Rhamnosus lactis strain for improving oral health, product and application thereof

Abstract

The present application is named a strain of rhamnose lactose casein bacillus which can improve oral health, its product and application, and belongs to the field of microbial technology. The technical problem to be solved is that the existing strains have poor targeting for the core problems of inhibition of volatile sulfur compounds (VSCs) in the oral cavity, relief of gingival inflammation, etc., and poor adaptability to oral care products. Traditional chemical products also have side effects such as mucous membrane irritation. The technical solution points are as follows: a rhamnose lactose casein bacillus MH-LR13 (preservation number CGMCC No. 37262) is provided, which can specifically inhibit VSCs producing bacteria and gingival inflammation related pathogenic bacteria, and has excellent oral colonization ability; the application of the strain in improving oral health products is protected, and the core efficacy is clarified; a composition containing the strain, an oral freshening spray and a suitable preparation process are provided, and the product is verified by human population experiment, which has high safety and is significantly better than existing strains and commercially available products, providing a natural and mild long-term solution for oral health.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to probiotic strains and their applications. Specifically, it relates to a strain of Lactobacillus rhamnosus MH-LR13 that has the function of improving oral health, as well as oral care products containing this strain and their preparation methods and applications. Background Technology

[0002] Oral health is a crucial component of overall health. Halitosis, gingivitis, bleeding gums, and swollen gums are common oral health issues that not only affect daily social interactions and quality of life but can also lead to more serious oral diseases in the long run. The core cause of halitosis is the breakdown of sulfur-containing amino acids by anaerobic bacteria in the mouth, producing volatile sulfur compounds (VSCs). Gum-related problems are closely related to an imbalance in the oral microecology and the excessive proliferation of harmful bacteria, leading to local inflammatory responses. With increasing consumer health awareness, the demand for safe, gentle products that address the root causes of oral health problems is growing rapidly.

[0003] Currently, oral care products are mainly divided into two categories: one is traditional chemical antibacterial products, which directly kill bacteria in the mouth by adding chemical ingredients such as cetylpyridinium chloride and chlorhexidine. Although they can temporarily relieve bad breath or inflammation symptoms, long-term use can easily disrupt the normal balance of oral flora, leading to side effects such as oral mucosal irritation and taste changes, and cannot achieve long-term improvement; the other is probiotic oral care products, which use probiotics to regulate the balance of oral microecology and solve oral problems from the root. They have the advantages of being gentle and safe, and have become an important direction for industry research and development.

[0004] Among the relevant patent documents retrieved, Chinese patent publication number CN120485077A discloses *Lactobacillus rhamnosus* LRa37 with accession number CGMCC No. 34558. The core function of this strain is to target and inhibit oral pathogens such as *Prevotella intermedius*, *Streptococcus pasteurellae*, and *Streptococcus parahaemolyticus*. It can also co-aggregate with and remove *Streptococcus mutans* and *Porphyromonas gingivalis*, and is resistant to lysozyme in the oral environment, adhering to human gingival epithelial cells for stable colonization, thereby improving oral microecological balance and alleviating periodontitis-related symptoms. Furthermore, this strain can regulate gut microbiota balance through the oral-gut axis, reducing serum pro-inflammatory factor levels. However, it does not provide targeted optimization and efficacy verification for the core cause of halitosis (VSCs), nor does it provide quantitative data on improving specific symptoms such as gingival bleeding and swelling, and it does not specify a formula and process for use in dedicated products such as oral freshening sprays.

[0005] Another related patent is Chinese Patent Publication No. CN120310704A, which discloses *Lactobacillus rhamnosus* CBBM005 with accession number CGMCC No. 33904. This strain is derived from human intestinal secretions and has functions of balancing intestinal flora, improving immunity, and oral health. It has a certain tolerance to lysozyme in the oral environment, and its fermentation products can inhibit the proliferation of oral flora. It also has a significant antibacterial effect on harmful oral bacteria such as *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Streptococcus mutans*. However, the research and development of this strain focuses on intestinal flora regulation and immune improvement, with oral care being only one of its multiple functions. Targeted verification was not conducted on core oral health issues such as reduced VSC concentration and improved gingival bleeding index, nor was the colonization efficiency and activity protection scheme of the strain optimized based on the morphological characteristics of oral care products.

