Streptococcus salivarius Ss-08 and application thereof in preventing and treating oral diseases

Abstract

The application discloses a Streptococcus salivarius Ss-08 and application thereof in prevention and treatment of oral diseases, and belongs to the technical field of microorganisms. The technical problem to be solved is to provide the Streptococcus salivarius Ss-08 and application thereof in prevention and / or treatment of oral diseases. The technical solution is to provide the Streptococcus salivarius Ss-08 and application thereof in oral submucosal fibrosis and extracellular vesicles thereof in oral squamous cell carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to Streptococcus salivarius Ss-08 and its application in the prevention and treatment of oral diseases, specifically to Streptococcus salivarius Ss-08 and its application in the prevention and / or treatment of oral submucosal fibrosis and the progression of oral squamous cell carcinoma. Background Technology

[0002] Oral submucous fibrosis (OSF) is a chronic disease characterized by progressive fibrosis of the oral mucosa. Its main clinical manifestations include pain upon consuming irritating foods, whitening and stiffening of the mucosa, palpable fibrous strands, progressive limitation of mouth opening, and decreased flexibility, severely impacting patients' oral function and quality of life. Existing research data indicates that OSF carries a certain risk of malignant transformation, with the rate of conversion to oral squamous cell carcinoma varying across different regions and populations. The hypoxic, chronic inflammatory microenvironment formed by fibrotic tissue is believed to promote increased genetic instability in epithelial cells, thereby inducing abnormal proliferation and malignant transformation. Furthermore, persistent inflammatory stimulation and abnormal immune regulation may weaken the body's ability to clear abnormal cells, making OSF an important "precursor state" for oral cancer. Therefore, in-depth research into the pathogenesis of OSF is of great significance for the early prevention and intervention of oral cancer.

[0003] Oral squamous cell carcinoma (OSCC) is the main pathological type of head and neck squamous cell carcinoma (HNSCC) and the leading cause of death in HNSCC patients. Although genetic factors, smoking, betel nut chewing, alcohol consumption, and poor oral hygiene are considered major risk factors for OSCC, its exact pathogenesis remains incompletely understood. OSCC can occur in multiple anatomical locations of the oral cavity, including the tongue, gingiva, floor of mouth, buccal mucosa, palate, and jawbone, with tongue cancer and floor of mouth cancer being the most common. Its early clinical manifestations are often nonspecific, with patients frequently presenting with lesions such as oral ulcers, leukoplakia, or erythema, which are easily overlooked or misdiagnosed, leading to a significant proportion of patients being diagnosed at an advanced stage. Faced with these serious challenges, there is an urgent need to establish targeted therapy strategies based on molecular subtyping and a personalized comprehensive prevention and treatment system. Therefore, exploring the molecular mechanisms of the occurrence, development, and poor prognosis of oral squamous cell carcinoma is of great significance for discovering potential new preventive and therapeutic targets for oral squamous cell carcinoma.

[0004] Extracellular vesicles (EVs), as nanoscale secretory structures ubiquitous in prokaryotes and eukaryotes, have been recognized as important mediators of intercellular communication. Eukaryotic EVs can generally be classified into subtypes such as exosomes, microvesicles, and apoptotic bodies based on their mode of development and size. Bacterial EVs are typically nanoscale (approximately 20–400 nm) lipid bilayer vesicles, carrying various biomolecules from the parent bacteria, including proteins, lipids, nucleic acids, and metabolites. The functional molecules they carry can spread between cells and even across species and tissues, regulating various physiological and pathological processes in recipient cells, such as immune responses, proliferation, apoptosis, and metabolism. These mechanisms not only reveal the key role of microorganisms in maintaining host homeostasis and disease regulation, but also provide a theoretical basis and application prospects for EVs as novel bioactive carriers in the prevention and treatment of diseases such as tumors.

