 2] × 100%, where x and y represent two strains, respectively, and (x + y) represents the mixture.

[0089] As shown in Table 2, the coaggregation ability of Lactobacillus rhamnosus LRa37 with S. mutans gradually increased over time, reaching 17.61±1.86% at 2 hours and significantly increasing to 65.10±1.71% after 21 hours of coculture. These data demonstrate that Lactobacillus rhamnosus LRa37 has a high coaggregation ability with S. mutans and can effectively remove S. mutans through coagulation, demonstrating its potential for preventing dental caries.

[0090] Table 2 Results of the coaggregation test of Lactobacillus rhamnosus LRa37 and Streptococcus mutans

[0091] Time (h) 2 5 21 24 Cohesion (%) 17.61±1.86 28.63±4.18 65.10±1.71 65.30±3.17

[0092] 5. Coaggregation ability test of Lactobacillus rhamnosus LRa37 and Porphyromonas gingivalis

[0093] Lactobacillus rhamnosus LRa37 was inoculated into MRS liquid medium at a 2% inoculum and cultured at 37°C for 24 hours. Porphyromonas gingivalis was inoculated into TSA solid medium containing 5% defibrinated sheep blood and cultured anaerobically at 37°C for 72 hours. The cultured Lactobacillus rhamnosus LRa37 and Porphyromonas gingivalis were centrifuged, the cells were collected, washed twice with PBS buffer, and then resuspended with PBS. The OD600 value of the Lactobacillus rhamnosus LRa37 bacterial solution was adjusted to 0.6 and the OD600 value of the Porphyromonas gingivalis bacterial solution was 0.4. The cultures were fully shaken and the initial OD600 values Ax and Ay were measured respectively. 20 mL of each of Lactobacillus rhamnosus LRa37 and Porphyromonas gingivalis were added to a 50 mL centrifuge tube and mixed. After vortexing for 20 seconds, the cells were incubated at 37°C. After incubation for 2 h, 5 h, 21 h, and 24 h, 2.5 mL of the upper liquid was carefully aspirated, and the absorbance (A) of the bacterial suspension at 600 nm was measured to calculate the coagulation force between the strain and Porphyromonas gingivalis. Coagulation force = [(Ax + Ay) / 2 - A(x + y)] / [Ax + Ay / 2] × 100%, where x and y represent two strains, respectively, and (x + y) represents the mixture.

[0094] Table 3 Results of the coaggregation test of Lactobacillus rhamnosus LRa37 and Porphyromonas gingivalis

[0095] Time (h) 2 5 21 24 Cohesion (%) 21.03±5.61 39.85±4.19 66.41±0.45 67.55±0.83

[0096] As shown in Table 3, the copolymerization ability of Lactobacillus rhamnosus LRa37 and Porphyromonas gingivalis was 21.03±5.61% at 2 hours, significantly increasing to 66.41±0.45% at 21 hours. Porphyromonas gingivalis is a recognized pathogen of periodontitis. These results suggest that Lactobacillus rhamnosus LRa37 has the potential to prevent and alleviate periodontitis by removing Porphyromonas gingivalis through copolymerization.

[0097] 6. Lysozyme tolerance test

[0098] MRS liquid culture medium containing 3 mg / mL, 2.5 mg / mL, 2 mg / mL, 1.5 mg / mL, 1 mg / mL and 0 mg / mL lysozyme was prepared respectively, and a final concentration of 5×10 9 CFU / mL of Lactobacillus rhamnosus LRa37, the number of viable Lactobacillus rhamnosus LRa37 was counted after 10 h of culture, and the tolerance rate under different concentrations of lysozyme was calculated. The tolerance rate was the percentage of the number of viable bacteria after culture to the number of viable bacteria in MRS liquid medium with 0 mg / mL lysozyme.