[0006] Therefore, developing a new strain of Lactobacillus rhamnosus that is highly specific for inhibiting VSCs and precisely improving gum health, and designing suitable oral care products based on this strain to achieve efficient, gentle, and long-lasting improvement of halitosis, gingivitis, and other problems, has become a pressing technical need in the field of oral care. Summary of the Invention

[0007] The purpose of this invention is to provide a novel strain of Lactobacillus rhamnosus MH-LR13 with strong targeting and excellent oral colonization ability, and its application in the preparation of products to improve oral health. At the same time, it provides special products such as oral freshening sprays containing this strain and corresponding preparation methods.

[0008] Terminology Explanation:

[0009] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0012] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0013] The term "oral volatile sulfur compounds (VSCs)" used in this article refers to the collective term for volatile sulfur-containing gases produced by anaerobic bacteria in the oral cavity (such as Porphyromonas gingivalis and Fusobacterium nucleatum) decomposing sulfur-containing amino acids (cysteine ​​and methionine). The main components are hydrogen sulfide (rotten smell), methanethiol (rotten cabbage smell), and dimethyl sulfide (rotten onion smell). They are key substances that cause physiological halitosis, and their concentration can be quantitatively detected by a Halimeter sulfide detector (unit: ppb).

[0014] The term "gingival bleeding index (GBI)" used in this article refers to a quantitative indicator for assessing the degree of gingival inflammation. The Loe-Silness scoring method is used: a dental probe is used to gently touch the gingival sulcus, and bleeding is graded according to the degree of bleeding (0 = no bleeding; 1 = mild inflammation, no bleeding on probing; 2 = moderate inflammation, punctate bleeding on probing; 3 = severe inflammation, bleeding spreads along the gingival margin; 4 = very severe inflammation, spontaneous bleeding). Four points are measured on each of the teeth in the entire mouth, and the average value is calculated.

[0015] The term "Visual Analog Scale (VAS)" used in this article refers to an assessment tool used to quantify the subjective feelings of test takers. It is a 10cm long straight line with "0 points" (corresponding to the worst state, such as extremely bad breath or severe gum swelling and pain) and "10 points" (corresponding to the best state, such as excellent breath or no gum discomfort) marked at the two ends. Test takers express their feelings by marking the corresponding positions on the line, and the distance of the marked point from the 0-point end is the specific score.

[0016] In a first aspect, the present invention provides a strain of Lactobacillus rhamnosus MH-LR13, the preservation number of which is CGMCC No. 37262.

[0017] Secondly, the present invention provides the application of the aforementioned Lactobacillus rhamnosus MH-LR13 in the preparation of products that improve oral health.

[0018] Specifically, the oral health includes, but is not limited to: reducing the concentration of volatile sulfur compounds in the oral cavity, improving halitosis, relieving gingival inflammation, reducing gingival bleeding, and / or alleviating gingival swelling and pain.

[0019] Specifically, the products include, but are not limited to, one or more of the following: oral freshening spray, mouthwash, toothpaste, or oral patches.

[0020] Thirdly, the present invention provides a composition comprising the aforementioned *Lactobacillus rhamnosus* MH-LR13.

[0021] Specifically, the composition comprises the aforementioned fermentation broth of Lactobacillus rhamnosus MH-LR13, fermentation broth supernatant, fermentation broth precipitate, lyophilized powder, and / or bacterial suspension.

[0022] More specifically, the fermentation broth refers to the liquid in which the microbial strain is inoculated into a culture medium and cultured for a period of time.

[0023] More specifically, the supernatant of the fermentation broth refers to the clear liquid at the top after centrifugation of the fermentation broth; it contains abundant metabolic products from the bacterial growth and reproduction process, as well as some bacterial cell fragments. The acidic substances and bacteriocins secreted by the bacteria have antagonistic and killing effects on harmful bacteria. The amino acids and vitamins synthesized by the bacteria after decomposing food are also in the culture medium, as well as enzymes secreted by the bacteria that are useful to the human body. Some of the bacterial cell components also have an immune-boosting effect on the human body.