[0005] Salinomyces ( Streptococcus salivarius Streptococcus salivarius (Salivarius) is one of the earliest and most representative symbiotic bacteria to colonize the human oral cavity. Primarily distributed on the dorsum of the tongue and in saliva, it is a crucial core member in maintaining oral mucosal homeostasis. Within hours of birth, it can establish persistent colonization on the upper respiratory tract and oral surface, exhibiting a high affinity for the host epithelium and a stable ability to occupy a colony. Streptococcus salivarius does not rely on lactic acid fermentation and does not produce strongly acidic metabolites that cause enamel demineralization, thus possessing a significant "non-cariogenic advantage." In the oral ecosystem, it tends to maintain a neutral and symbiotic homeostasis. This characteristic makes it a safer and more suitable candidate for developing oral probiotics. Research by MacDonald et al. found that… S. salivarius K12 and M18 can significantly inhibit the inflammatory response of human gingival fibroblasts induced by periodontal pathogens, manifested by reduced IL-6 and IL-8 secretion, and the probiotics themselves do not induce inflammation. Human studies have further confirmed this. S. salivarius K12 does not disrupt salivary microbiota homeostasis, and its periodontal protective effect is mainly achieved through immune regulation. Stašková et al.'s study systematically evaluated... S. salivariusThe K12 secretion products exhibited antibacterial and antibiofilm effects against various potential oral pathogens under in vitro conditions. Results showed that the cell-free supernatant significantly inhibited the growth of *Streptococcus mutans* (Sm) and effectively weakened bacterial biofilm formation without the presence of live bacteria, suggesting that its biological effects are mainly mediated by secretible functional factors. Whether the extracellular vesicles (EVs) contained in its secretion system can act as functional signal carriers to participate in regulating the oral tumor microenvironment and influencing tumor-related biological processes remains a subject of insufficient systematic research. Currently, regarding… S. salivarius The direct mechanism of action of derived EVs in tumor cell behavior and tumor immune regulation remains unclear and needs further clarification.

[0006] Relevant patent documents retrieved: The country of publication is China, publication number CN 121487740 A, publication date 2026.02.06, this document discloses the use of Streptococcus salivarius for the treatment or prevention of ear, nose and throat (ENT) diseases induced or associated with cancer therapy.

[0007] Relevant non-patent literature retrieved: Li Yuanyuan, Zhu Pingyi, Chen Xin, et al. Study on the preventive effect and mechanism of Streptococcus salivarius on chemotherapy-induced oral mucositis [C] / / Chinese Stomatological Association Oral Mucosal Disease Committee, Chinese Stomatological Association Integrative Medicine Committee. Abstracts of the 16th Academic Conference of the Chinese Stomatological Association Oral Mucosal Disease Committee and the 14th Academic Conference of the Chinese Stomatological Association Integrative Medicine Committee, 2024:61.DOI:10.26914 / c.cnkihy.2024.076661. This article discloses the preventive effect of Streptococcus salivarius on chemotherapy-induced oral mucositis.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The prior art does not disclose Ss-08 of *Streptococcus salivarius*, and S. salivarius Its role and related mechanisms in oral submucosal fibrosis S. salivarius The direct mechanism of action of derived EVs in tumor cell behavior and tumor immune regulation remains unclear. Summary of the Invention

[0009] The purpose of this invention is to provide: Streptococcus salivarius Ss-08 and its application in the prevention and / or treatment of oral diseases, and related technologies, to address technical issues such as the unclear direct mechanism of action of Streptococcus salivarius in oral submucosal fibrosis and oral squamous cell carcinoma, or combinations thereof.

[0010] Terminology Explanation: 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.

[0011] 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.

[0012] Definitions of standard chemical terms can be found in the references “Modern Molecular Biology” (6th edition, by Zhu Yuxian, published by Higher Education Press), “Genetic Engineering” (3rd edition, edited by Sun Ming, published by Higher Education Press), or “Experimental Guide to Clinical Molecular Biology Testing Techniques” (2nd edition, edited by Li Yan, published by People’s Medical Publishing House).

[0013] Unless otherwise specified, conventional methods within the scope of the art, such as centrifugation, membrane filtration, RT-qPCR, Western blot, etc., shall be used.

[0014] 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.

[0015] The term "EMT" used in this article refers to epithelial-mesenchymal transition, the process by which epithelial cells, under specific physiological or pathological conditions, lose their epithelial phenotypes such as polarity and intercellular connections, and acquire mesenchymal cell phenotypes such as migration, invasion, and anti-apoptosis. EMT is a key mechanism in tumor metastasis, organ fibrosis, and embryonic development, and its markers include downregulation of E-cadherin and upregulation of vimentin and N-cadherin.

[0016] The term "OSF" used in this article refers to Oral Submucous Fibrosis. This is a chronic, progressive oral mucosal disease characterized by fibrosis of the submucosal tissue (i.e., normal tissue is replaced by fibrous tissue), leading to hardening and loss of elasticity of the oral mucosa, ultimately causing limited mouth opening.

[0017] The term "CAL-27" used in this article refers to the human tongue squamous cell carcinoma cell line, which is widely used in oral cancer research. It is essentially a group of cancer cells cultured in vitro. Because it originates from the tumor tissue of the same patient and can proliferate indefinitely in the laboratory, it is considered a standard "research tool" and is often used to explore the pathogenesis of oral cancer and the effects of drugs.

[0018] The term "EVs" used in this article refers to membrane-bound vesicles actively secreted by cells into the extracellular space. These vesicles, approximately 20-1000 nm in diameter, carry biomolecules such as proteins, nucleic acids (mRNA, miRNA, DNA), and lipids, and mediate intercellular communication. Bacterial EVs play an important role in pathogen-host interactions, carrying virulence factors and regulating host immunity and signaling pathways.