[0099] Table 4 Lysozyme tolerance test results of Lactobacillus rhamnosus LRa37

[0100] Lysozyme concentration (mg / mL) 1 1.5 2 2.5 3 Tolerance rate (%) 82.09±5.38 75.62±3.76 66.67±3.76 48.91±2.05 39.45±1.69

[0101] As shown in Table 4, the survival rate of Lactobacillus rhamnosus LRa37 was 82.09 ± 5.38% at 1 mg / ml of lysozyme, 66.67 ± 3.76% at 2 mg / ml, and 39.45 ± 1.69% at 3 mg / mL. These data indicate that Lactobacillus rhamnosus LRa37 has a strong tolerance to lysozyme and a strong ability to survive in the oral cavity. When used in oral health products such as toothpaste, mouthwash, chewing gum, and chewable tablets, it can better survive and exert its effects in the oral cavity.

[0102] 7. Surface hydrophobicity test

[0103] Lactobacillus rhamnosus LRa37 was inoculated into LMRS liquid medium at a 2% inoculum and cultured at 37°C for 24 hours. The culture was then set aside. The activated culture was centrifuged at 5000 rpm for 5 minutes to collect the cells. The cells were washed twice with an equal volume of PBS buffer and resuspended in PBS buffer. The OD600 value of the suspension was adjusted to approximately 1.0, and the absorbance at 600 nm (A0) was measured. 12 mL of the suspension was mixed with 4 mL of xylene in a test tube, which was capped and thoroughly mixed (vibrating with a homogenizer for 60 seconds). The tube was allowed to stand at room temperature for 10 minutes, then vortexed again (vibrating with a homogenizer for 60 seconds), and allowed to stand at room temperature for 20 minutes. The aqueous phase was removed and the absorbance at 600 nm (A1) was measured. Buffer was used as a blank control. Surface hydrophobicity was expressed as the percentage of bacterial adhesion to organic solvents: surface hydrophobicity (%) = (1-A1 / A0) × 100%.

[0104] Table 5 Surface hydrophobicity test results of Lactobacillus rhamnosus LRa37

[0105] strain number Surface hydrophobicity% LRa37 91.33±1.15

[0106] As shown in Table 5, Lactobacillus rhamnosus LRa37 has a high surface hydrophobicity, indicating its strong ability to colonize the oral cavity. Furthermore, when oral products made with Lactobacillus rhamnosus LRa37 are ingested, such as toothpaste, mouthwash, chewing gum, and chewable tablets, Lactobacillus rhamnosus LRa37 can better adhere to the intestinal mucosa through the oral-gut axis, prolonging its colonization time and enhancing its probiotic effects.

[0107] 8. Adhesion test of Lactobacillus rhamnosus LRa37 and HGE cells

[0108] (1) HGE cell culture

[0109] Human gingival epithelial cells (HGE cells, catalog number CP-H178, Wuhan Punosai) were cultured in DMEM medium supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS), 1% penicillin and streptomycin, and cultured in a 37°C, 90% humidity, 5% CO2 incubator.

[0110] (2) Adhesion test plan

[0111] HGE cells are the main cell type of human gingival epithelium, and their adhesion ability directly reflects the colonization potential of the strain in the oral cavity. 5 The cells were inoculated into 12-well plates at a density of 1×10 cells / mL and the medium was changed every other day until the HGE cells in the 12-well plates reached 90% confluency. At the same time, the cultured Lactobacillus rhamnosus LRa37 bacteria were collected by centrifugation at 10,000 rpm for 1 minute at room temperature. The cells were then washed twice with sterile PBS and resuspended in DMEM medium to adjust the concentration of the bacteria to 1×10 8 CFU / mL. Remove the 12-well plate, aspirate the culture medium, rinse twice with PBS buffer, aspirate the buffer, then add 1 mL / well of the prepared bacterial suspension, mix thoroughly, and incubate in a 5% CO2, 37°C incubator for 2 hours. After completion, carefully remove the culture supernatant, rinse five times with sterile PBS to remove unadhered bacteria, and add 0.2 mL / well of trypsin cell digestion solution for 5 minutes to elute the cells from the culture plate wells. The collected solution is the sample, which is then serially diluted and viable bacteria counted.