[0024] More specifically, the fermentation broth sediment refers to the liquid sediment obtained after centrifugation, which includes free protein, residual bacterial cells, broken cells, and culture medium residue, mainly protein and intracellular matrix.

[0025] More specifically, the lyophilized powder is obtained by freeze-drying culture medium; the lyophilized powder generally also includes a lyophilization protectant. The lyophilization protectant includes, but is not limited to: pH buffers, fillers, sugars, nonionic surfactants, ligands, etc. The pH buffers include, but are not limited to, any one or more of Tris, amino acids or their salts, citric acid or its salts, acetic acid or its salts. The fillers include, but are not limited to, any one or more of mannitol, glycine, and bovine serum albumin. The sugars can be disaccharides, such as sucrose or trehalose, any one or more. The nonionic surfactants include, but are not limited to, Tween, and Tween can be selected from Tween-20, Tween-60, Tween-80, etc. The lyophilization protectant may also include antioxidants, etc. Specifically, the lyophilization protectant may also include albumin, polyethylene glycol, etc.

[0026] More specifically, the bacterial suspension is a homogeneous suspension formed by discarding the supernatant after centrifuging the culture medium, adding water, culture medium or buffer solution, and suspending the lower layer of bacteria by shaking or blowing.

[0027] Preferably, it includes the aforementioned lyophilized powder of Lactobacillus rhamnosus MH-LR13.

[0028] Specifically, the composition further includes one or more of the following: sweeteners, acidity regulators, flavor regulators, fillers, binders, plant-based functional compound powders, and lubricants.

[0029] More specifically, it also includes isomaltitol, sorbitol, citric acid, flavor powder, sodium bicarbonate, microcrystalline cellulose, green coffee compound powder, natural menthol, magnesium stearate, and sucralose.

[0030] More specifically, the ingredients include 30%-40% isomalt, 25%-35% sorbitol, 8%-15% citric acid, 8%-12% flavor powder, 6%-10% sodium bicarbonate, 1%-3% microcrystalline cellulose, 0.05%-0.2% green coffee compound powder, 0.03%-0.1% natural menthol, 0.8%-1.2% magnesium stearate, and 0.1%-0.2% sucralose (by weight); the amount of Lactobacillus rhamnosus MH-LR13 freeze-dried powder added is 1.0%-3.0% (by weight).

[0031] Preferably, the composition comprises 35% isomaltitol, 29.5% sorbitol, 12% citric acid, 10% flavor powder, 8% sodium bicarbonate, 2% microcrystalline cellulose, 0.1% green coffee compound powder, 0.06% natural menthol, 1% magnesium stearate, and 0.14% sucralose (by weight); the amount of Lactobacillus rhamnosus MH-LR13 freeze-dried powder added is 2.2% (by weight).

[0032] Specifically, the method for preparing the aforementioned composition is characterized by comprising taking each component of the aforementioned composition, mixing the components, and then compressing them into tablets using a conventional tableting process.

[0033] Fourthly, the present invention provides an oral freshening spray comprising the aforementioned Lactobacillus rhamnosus MH-LR13.

[0034] Specifically, this includes the fermentation broth, fermentation broth supernatant, fermentation broth precipitate, lyophilized powder, and / or bacterial suspension of the aforementioned Lactobacillus rhamnosus MH-LR13.

[0035] Specifically, it also includes sweeteners, antimicrobial enzymes, moisturizing solubilizers, acidity regulators, flavor regulators, emulsifiers, and solvents.

[0036] More specifically, it also includes xylitol, erythritol, lysozyme, trehalose, glycerin, sodium citrate, citric acid, peppermint oil, steviol glycosides, Tween-80, and water.