[0019] The term "Fn" used in this article refers to a Gram-negative anaerobic bacillus, a common commensal bacterium in the oral cavity, and also associated with periodontal disease, colorectal cancer, and oral squamous cell carcinoma. It promotes bacterial aggregation and host cell inflammatory responses by secreting adhesins and extracellular vesicles, and is involved in immunosuppression of the tumor microenvironment.

[0020] The term "HPV" as used in this article refers to a non-enveloped, double-stranded DNA virus that primarily infects epithelial cells. Persistent infection with high-risk types (such as HPV16 and HPV18) can lead to cervical cancer, squamous cell carcinoma of the head and neck (including oral cancer), and other cancers. HPV interferes with cell cycle and apoptosis by degrading p53 and Rb through the E6 / E7 oncoprotein.

[0021] The term "HSV-1" as used in this article refers to an enveloped double-stranded DNA virus that primarily causes cold sores (herpes labialis), but can also cause keratitis, encephalitis, etc. HSV-1 can establish latent infection in ganglia and reactivate in immunocompromised individuals.

[0022] The term "NICD" as used in this article refers to the intracellular fragment released by γ-secretase after the Notch signaling pathway is activated by ligands. NICD enters the cell nucleus, binds to the transcription factor CSL, and activates the transcription of downstream target genes (such as Hes and Hey), participating in cell differentiation, proliferation, and tumorigenesis.

[0023] The term "NTA" as used in this article refers to a particle characterization instrument based on laser scattering and Brownian motion tracking, used to measure the particle size distribution and concentration of nanoparticles (such as EVs). Typical devices, such as ZetaView and NanoSight, can track particle motion in real time and convert it into particle size and quantity, and are commonly used characterization tools in EV research.

[0024] As used in this article, "OSCC" refers to squamous cell carcinoma originating from the oral mucosal epithelium, accounting for more than 90% of oral cancers. Major risk factors include tobacco, alcohol, betel nut consumption, and HPV infection. It is characterized by local invasion and lymph node metastasis, with a 5-year survival rate of approximately 60-70%.

[0025] The term "PDEVs" used in this article refers to EVs secreted by probiotics (such as Lactobacillus and Bifidobacterium), which carry proteins, RNA, and metabolites derived from the bacteria. PDEVs possess biological activities such as anti-inflammatory effects, immunomodulation, promotion of intestinal barrier function, and induction of tumor cell apoptosis, and are one of the important mediators through which probiotics exert their effects.

[0026] The term "Pg" as used in this article refers to a Gram-negative anaerobic coccus that is a major pathogen of chronic periodontitis. It secretes gingipains and virulence factors, damaging host tissues and evading immune clearance. It is associated with a variety of systemic diseases, including oral cancer, Alzheimer's disease, and cardiovascular disease.

[0027] The term "TEM" as used in this article refers to a microscope that uses a high-energy electron beam to image ultrathin samples, achieving a resolution of 0.1-0.2 nm. It is used to observe the ultrastructure of cells, viral particles, and the morphology of EVs. It is the "gold standard" method for verifying sample purity and morphology in EV research.

[0028] The term "Sm" used in this article refers to a Gram-positive facultative anaerobic coccus, a major pathogenic bacterium of dental caries. It possesses strong acid-producing, acid-resistant, and extracellular polysaccharide-synthesizing abilities, enabling it to form cariogenic biofilms on tooth surfaces. Recent studies have found that its endothelial cells (EVs) can activate the β-catenin pathway, promoting the progression of oral squamous cell carcinoma.

[0029] The term "Ss" used in this article refers to a Gram-positive facultative anaerobic coccus, one of the earliest symbiotic bacteria to colonize the human oral cavity. It primarily inhabits the dorsum of the tongue and saliva, does not produce acid or cause tooth decay, and instead secretes bacteriocins to inhibit pathogenic bacteria (such as...). S. mutans (This is considered a marker of oral health and a potential probiotic.)

[0030] The term "4NQO" as used in this article refers to a carcinogenic chemical widely used to induce oral cancer animal models. Its metabolites can form adducts with DNA, leading to mutations such as G→T transversions. Administering 4NQO dissolved in mouse drinking water (e.g., 50 µg / mL) for 8–16 weeks can induce tongue squamous cell carcinoma highly similar to human OSCC, making it a classic tool for studying the occurrence and progression of oral cancer.

[0031] As used herein, the term "prevention and / or treatment" means that a substance, composition, or method has one or more functions of "preventing" the occurrence of a disease, or "treating" a disease that has already occurred, or "having both preventive and therapeutic functions."