[0112] As shown in Table 6, the adhesion ability of Lactobacillus rhamnosus LRa37 to HGE cells was 26.69±2.15 CFU / cell, indicating that Lactobacillus rhamnosus LRa37 has a strong ability to adhere to and colonize on gingival epithelial cells.

[0113] Table 6 Adhesion test results of Lactobacillus rhamnosus LRa37 and HGE cells

[0114] Group Adhesion capacity CFU / cell Lactobacillus rhamnosus LRa37 26.69±2.15

[0115] 9. In vivo periodontitis test

[0116] (1) In vivo experimental animals and test solutions

[0117] Eighteen male, 6-8-week-old, SPF-grade Sprague-Dawley rats weighing 180-200 g were randomly divided into three groups of six rats each, based on the principle of consistent average weight within each group. The three groups were blank group (Control group), model group (Model group), and Lactobacillus rhamnosus LRa37 group (LRa37 group).

[0118] Preparation of Lactobacillus rhamnosus LRa37 suspension: Lactobacillus rhamnosus LRa37 cells were inoculated into MRS medium at an inoculum volume of 2% of the total volume of MRS medium, and cultured anaerobically at 37°C for 24 h. The bacterial suspension concentration was adjusted to 8 × 10 10 CFU / mL and resuspend in 2% CMC.

[0119] Preparation of Porphyromonas gingivalis suspension: Porphyromonas gingivalis cells were inoculated into BHI medium at an inoculum volume of 2% of the total volume of BHI medium, and cultured anaerobically at 37°C for 24 h. The bacterial suspension concentration was adjusted to 1×10 9 CFU / mL and resuspend in 2% CMC.

[0120] (2) Periodontitis modeling and group experiments

[0121] Silk ligation: Except for the blank group (no modeling treatment and normal diet), rats in the Model and LRa37 groups were anesthetized with an intraperitoneal injection of 0.2 ml / 100 g ketamine hydrochloride. The right maxillary second molars of the rats were ligated with 3-0 silk suture. After successful ligation, the rats entered a three-day recovery period.

[0122] Infection and prevention: After the 3-day recovery period, the cells were infected with a suspension of Porphyromonas gingivalis, while the intervention group was treated with Lactobacillus rhamnosus LRa37. Infection was performed every 2 days, and prevention was performed once a day for 21 days. The specific methods are as follows:

[0123] Among them, the specific operation methods for infection and prevention are: after the rat is anesthetized, use a sterile syringe to draw 50μl of bacterial suspension and inject it into the ligation site, and apply it evenly with a medical dental brush. After completion, fast and abstain from water for half an hour.

[0124] Model group: Use a sterile syringe to draw 50 μl of Porphyromonas gingivalis suspension and inject it into the ligature. Apply it evenly with a medical dental brush. After that, fast and deprive of water for half an hour.

[0125] LRa37 group: Use a sterile syringe to draw 50μl of Porphyromonas gingivalis suspension and apply it to the ligature site, then apply it evenly with a medical dental brush. After that, fast and drink for half an hour. After that, use a sterile syringe to draw 50μl of Lactobacillus rhamnosus LRa37 suspension and apply it to the ligature site, then apply it evenly with a medical dental brush. After that, fast and drink for half an hour.

[0126] During the experiment, the Model group and the LRa37 group were supplemented with 10% (m / m) sucrose in distilled water, and the blank control group was fed a normal diet.

[0127] Table 7 Rats grouping

[0128] Group quantity diet Blank group (NC) 6 Normal diet Model group (MC) 6 10% sucrose water Intervention group (LRa37) 6 10% sucrose water

[0129] (3) Effect of Lactobacillus rhamnosus LRa37 on body weight in rats with periodontitis

[0130] Rats in the blank, model, and intervention groups were weighed every three days to compare their growth. Weight was measured seven times in total, and the results were expressed as weight gain ratio. Weight 2 / weight 1 = weight gain ratio (%). Note: Weight 1 is the weight before the first intervention; weight 2 is the weight during the invasion prevention and treatment process.