[0037] More specifically, the addition amount of *Lactobacillus rhamnosus* MH-LR13 lyophilized powder is 1.0%-3.0%, xylitol 2%-4%, erythritol 1%-3%, lysozyme 0.05%-0.2%, trehalose 3%-5%, glycerol 4%-6%, sodium citrate 0.1%-0.2%, appropriate amount of citric acid (to adjust pH to 6.0-6.5), peppermint essential oil 0.2%-0.4%, steviol glycosides 0.03%-0.07%, Tween-80 0.1%-0.3%, and water to 100% (by weight).

[0038] Preferably, the added amount of *Lactobacillus rhamnosus* MH-LR13 lyophilized powder is 2.0%, xylitol 3.0%, erythritol 2.0%, lysozyme 0.1%, trehalose 4.0%, glycerol 5.0%, sodium citrate 0.15%, citric acid (to adjust pH to 6.2), peppermint oil 0.3%, steviol glycosides 0.05%, Tween-80 0.2%, and water to make up to 100% (by weight).

[0039] Specifically, the preparation method of the aforementioned oral freshening spray includes the following steps: (1) Add xylitol, erythritol, trehalose, glycerol and sodium citrate to water, heat to dissolve and then cool. Add lysozyme and adjust the pH to 6.0-6.5 to obtain system A. (2) Reconstitute the lyophilized powder of Lactobacillus rhamnosus MH-LR13 with water to obtain a bacterial suspension, add it to system A in step (1) and mix well to obtain system B; (3) Mix peppermint oil, steviol glycosides, Tween-80 and remaining water to emulsify and obtain an emulsion. Add the emulsion to system B in step (2) and stir evenly to obtain a liquid. (4) Fill and seal the liquid in a sterile environment to obtain the product.

[0040] The present invention has at least the following beneficial effects: (1) Compared with the prior art, the Lactobacillus rhamnosus MH-LR13 of the present invention is a novel strain that has significantly superior technical effects in the core aspects of improving oral health, such as reducing the concentration of volatile sulfur compounds in the oral cavity, improving halitosis, relieving gingival inflammation, reducing gingival bleeding, and alleviating gingival swelling and pain. Experimental data confirm that the composition and oral freshening spray prepared based on this new strain show significant improvement in various oral health-related indicators after a period of intervention, and the effect is better than that of similar commercially available products and existing known control strains.

[0041] (2) Compared with the prior art, the present invention provides a technical solution based on a novel strain with a different technical concept, and the technical effect is significantly better. The core difference between the two is that the prior art mostly relies on chemical antibacterial components to directly kill bacteria or uses known conventional probiotic strains, which have problems such as limited effect and easy to cause side effects; while the present invention takes the unpublished Lactobacillus rhamnosus MH-LR13 strain as the core, and regulates the microecological balance through its stable colonization in the oral cavity. Combined with natural adaptable components, it forms a synergistic effect, which not only achieves multiple oral health improvement effects, but also avoids the risks of chemical stimulation and flora imbalance. The technical concept is more innovative and groundbreaking.

[0042] Preservation Instructions Preserved strain: MH-LR13; Classification and nomenclature: Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ); Accession number: CGMCC No. 37262; Preservation period: January 4, 2026; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0043] Figure 1 The preparation process is shown in Example 3. Detailed Implementation

[0044] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0046] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0047] Example 1 The preparation method of Lactobacillus rhamnosus MH-LR13 lyophilized powder is as follows: (1) Inoculate the bacterial strain into the culture medium and incubate at 37°C to obtain the primary seed culture; (2) Inoculate the primary seed culture into the culture medium at a volume ratio of 2%-5% and incubate at 37°C to obtain the secondary seed culture; (3) Inoculate the secondary seed liquid into the fermenter at a volume ratio of 2%-5% and ferment at 37°C. Separate the bacterial sludge from the fermentation liquid by centrifugation. (4) The mycelium sludge was resuspended with a freeze-drying protectant, mixed with the freeze-drying protectant, and then freeze-dried to obtain a viable count of 1.5 × 10⁻⁶. 11 Freeze-dried powder with CFU / g.