[0032] In a first aspect, the present invention provides: a Streptococcus salivarius Ss-08, wherein the Streptococcus salivarius Ss-08 has the accession number CCTCC NO:M20261361, the accession location is China Center for Type Culture Collection, and the accession date is June 17, 2026.

[0033] Secondly, the present invention provides a preparation of Streptococcus salivarius Ss-08, the preparation comprising: fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, and extracellular vesicles.

[0034] Specifically, the fermentation broth comprises a mixed liquid system of the cells themselves, intracellular and extracellular metabolites, unused culture medium components, and fermentation byproducts.

[0035] Specifically, the preparation method of the fermentation broth is, for example, to culture Streptococcus salivarius Ss-08 in a culture medium under artificially controlled fermentation conditions.

[0036] Specifically, the fermentation broth precipitate includes live / dead cells of the strain, cell fragments, and insoluble substances produced in the fermentation system.

[0037] Specifically, the preparation method of the fermentation broth precipitate is, for example, the solid phase component separated after the fermentation broth of the strain is subjected to treatment such as standing, centrifugation or filtration.

[0038] Specifically, the fermentation broth supernatant contains a clarified liquid phase component consisting of the strain's extracellular metabolites, soluble culture medium residues, and soluble fermentation byproducts.

[0039] Specifically, the method for preparing the supernatant of the fermentation broth is, for example, to obtain it by allowing the fermentation broth of the strain to stand, centrifuge, or filter to remove solid phase precipitates such as bacterial cells.

[0040] Specifically, the live bacteria are bacteria with normal physiological activity that can carry out life activities such as metabolism and reproduction.

[0041] According to some embodiments of the present invention, the method for preparing live Streptococcus salivarius Ss-08 includes the following steps: (1) Culture of Streptococcus salivarius Ss-08; (2) Isolate bacterial cells.

[0042] Furthermore, in step (1), the inoculum size is 10. 4 -10 6 CFU / mL of Streptococcus salivarius culture; The separation described in step (2) is centrifugation, with centrifugation conditions of 4℃ at 4000-7000 rpm for 15-20 minutes.

[0043] Furthermore, in step (1), the inoculum size is 1.5 × 10⁻⁶. 4 CFU / mL of Streptococcus salivarius culture; The separation described in step (2) is centrifugation, with centrifugation conditions of 4°C at 5000 rpm for 20 min.

[0044] Specifically, the method for preparing the extracellular vesicles of Streptococcus salivarius Ss-08 includes the following steps: (1) Resuscitate Streptococcus salivarius Ss-08; (2) Culture of Streptococcus salivarius Ss-08; (3) Obtain the supernatant from the bacterial culture; (4) Obtain the precipitate from the supernatant.

[0045] Furthermore, step (1) includes taking 10 4 -10 6 Ss-08 bacterial suspensions at CFU / mL were inoculated into culture media and incubated at 37℃ for 16–18 h. Step (2) involves collecting the bacterial culture after incubation at 37°C for 16–18 h, resuspending the bacterial cells, and continuing the incubation. Step (3) involves centrifuging the bacterial culture to remove the precipitate and retaining the supernatant; Step (4) involves centrifuging the supernatant obtained in the previous step, discarding the supernatant, and collecting the resulting precipitate.

[0046] Furthermore, step (1) includes taking 1.5 × 10 4 Ss-08 bacterial suspension with CFU / mL was inoculated into 45 mL BHI medium and incubated at 37℃ for 16–18 h. Step (2) involves centrifuging the bacterial culture at 5000 rpm for 20 min at 4℃ after static incubation at 37℃ for 16–18 h, discarding the supernatant, collecting the precipitate, resuspending it, and inoculating it into culture flasks containing 400 mL of fresh BHI medium, and continuing static incubation at 37℃ for 16–18 h. Step (3) involves centrifuging the bacterial culture at 4°C and 5000 rpm for 20 min, discarding the precipitate, and retaining the supernatant; Step (4) involves aliquoting the supernatant from which the bacterial cells have been filtered into ultracentrifuge tubes and centrifuging at 120,000 × g and 4°C for 60 min. The supernatant is discarded, and the precipitate obtained from centrifugation is collected.

[0047] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: The first preferred solution involves preparing extracellular vesicles and performing multiple centrifugations when obtaining the supernatant from the bacterial culture. This solution not only solves the technical problem of "isolating live bacteria" but also further addresses the technical problem of "low purity of extracellular vesicles".

[0048] The second preferred solution involves preparing extracellular vesicles, centrifuging the supernatant from the bacterial culture multiple times, filtering it through a membrane, and then spreading it onto BHI agar plates. This solution not only solves the technical problem of "isolating live bacteria" but also further addresses the technical problem of "testing whether live bacteria are present in the supernatant."