[0131] Literature reports that periodontitis can cause weight changes in rats. The main reason is that it causes pain and discomfort in the affected teeth, leading to difficulty chewing, which affects food intake and the ability to digest and absorb nutrients. In addition, the inflammatory response in the affected area may increase the metabolic rate, leading to energy consumption and subsequent weight loss in rats. Figure 2 As shown in the results, the body weight of rats in the model group was significantly lower than that in the control group (P<0.01). Under the intervention of Lactobacillus rhamnosus LRa37, the body weight of the intervention group (LRa37) increased significantly and was close to that of the control group at the end of the experiment; this indicates that Lactobacillus rhamnosus LRa37 can alleviate the weight loss caused by periodontitis.

[0132] (4) Effect of Lactobacillus rhamnosus LRa37 on alveolar bone resorption in rats with periodontitis

[0133] After the experiment, the right maxilla of rats in the blank group, model group and intervention group were taken and fixed in 10% paraformaldehyde solution for 3 days. Then they were rinsed with clean water and the soft tissue around the alveolar bone was removed, and the hard tissue was retained for Micro-CT scanning.

[0134] The original image was reconstructed using the 3D reconstruction software Recon. The distance from the cementoenamel junction to the alveolar crest (CEJ-ABC) of the second molar was measured using the measurement software ImageJ. The Micro-CT results are shown in Figure 2. Figure 3 As shown in the figure, the alveolar bone of the second molar in the model group was severely absorbed, and the root was obviously exposed, while there was a significant improvement in the intervention group.

[0135] The amount of alveolar bone loss is a key indicator for evaluating the severity of periodontitis. The statistical results are shown in Table 8. The values are expressed as mean and standard deviation, and the differences between groups are expressed by differentiated letters.

[0136] As shown in Table 8, the distance from the cementoenamel junction to the alveolar crest (CEJ-ABC) of the second molar in the model group was approximately 1.0 mm, indicating that the periodontitis model was successfully constructed. Under the intervention of Lactobacillus rhamnosus LRa37, at the end of the experiment, the distance from the cementoenamel junction to the alveolar crest (CEJ-ABC) of the second molar in the LRa37 group was significantly lower than that in the MC group (P<0.05), indicating that LRa37 alleviated the alveolar bone resorption caused by periodontitis.

[0137] Table 8 Alveolar bone resorption in rats

[0138] Group CEJ-ABC (mm) Blank group (NC) 0.52±0.08b Model group (MC) 1.00±0.11a Intervention group (LRa37) 0.67±0.09b

[0139] (5) Effects of Lactobacillus rhamnosus LRa37 on the colon and periodontal tissues of rats with periodontitis

[0140] After the experiment, 1 cm of the colon and the right maxilla of the rats in the blank group, model group and intervention group were fixed in 10% paraformaldehyde solution, and paraffin sections were made and stained with HE.

[0141] like Figure 4 As shown, the gingival epithelium of rats in the control group was intact, without inflammation or cellular infiltration, and the collagen fibers in the periodontal ligament were neatly arranged. In the model group, the periodontal ligament fibers were disordered, with fibrosis and dissolution. Numerous inflammatory cells infiltrated the edges of the connective tissue, and significant alveolar bone resorption was observed. These results indicate that periodontitis causes inflammation in the rat periodontal tissue. Compared with the model group, rats in the Lactobacillus rhamnosus LRa37 intervention group showed less gingival epithelial damage, a smaller area of inflammatory cell infiltration, and more neatly arranged collagen fibers in the periodontal ligament. These results indicate that Lactobacillus rhamnosus LRa37 intake improves the pathological damage to periodontal tissue caused by periodontitis.

[0142] like Figure 5 As shown, the model group showed a significant decrease in colonic crypts, accompanied by extensive inflammatory infiltration and numerous hemorrhagic spots, indicating a loss of normal intestinal architecture. This suggests that periodontal pathogens may compromise intestinal health through the oral-gut axis. In contrast, the Lactobacillus rhamnosus LRa37-treated group showed only partial crypt loss and minimal inflammatory infiltration, suggesting that Lactobacillus rhamnosus LRa37 has the potential to mitigate intestinal inflammation caused by periodontal pathogens.