[0048] Example 2 2.1 Formulation and preparation process of oral freshening spray (1) Prepare 1000g of oral freshening spray Weigh each component according to the following proportions: Lactobacillus rhamnosus MH-LR13 lyophilized powder (live count 1.5 × 10⁻⁶) 10 CFU / g): 20g (2.0%); Xylitol: 30g (3.0%); Erythritol: 20g (2.0%); Lysozyme (Ningxia Xiasheng Industrial Group Co., Ltd., enzyme activity > 5000 U / mg): 1g (0.1%); Trehalose: 40g (4.0%); Glycerin: 50g (5.0%); Sodium citrate: 1.5g; Citric acid: appropriate amount (for pH adjustment); Peppermint essential oil: 3g (0.3%); Steviosides: 0.5g (0.05%); Purified water: Add to a total of 1000g.

[0049] (2) Preparation process (a) Add 700g of purified water to a sterile mixing tank, heat to 37°C, and add xylitol, erythritol, trehalose, glycerol and sodium citrate while stirring. After complete dissolution, cool to 25°C.

[0050] (b) Add lysozyme and stir to dissolve. Slowly adjust the pH to 6.2 with a 10% (m / v) citric acid solution.

[0051] (c) In another sterile container, gently stir and reconstitute the MH-LR13 lyophilized powder with 100g of purified water to form a homogeneous bacterial suspension.

[0052] (d) Slowly pour the bacterial suspension into the main material tank and mix at a low speed (<100 rpm) for 15 minutes.

[0053] (e) Mix peppermint essential oil, stevia glycosides, 2g Tween-80 and the remaining 200g purified water, and homogenize and emulsify at high speed for 2 minutes to obtain an emulsion.

[0054] (f) Add the emulsion to the main material tank and stir at low speed until homogeneous.

[0055] (g) Add purified water to 1000g and continue stirring for 10 minutes.

[0056] (h) Transfer the liquid to the aseptic filling line.

[0057] (i) In a Class A clean environment, fill the liquid into a 10mL sterile spray bottle and immediately cap and seal it.

[0058] (g) Sampling and testing showed that the initial viable count of the product reached 3.0 × 10⁻⁶. 8 The concentration of CFU / mL and the pH value of 6.2 meet the microbial limits.

[0059] 2.2 Population Experiment To verify the effectiveness of this product compared to a placebo control and commercially available ordinary mouthwash in reducing the concentration of volatile sulfur compounds (VSCs, the main components of halitosis) in the oral cavity of subjects; to evaluate the effect of this product on improving subjects' perceived freshness of breath; and to evaluate the safety and tolerability of this product (record all adverse events).

[0060] 2.2.1 Research Design Design type: randomized, double-blind, placebo and positive control parallel group design.

[0061] Study population: 75 healthy adult volunteers aged 18-60 years are planned to be recruited. Inclusion criteria: no systemic diseases, not pregnant, not taking antibiotics within the past month, and halitosis group with a halitosis value of 200 ppb or higher measured by Halimeter (sulfide detector).

[0062] Exclusion criteria: Individuals with periodontitis, gingivitis, oral mucositis, smokers, and pregnant women.

[0063] 2.2.2 Grouping and Intervention: Experimental group (25 people): used the compound probiotic oral freshening spray containing Lactobacillus rhamnosus MH-LR13 of this invention.

[0064] Placebo control group (25 people): used a spray that looked and tasted exactly the same but did not contain MH-LR13 bacterial powder and lysozyme (containing only buffer, flavoring agent and protectant).

[0065] Positive control group (25 people): used a commercially available Colgate mouthwash (Colgate Jasmine Green Tea Flavored Mouthwash, Colgate-Palmolive (China) Co., Ltd.) mainstream chemical antibacterial mouthwash (such as one containing cetylpyridinium chloride).

[0066] Instructions for use: All subjects should spray this product or control product into their mouths 3-4 times after brushing their teeth in the morning and evening, rinse for 30 seconds, and then spit it out. Do not rinse with water during this period. Use the provided toothpaste without antibacterial properties during the study period.

[0067] The study lasted for 4 weeks, with a baseline visit (day 0), a mid-term visit (day 14), and a final visit (day 28).