[0049] The third preferred solution involves preparing extracellular vesicles and centrifuging multiple times when obtaining the precipitate from the supernatant. This solution not only addresses the technical problem of "obtaining extracellular vesicles" but also further solves the technical problem of "purifying extracellular vesicles."

[0050] Thirdly, the present invention provides a microbial agent comprising the above-mentioned Streptococcus salivarius Ss-08 or the above-mentioned preparation.

[0051] The microbial agent may be a solid, liquid, semi-solid, or any physical form containing the necessary active ingredients to achieve any of the applications, functions, or effects described in this invention, which can be obtained by those skilled in the art through current or future technologies.

[0052] Specifically, the bacterial agent is a liquid preparation, including one of the following: injection, oral solution, mixture, tincture, lotion, suspension, and gel.

[0053] Specifically, the liquid preparation is an injection solution.

[0054] The microbial agent also includes nutritionally acceptable nutrient additives.

[0055] The nutritional additives mentioned herein include, but are not limited to, any one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0056] Fourthly, the present invention provides the use of the above-mentioned Streptococcus salivarius Ss-08 or the above-mentioned preparation in the preparation of medicaments for the prevention and / or treatment of oral diseases.

[0057] The viable count of Streptococcus salivarius Ss-08 in the drug is 1×10⁻⁶. 6 -1×10 8 CFU / mL or 1×10 6 -1×10 8 CFU / mg.

[0058] Furthermore, the viable count of *Streptococcus salivarius* Ss-08 in the drug is 1 × 10⁻⁶. 7 CFU / mL.

[0059] The drug contains 300-600 μg / mL of extracellular vesicles of Streptococcus salivarius Ss-08.

[0060] Furthermore, the drug contains 400 μg / mL of extracellular vesicles of Streptococcus salivarius Ss-08.

[0061] The oral diseases mentioned herein are selected from one of the following: dental caries and pulp diseases, periodontal tissue diseases, oral mucosal diseases, oral and maxillofacial surgical and developmental diseases, and dentition and occlusion abnormalities.

[0062] Furthermore, the oral and maxillofacial surgery and developmental diseases mentioned are selected from one of the following: dental and alveolar surgical diseases, oral and maxillofacial infections, oral and maxillofacial injuries, oral and maxillofacial tumors, salivary gland diseases, and maxillofacial deformities and developmental diseases; the oral mucosal diseases mentioned are selected from one of the following: oral mucosal infectious diseases, oral mucosal immune diseases, oral mucosal ulcer diseases, potential oral malignant diseases, oral mucosal allergic diseases, and oral mucosal traumatic diseases.

[0063] Furthermore, the oral and maxillofacial tumor is oral squamous cell carcinoma; the potential malignant disease of the oral cavity is oral submucosal fibrosis.

[0064] The drug has at least one of the following effects: (1) Improve oral mucosal pathological damage caused by oral submucosal fibrosis; (2) Inhibit abnormal activation of gene signaling pathways in the tongue; (3) Promote the recovery of expression of molecules related to tight junctions in oral epithelium; (4) Reduce the progression of oral fibrosis; (5) Regulating oral and intestinal flora to participate in the regulation of oral submucosal fibrosis; (6) Inhibits transcription of oral squamous cell carcinoma cells; (7) Inhibits the progression of oral squamous cell carcinoma cells.

[0065] Fifthly, the present invention provides a product for the prevention and / or treatment of oral diseases, the product comprising the above-mentioned Streptococcus salivarius Ss-08 or the above-mentioned preparation or the above-mentioned bacterial agent.

[0066] The product is a drug, and the viable count of Streptococcus salivarius Ss-08 in the drug is 1×10⁻⁶. 6 -1×10 8 CFU / mL or 1×10 6 -1×10 8 CFU / mg.

[0067] Furthermore, the viable count of *Streptococcus salivarius* Ss-08 in the drug is 1 × 10⁻⁶. 7 CFU / mL.

[0068] The drug contains 300-600 μg / mL of extracellular vesicles of Streptococcus salivarius Ss-08.

[0069] Furthermore, the drug contains 400 μg / mL of extracellular vesicles of Streptococcus salivarius Ss-08.

[0070] Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0071] Embodiments 1-2 of this invention at least support the protection scope of claim 1.

[0072] Regarding the claim 1: The technical feature "Salinomyces salinomyces Ss-08" is derived from the collection and pretreatment of samples, isolation and purification of Streptococcus salinomyces, preliminary identification of Streptococcus salinomyces, and molecular biological identification of Streptococcus salinomyces as explained above or in Examples 1-2, summarized by the common feature "Salinomyces salinomyces". Therefore, those skilled in the art can reasonably presume that the subordinate concept of the technical feature "Salinomyces salinomyces Ss-08", the technical means that are basically equivalent to "Salinomyces salinomyces Ss-08", and the technical means that can replace "Salinomyces salinomyces Ss-08" based on the existing technical level and within the scope of conventional technical means and common knowledge should all fall within the protection scope of claim 1.