[0143] (6) Effects of Lactobacillus rhamnosus LRa37 on inflammatory factors in the serum of rats with periodontitis

[0144] Studies have reported that TNF-α is the first inflammatory mediator produced by the body after stimulation. Its expression increases during various inflammatory diseases, inducing the production of IL-1, IL-6, and IL-8, exacerbating the inflammatory response. Furthermore, research has shown that TNF-α can serve not only as a diagnostic marker for periodontitis but also as a marker of risk and susceptibility to the disease.

[0145] After the experiment, blood was collected from the rats in the control, model, and intervention groups via the abdominal aorta using a disposable blood collection needle and a vacuum tube. After centrifugation, serum was collected and assayed for inflammatory cytokines (IL-6, TNF-α, MCP-1, and IL-12p70) using a kit. The results are summarized in Table 9. Values are presented as mean and standard deviation. Differences between groups are indicated by letters indicating differences.

[0146] As shown in Table 9, the levels of proinflammatory factors (IL-6, TNF-α, MCP-1, and IL-12p70) in the serum of rats in the model group were significantly higher than those in the intervention and control groups (P < 0.05). The levels of TNF-α and other proinflammatory factors in the serum of rats significantly decreased after ingestion of Lactobacillus rhamnosus LRa37 (P < 0.05), indicating that Lactobacillus rhamnosus LRa37 can reduce the expression of inflammatory factors in the serum.

[0147] Table 9. Levels of inflammatory factors in rat serum

[0148] Group IL-6 (pg / ml) TNF-α (pg / ml) MCP-1 (pg / ml) IL-12P70 (pg / ml) Blank group (NC) 74.96±17.939c 102.58±18.50c 130.68±11.68c 81.08±14.75c Model group (MC) 143.88±16.54a 193.40±24.68a 296.90±29.74a 149.44±12.01a Intervention group (LRa37) 110.62±4.79b 136.48±20.07b 220.38±16.38b 107.93±12.21b

[0149] (7) Effects of Lactobacillus rhamnosus LRa37 on the intestinal flora of rats with periodontitis

[0150] After the experiment, the cecum of rats in the blank, model, and intervention groups was collected and placed in 50ml centrifuge tubes. The tubes were immediately refrigerated in liquid nitrogen / dry ice and then stored in a -80°C freezer. 16S amplicon analysis of the microbiome in the rat cecum contents was performed to analyze the intestinal flora.

[0151] α diversity can reflect the species richness and species diversity of each group. Figure 6As shown in the figure, the Chao 1 index of α diversity in the model group was significantly lower than that in the control group (P<0.05), which indicated that the intestinal microbial richness in the model group was significantly decreased. After rats ingested Lactobacillus rhamnosus LRa37, the Chao 1 index and Simpson index were significantly higher than those in the model group (P<0.05). Figure 7 As shown in the figure, there are significant differences in the microbiota between the model group and the control group, and the LRa37 group is closer to the control group. This shows that LRa37 strain intervention can improve the richness of the intestinal microbiota in rats with periodontitis.

[0152] To further understand the differences in microbial communities between the periodontitis model group and the control group as well as the Lactobacillus rhamnosus LRa37 group, OTUs were classified by database comparison, and the phylum (e.g. Figure 8 ) and genus (e.g. Figure 9 ) level by species richness at the phylum level. At the phylum level, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria accounted for more than 80% of the total, with Firmicutes being the dominant phylum. Compared with the control group, the Firmicutes community in the periodontitis model group was significantly reduced (P<0.05), while the Bacteroidetes and Actinobacteria were significantly increased (P<0.05).