[0068] Oral VSCs concentration: The concentration (ppb) of gases such as hydrogen sulfide and methanethiol in the oral cavity was measured directly on days 0, 14, and 28 using a breath analyzer.

[0069] Self-assessment: Using a visual analog scale, participants rated their breath freshness on a daily basis (0-10, 0 being very poor and 10 being excellent). Record the results at each visit.

[0070] Safety: Record all adverse events (such as irritation, allergies, etc.) and check the condition of the oral mucosa.

[0071] 2.2.3 Results and Analysis Oral VSCs concentrations are shown in Table 1: Table 1 Oral VSCs Concentration

[0072] As shown in Table 1, both the experimental group and the positive control group showed extremely significant reductions in VSCs at 14 and 28 days (p<0.05), while the placebo group showed virtually no change. Simultaneous point comparisons showed that both active groups were significantly superior to the placebo; furthermore, the experimental group showed significantly lower levels of VSCs than the positive control at both 14 and 28 days, indicating that the intervention had an excellent effect in reducing VSCs.

[0073] Self-assessment VAS scores are shown in Table 2: Table 2 Self-Assessment VAS Scores

[0074] As shown in Table 2, the experimental group showed the most significant improvement in VAS score. On day 14, both active groups were significantly better than placebo, but there was no significant difference between the experimental and positive control groups. On day 28, the experimental group was not only better than placebo, but also significantly better than the positive control, suggesting that the self-assessment improvement effect was further amplified over time.

[0075] Example 3 This embodiment provides a Lactobacillus rhamnosus composition with high bacterial survival rate, excellent taste, and clear experimental data supporting its ability to improve oral health.

[0076] 3.1 Formulation and Preparation Process 3.1.1 Formula The grape flavor formula (by weight percentage) is shown in Table 3: Table 3 Grape Flavor Recipe

[0077] Lemon flavor is shown in Table 4: Table 4 Lemon Flavor

[0078] 3.1.2 Preparation process The raw materials of the composition are mixed, and Lactobacillus rhamnosus MH-LR13 bacterial powder is added during the mixing process. After the raw materials and Lactobacillus rhamnosus MH-LR13 bacterial powder are mixed evenly, a conventional tableting process is used. Figure 1 This can be used to prepare a probiotic composition (0.7g / tablet).

[0079] 3.2 Evaluation of the effects of consumption on different population groups 3.2.1 Experimental Design Experiment type: randomized, double-blind, placebo-controlled clinical trial.

[0080] Subjects: Recruit 50 volunteers who meet the following criteria: (1) Age 18-60 years old.

[0081] (2) Mild to moderate halitosis (VASc > 200 ppb) and / or subjective gum discomfort (such as bleeding when brushing teeth).

[0082] (3) There are no major oral treatment plans in the next two months.

[0083] Sign the informed consent form.

[0084] Grouping: Participants were randomly divided into an experimental group (receiving the composition in Table 3) and a control group (receiving a placebo composition with the same appearance and taste as the experimental group, the only difference being that it did not contain the bacterial powder), with 25 people in each group.

[0085] 3.2.2 Experimental Scheme Product usage: Take 1 tablet 3 times daily after meals for 4 weeks.

[0086] Prohibited items: During the experiment, the use of other probiotic supplements, medicated mouthwashes, and medications for treating halitosis / gingivitis is prohibited.

[0087] 3.2.3 Evaluation Indicators and Scoring Methods The following indicators were measured and scored at the beginning of the experiment, in weeks 0, 2, and 4.

[0088] (1) Oral odor assessment Sensory evaluation: Trained professionals (olfactory evaluators) score the breath exhaled by the subject from a distance of 10cm from the subject's mouth. A 0-5 scale is used: 0 = no odor, 1 = slight odor, 2 = moderate odor, 3 = strong odor, 4 = severe odor, 5 = extremely foul odor.

[0089] The results are shown in Table 5: Table 5 Oral Odor Assessment

[0090] As shown in Table 5, the effects began to appear in week 2, peaking in week 4, and exhibiting a highly statistically significant difference and a large effect size compared to the control group. This demonstrates that the experimental intervention has a rapid and sustained effect on reducing oral odor scores.