[0073] Embodiments 1, 11, and 12 of this invention at least support the protection scope of claims 2-3.

[0074] Regarding claims 2-3: The technical feature "preparation of *Streptococcus salivarius* Ss-08" is derived from the common feature "*Streptococcus salivarius* Ss-08" in the foregoing explanation or Examples 1, 11, and 12. Therefore, those skilled in the art can reasonably infer that the technical feature "preparation of *Streptococcus salivarius* Ss-08," the subordinate concept of "preparation of *Streptococcus salivarius* Ss-08," the essentially equivalent technical means of "preparation of *Streptococcus salivarius* Ss-08," and the technical means that can replace "preparation of *Streptococcus salivarius* Ss-08" within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of claims 2-3. For example, replacing "preparation of *Streptococcus salivarius* Ss-08" with extracellular vesicles of *Streptococcus salivarius* Ss-08, fermentation broth of *Streptococcus salivarius* Ss-08, etc., while keeping other technical features unchanged, still falls within the protection scope of claims 2-3 of this invention.

[0075] Embodiments 3-10 and 13-19 of this invention at least support the protection scope of claim 4.

[0076] Regarding claim 4: The technical feature "a bacterial agent" is derived from the corresponding technical features of fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, extracellular vesicles, etc., as explained above or in Examples 3-10 and 13-19, summarized by the common feature "Streptococcus salivarius Ss-08". Therefore, those skilled in the art can reasonably infer that the preparation of the technical feature Streptococcus salivarius Ss-08, the subordinate concept of the preparation of Streptococcus salivarius Ss-08, the technical means that are basically equivalent to the preparation of Streptococcus salivarius Ss-08, and the technical means that can replace the preparation of Streptococcus salivarius Ss-08 within the scope of conventional technical means and common knowledge based on the existing technical level, should all fall within the protection scope of claim 4. For example, if the preparation of Streptococcus salivarius Ss-08 is replaced with extracellular vesicles of Streptococcus salivarius Ss-08, fermentation broth of Streptococcus salivarius Ss-08, etc., while other technical features remain unchanged, it still falls within the protection scope of claim 4 of this invention.

[0077] Embodiments 3-10 and 13-19 of this invention at least support the protection scope of claims 5-8.

[0078] Regarding claims 5-8: The technical feature "application in drugs for the prevention and / or treatment of oral diseases" is summarized from the corresponding technical features in the foregoing explanation or Examples 3-10 and 13-19 of this document: improving OSF oral mucosal pathological damage; inhibiting abnormal activation of tongue gene signaling pathways; promoting the recovery of expression of molecules related to tight junctions in oral epithelium; significantly reducing the process of oral fibrosis; regulating the participation of oral flora and gut flora in the regulation of oral submucosal fibrosis; systematically inhibiting signaling pathways transcribed by CAL-27 cells; inhibiting CAL-27 cell activity in vitro; and inhibiting CAL-27 cell growth in vivo. Therefore, those skilled in the art can reasonably presume that the application of technical features in drugs for the prevention and / or treatment of oral diseases, the subordinate concepts of the application of drugs for the prevention and / or treatment of oral diseases, the technical means that are essentially equivalent to the application of drugs for the prevention and / or treatment of oral diseases, and the technical means that can replace the application of drugs for the prevention and / or treatment of oral diseases based on the existing level of technology and conventional technical means and common knowledge should all fall within the protection scope of claims 5-8. For example, if the application of drugs for the prevention and / or treatment of oral diseases is replaced with the application of drugs for the prevention and / or treatment of oral and maxillofacial tumors, or the application of drugs for the prevention and / or treatment of potential malignant oral diseases, while other technical features remain unchanged, it still falls within the protection scope of claims 5-8 of this invention.

[0079] Embodiments 3-10 and 13-19 of this invention at least support the protection scope of claims 9-10.

[0080] Regarding claims 9-10: The technical feature "drugs for the prevention and treatment of oral diseases" is summarized from the corresponding technical features explained above or in Examples 3-10 and 13-19: improving the pathological damage of the oral mucosa in OSF; inhibiting the abnormal activation of gene signaling pathways in the tongue; promoting the recovery of expression of molecules related to tight junctions in the oral epithelium; significantly reducing the process of oral fibrosis; regulating the oral flora and gut flora in the regulation of oral submucosal fibrosis; systematically inhibiting the signaling pathways of CAL-27 cell transcription; inhibiting CAL-27 cell activity in vitro; and inhibiting CAL-27 cell growth in vivo. Therefore, those skilled in the art can reasonably presume that drugs for preventing and treating oral diseases, subordinate concepts of drugs for preventing and treating oral diseases, technical means that are essentially equivalent to drugs for preventing and treating oral diseases, and technical means that can replace drugs for preventing and treating oral diseases based on existing technical levels and conventional technical means and common knowledge should all fall within the protection scope of claims 9-10. For example, if other technical features remain unchanged, replacing drugs for preventing and treating oral diseases with drugs for preventing and treating oral and maxillofacial tumors or drugs for preventing and treating potential malignant oral diseases still falls within the protection scope of claims 9-10 of this invention.