[0153] Furthermore, the dominant genera in the periodontitis model group were Escherichia (22.98%) and Bacteroides (10.86%), which differed from the dominant microbial population in the control group. These two genera contained a high proportion of harmful bacteria. Bacteroides is a common clinical pathogen. Studies have reported that excessive accumulation of Bacteroides can promote intestinal inflammation, and severe colon inflammation was also observed in model rats using HE staining of colon sections. Escherichia is a Gram-negative bacterium, which includes the well-known Escherichia coli. Under healthy conditions, Escherichia maintains a balance with the host immune system and does not trigger an excessive inflammatory response. However, when the intestinal microbiome is dysbiotic, Escherichia can cause an excessive inflammatory response, negatively impacting intestinal and overall health. Escherichia accounted for a high genus-level proportion of 22.98% of the species composition in the model group, making it a contributing factor to intestinal inflammation in model rats. These results suggest that the intestinal microbiome undergoes changes during the development of periodontitis.

[0154] After Lactobacillus rhamnosus LRa37 treatment, the Firmicutes, Bacteroidetes, and Proteobacteria groups approached those in the control group, with significant differences compared to the model group (P < 0.05). Furthermore, at the genus level, the abundance of Escherichia and Bacteroidetes decreased significantly compared to the model group (P < 0.05), approaching that of the control group. Furthermore, Lactobacillus rhamnosus LRa37 treatment also restored the abundance of other bacterial genera to levels comparable to those in the control group.

[0155] In summary, it is shown that after the onset of periodontitis, oral pathogens affect the composition of intestinal flora through the oral-gut axis, and the intake of Lactobacillus rhamnosus LRa37 can effectively alleviate the intestinal flora imbalance caused by the periodontitis model, increase the abundance and diversity of intestinal flora microorganisms to normal levels, and slow down the accumulation of intestinal pathogenic microorganisms caused by periodontitis, promoting the restoration of intestinal microbial status and composition to a healthy state.

[0156] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. A strain of Lactobacillus rhamnosus, characterized in that The Lactobacillus rhamnosus LRa37 has a deposit number of CGMCC No. 34558.

2. A composition, characterized in that Comprising the living cells of Lactobacillus rhamnosus LRa37 according to claim 1; the dead cells of Lactobacillus rhamnosus LRa37 according to claim 1; The inactivated bacteria of Lactobacillus rhamnosus LRa37 according to claim 1; One or more lysates of Lactobacillus rhamnosus LRa37 according to claim 1; One or more metabolites of Lactobacillus rhamnosus LRa37 according to claim 1; One or more analogs of Lactobacillus rhamnosus LRa37 according to claim 1, one or more fragments of Lactobacillus rhamnosus LRa37 according to claim 1, or a combination thereof.

3. The composition according to claim 2, characterized in that The concentration of the live bacteria, inactivated bacteria or dead bacteria of the Lactobacillus rhamnosus LRa37 according to claim 1 is 10 3 to 10 16 within the range of colony-forming units.

4. A preparation, characterized in that The preparation contains the Lactobacillus rhamnosus LRa37 as claimed in claim 1 as an active ingredient and auxiliary materials for forming the preparation.

5. The preparation according to claim 4, characterized in that The preparation uses a mixture of one or more live bacteria, inactivated bacteria and dead bacteria of rhamnosus casei LRa37, one or more lysates of rhamnosus casei LRa37, or one or more metabolites of rhamnosus casei LRa37, one or more analogs of rhamnosus casei LRa37, one or more fragments of rhamnosus casei LRa37, or a combination thereof as an active ingredient.

6. The preparation according to claim 5, characterized in that The concentration of the active ingredient is 0.0001% (w / w) to 99% (w / w).

7. The preparation according to claim 4, characterized in that The preparation is selected from at least one of an oral probiotic preparation, an intestinal probiotic preparation, a preparation for maintaining physiological balance, a preparation for regulating intestinal microbial flora, a food, a health product, and a dietary supplement.

8. A probiotic composition, characterized in that The invention contains the Lactobacillus rhamnosus LRa37 according to claim 1 as an effective ingredient, and one or more prebiotics.

9. Use of Lactobacillus rhamnosus as claimed in claim 1 in preparing a preparation.

10. The use according to claim 9, characterized in that The preparation is selected from at least one of an oral probiotic preparation, an intestinal probiotic preparation, a preparation for maintaining physiological balance, a preparation for regulating intestinal microbial flora, a food, a health product, and a dietary supplement.

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