[0091] (2) Gingival health score Gingival Bleeding Index: The dentist uses a professional probe to gently touch the subject's gingival sulcus to observe bleeding. Four points on each tooth are scored (0 = healthy gums, no bleeding; 1 = mild inflammation, no bleeding on probing; 2 = moderate inflammation, punctate bleeding on probing; 3 = severe inflammation, bleeding spreading along the gingival margin on probing; 4 = very severe inflammation, spontaneous bleeding). The overall average is calculated.

[0092] Self-assessment questionnaire: The visual analog scale was used, and the subjects marked their self-perception of the degree of "gingival bleeding" and "gingival swelling and pain" on a 10cm line (0 = none, 10 = very serious).

[0093] The results are shown in Table 6: Table 6. Gingival health score

[0094] As shown in Table 6, the composition can rapidly (within 2 weeks) and sustainably (up to 4 weeks) improve the gingival inflammation state, while also improving the patient's subjective symptom experience, thus demonstrating clear clinical value.

[0095] (4) Safety indicators Record any adverse events that occur during the entire experiment (such as gastrointestinal discomfort, allergies, etc.).

[0096] No adverse events occurred.

[0097] Example 4 Plaque Index (PI) 4.1 Subject source and screening criteria: Inclusion criteria: 1. Age requirement: Adults aged 18-60.

[0098] 2. Health status: Good overall health, with no major systemic diseases.

[0099] 3. Oral condition: (1) At least 20 detectable natural teeth (excluding third molars); (2) Having an appropriate degree of plaque or gingivitis (baseline PI score requirement, such as PI≥1.95 or PI≥2). (3) Mild to moderate gingivitis.

[0100] 4. Special requirements: (1) Women who are not pregnant or breastfeeding; (2) Did not participate in other similar experiments at the same time; (3) Sign the informed consent form and be able to complete the experiment as required.

[0101] Exclusion criteria: (1) Oral condition: orthodontic appliances, implants, removable dentures; severe periodontitis; open caries; oral mucosal lesions; oral soft and hard tissue tumors, etc.

[0102] (2) Drug use: Antibiotics used within 1-3 months prior to the test; or drugs that may affect the test results.

[0103] (3) Physical factors: serious systemic diseases (heart, liver, kidney, etc.); uncontrolled diabetes; immune system diseases.

[0104] (4) Special populations: pregnant or lactating women; smokers (partially tested); those with a history of allergy to any of the test product ingredients.

[0105] (5) Other: Currently participating in other clinical trials; plans to receive dental treatment during the trial period.

[0106] Participants were randomly assigned to either an experimental group (receiving the composition listed in Table 3) or a control group (receiving a placebo composition with the same appearance and taste as the experimental group, differing only in that it did not contain the bacterial powder). Each group consisted of 25 participants. One tablet was taken three times daily after meals for four weeks. Tests were conducted at weeks 0, 2, and 4 at the start of the experiment.

[0107] 4.2 Quigley-Hein plaque index determination method: Using a plaque display agent containing 1% alkaline fuchsin, the plaque on the tooth surface representing the upper and lower jaws, left and right sides, and front and back teeth is stained. The oral hygiene status is quantitatively assessed based on the stained area and thickness.

[0108] (1) The subject rinsed his mouth with water once; (2) Apply the reagent evenly to all tooth surfaces, including buccal, lingual / palatal, mesial, and distal surfaces, using a cotton swab; (3) After waiting for 30 seconds, rinse your mouth twice with water until the mouthwash is basically colorless; (4) The remaining red area is where the plaque adheres. Check and record the plaque.

[0109] Table 7 Quigley-Hein Plaque Index (Q-HI) Staining Scoring Criteria

[0110] PI = Total score / Total number of tooth surfaces. The results are shown in Table 8.