[0081] The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in terms of the effects of Streptococcus salivarius on oral mucosal fibrosis, the effects of Streptococcus salivarius on collagen deposition, and the effects of extracellular vesicles of Streptococcus salivarius on the proliferation, healing and invasion of CAL-27 cells.

[0082] According to experimental tests, this invention changes the effect of saliva streptococci on oral mucosal fibrosis and collagen deposition from the existing technology where the abundance of streptococci in the oral flora of OSF patients is higher, to providing a saliva streptococcus that can reduce the degree of oral mucosal fibrosis and collagen deposition.

[0083] Experimental tests investigated the effects of extracellular vesicles (SmEVs) of Streptococcus salivarius on the proliferation, healing, and invasion of CAL-27 cells. Current technology shows that SmEVs can be absorbed by OSCC cells (CAL-27) and significantly enhance the proliferation, migration, and invasion of cancer cells, with a maximum proliferative concentration of 400 µg / mL, promoting CAL-27 cell proliferation in a concentration-dependent manner. In OSCC cells treated with SmEVs, Wnt / β-catenin was significantly upregulated (p<0.05), and the expression of downstream key molecules TCF7 and Fra-1 also increased accordingly (P<0.01, P<0.001). In animal (nude mouse) experiments, the tumor growth rate in the SmEV-injected experimental group was significantly faster than that in the control group (p<0.05), further confirming its in vivo pro-cancer effect, namely, promoting the malignant progression of oral squamous cell carcinoma. In this invention, SsEVs significantly inhibited CAL-27 cell proliferation at both 24h and 48h in a concentration-dependent manner, with a maximum inhibitory concentration of 400 µg / mL. Simultaneously, it significantly reduced cell scratch healing ability and decreased the number of Transwell-migrating and invasive cells. Nude mouse xenograft experiments showed that tumor volume growth was slowed in the SsEVs-treated group, with both terminal tumor volume and weight significantly lower than the control group (p<0.001). RNA-seq revealed widespread transcriptional remodeling in CAL-27 cells after SsEVs treatment, with downregulated genes significantly enriched in cell cycle, Wnt, Hippo, Ras / Rap1, TGF-β, and Notch pathways (p<0.01, p<0.001); the Notch signaling pathway showed significant negative enrichment. Further validation showed that Notch1, Jagged1, and HeyL were significantly downregulated at both mRNA and protein levels, and the immunohistochemical staining intensity of Notch1, Jagged1, and HeyL in xenograft tissue was also significantly reduced.

[0084] According to experimental tests, this invention isolated multiple strains of Streptococcus salivarius from saliva samples of healthy volunteers. After observation and identification, all strains conformed to the characteristics of the Streptococcus genus. Among them, strain Ss-08 showed strong antibacterial activity, low pathogenicity-related phenotype, and good safety.

[0085] According to experimental tests, this invention applied Ss-08 to OSF. Compared with the OSF group, the Ss intervention group showed improved oral mucosal pallor and limited mouth opening, reduced oral mucosal fibrosis, and decreased collagen deposition. GO functional enrichment analysis showed that differentially expressed genes related to OSF mainly participate in extracellular matrix remodeling and inflammatory immune responses; after Ss intervention, differentially expressed genes mainly involve immune regulation, barrier function maintenance, and innate immune-related signaling pathways. GSEA results showed that compared with the CON group, the OSF group showed up-regulation and enrichment of processes such as keratinization, Wnt signaling pathway, cell cycle, and ribosome biogenesis, and down-regulation and enrichment of T cell proliferation-related processes; after Ss intervention, down-regulation and enrichment of processes such as keratinization, cell cycle, ribosome biogenesis, and chromosome organization / separation were predominantly observed. KEGG enrichment results showed that Ss intervention can significantly inhibit Wnt signaling pathway activation in OSF and upregulate tight junction pathway gene expression. RT-qPCR results showed that after Ss intervention, the expression of Wnt3a, Ctnnb1 and Ccnd1 genes was downregulated, while the expression of Cldn3 was upregulated.

[0086] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for treating oral diseases, the method comprising administering to a subject the above-described Streptococcus salivarius Ss-08 or the above-described preparation or the above-described bacterial agent or the above-described product.