[0111] Table 8 PI Results

[0112] Significant improvement began to appear in the experimental group in week 2. P <0.001), with no significant change in the control group ( P >0.5 indicates that the effect is due to probiotic intervention rather than a placebo effect or natural fluctuations. Example 5: Saliva pH value Subject source and selection criteria: Inclusion criteria: 1. Age range: Adults aged 18-65.

[0113] 2. Number of teeth: ≥20 teeth in total.

[0114] 3. Overall good health: No systemic diseases (such as diabetes, Sjögren's syndrome, etc. that affect saliva secretion).

[0115] 4. Oral health: No severe untreated caries or pulpitis, and no active periodontal abscess.

[0116] 5. Informed consent: Voluntarily sign the informed consent form and cooperate with follow-up visits.

[0117] Exclusion criteria: 1. Medication history: Take antibiotics, hormones or immunosuppressants within the past 1-3 months.

[0118] 2. Treatment history: Currently undergoing orthodontic treatment (wearing braces) or having undergone teeth whitening or periodontal scaling within the past 3 months.

[0119] 3. Lifestyle habits: Long-term smokers.

[0120] 4. Pregnancy: Pregnant women or breastfeeding women.

[0121] Participants were randomly assigned to either an experimental group (receiving the composition listed in Table 3) or a control group (receiving a placebo composition with the same appearance and taste as the experimental group, differing only in that it did not contain the bacterial powder). Each group consisted of 25 participants. One tablet was taken three times daily after meals for four weeks. Tests were conducted at weeks 0, 2, and 4 at the start of the experiment.

[0122] Take fresh saliva into a clean container and measure the pH value with a pH electrode. Measure the same sample twice in parallel and take the average value. The results are shown in Table 9.

[0123] Table 9 pH Value Results

[0124] The saliva pH value in the experimental group significantly increased from 6.39 in week 0 to 7.05 in week 4. P <0.01), while the control group remained stable throughout the experiment ( P >0.05), and began to rise significantly in the second week ( P <0.05), and at week 4, the pH value of the experimental group was significantly higher than that of the control group ( P <0.01), indicating that the probiotic composition can effectively improve the oral pH environment.

[0125] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus MH-LR13, characterized in that, The preservation number of *Lactobacillus rhamnosus* MH-LR13 is CGMCC No. 37262.

2. The use of Lactobacillus rhamnosus MH-LR13 as described in claim 1 in the preparation of products for improving oral health; The oral health benefits include reducing the concentration of volatile sulfur compounds in the oral cavity, improving halitosis, relieving gingival inflammation, reducing gingival bleeding, and / or alleviating gingival swelling and pain.

3. The application according to claim 2, characterized in that, The product is one or more of the following: oral freshening spray, mouthwash, toothpaste, or oral patches.

4. A composition, characterized in that, Includes Lactobacillus rhamnosus MH-LR13 as described in claim 1.

5. The composition according to claim 4, characterized in that, It also includes one or more of flavor modifiers, fillers, binders, and lubricants.

6. A method for preparing the composition according to claim 4 or 5, characterized in that, The composition comprises taking each component of the composition according to claim 4 or 5, mixing the components, and then compressing them into tablets using a conventional tableting process.

7. A breath freshening spray, characterized in that, Includes Lactobacillus rhamnosus MH-LR13 as described in claim 1.

8. The oral freshening spray according to claim 7, characterized in that, It also includes lysozyme, emulsifiers, and solvents.

9. The method for preparing the oral freshening spray according to claim 7 or 8, characterized in that, Includes the following steps: (1) Add xylitol, erythritol, trehalose, glycerol and sodium citrate to water, heat to dissolve and then cool. Add lysozyme and adjust the pH to 6.0-6.5 to obtain system A. (2) Reconstitute the lyophilized powder of Lactobacillus rhamnosus MH-LR13 with water to obtain a bacterial suspension, add it to system A in step (1) and mix well to obtain system B; (3) Mix peppermint oil, steviol glycosides, Tween-80 and water to emulsify and obtain an emulsion. Add the emulsion to system B in step (2) and stir evenly to obtain a liquid. (4) The liquid is filled and sealed in a sterile environment to obtain the product.

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