[0087] Preservation Instructions Preserved strain: Streptococcus syringae Ss-08; Category Naming: Streptococcus salivarius Ss-08; Accession number: CCTCC NO: M 20261361; Preservation period: June 17, 2026; Depository: China Center for Type Culture Collection; Location of the collection: Wuhan University, Wuhan, China. Attached Figure Description

[0088] Figure 1 This image shows the colony morphology and characteristics of the isolated strain.

[0089] Figure 2 This image shows the results of Streptococcus salivarius on Columbia blood agar plates without hemolysis.

[0090] Figure 3 In the figure, A represents the biofilm formation ability of the *Streptococcus salivarius* strain. Figure 3 In the figure, B represents the inhibitory effect of *Streptococcus salivarius* on biofilm formation of *Streptococcus mutans*. Compared with the *Streptococcus mutans* group, P<0.05, P<0.01, P<0.001, P < 0.0001; t-test.

[0091] Figure 4 Figure A in the figure shows the results of arecoline-induced changes in body weight in OSF mice improved by Streptococcus salivarius. Figure 4 Figure B in the graph shows the results of arecoline-induced changes in water intake in OSF mice induced by Streptococcus salivarius. # Compared to the CON group, #### P<0.0001; Compared to the OSF group, P<0.05, P<0.01, one-way ANOVA.

[0092] Figure 5 Pathological changes in the oral mucosa of mice; Figure 5 The images in AC represent the results of HE staining in three groups. Figure 5 The DF diagram in the image shows the results of three groups of Masson staining.

[0093] Figure 6 This is a volcano plot analysis result of differentially expressed genes; Figure 6 In the figure, A represents the volcano plot showing the difference in oral mucosal tissue transcriptomes between the OSF group and the Con group; Figure 6 In the diagram, B represents the differentially expressed gene volcano plot between the OSF+Ss group and the OSF group; red dots indicate significantly upregulated genes, and green dots indicate significantly downregulated genes.

[0094] Figure 7 This is a graph showing the GO enrichment analysis results of biological processes (BP) items enriched in the comparison between the OSF group and the CON group; the x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0095] Figure 8 This is a graph showing the GO enrichment analysis results of enriched cellular components (CC) in the comparison between the OSF group and the CON group; the x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0096] Figure 9This is a graph showing the GO enrichment analysis results of the enriched molecular function (MF) items in the comparison between the OSF group and the CON group; the x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0097] Figure 10 This is a graph showing the GO enrichment analysis results of biological processes (BP) entries enriched in the comparison between the OSF+Ss group and the OSF group; the x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0098] Figure 11 This is a graph showing the GO enrichment analysis results of enriched cellular components (CC) between the OSF+Ss group and the OSF group; the x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0099] Figure 12 This is a graph showing the GO enrichment analysis results of the enriched molecular function (MF) items in the comparison between the OSF+Ss group and the OSF group; the x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0100] Figure 13 This is a graph showing the results of GSEA-GO bioprocess analysis based on the enrichment map; Figure 13 In this context, A represents the GOBP functional modules enriched in the comparison between the OSF group and the CON group; Figure 13 In the diagram, B represents the GOBP functional module enriched in the comparison between the OSF+Ss group and the OSF group. Each node represents a GO biological process, the node size represents the size of the gene set, the node color reflects the direction and degree of enrichment, and the edges represent the overlap between gene sets.

[0101] Figure 14 The KEGG pathway enrichment analysis is shown in the comparison between the OSF group and the CON group. The left side is the pathway-gene association diagram, and the right side is the enrichment bubble diagram. The x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0102] Figure 15 The KEGG pathway enrichment analysis was performed between the OSF+Ss group and the OSF group. The left side is a pathway-gene association diagram, and the right side is an enrichment bubble diagram. The x-axis represents the rich factor, the size of the point represents the number of genes, and the color represents statistical significance.

[0103] Figure 16 A heatmap showing differentially expressed genes related to tight junction pathways between the OSF and CON groups.

[0104] Figure 17 A heatmap showing differentially expressed genes related to tight junction pathways between the OSF+Ss group and the OSF group.

[0105] Figure 18 This is a GSEA enrichment analysis diagram of the Wnt signaling pathway compared to the control group in the OSF group.

[0106] Figure 19 A heatmap showing the core genes of the Wnt signaling pathway in comparison between the OSF and CON groups.

[0107] Figure 20 The GSEA enrichment analysis diagram of the Wnt signal pathway is shown for the OSF+Ss group compared with the OSF group.

[0108] Figure 21 A heatmap showing the core genes of the Wnt signaling pathway in comparison between the OSF+Ss group and the OSF group.

[0109] Figure 22 The figure shows the expression results of Wnt signaling pathway-related genes in the oral tissue of OSF mice after Ss intervention; Figure 22 In this context, A represents the Wnt3a gene; Figure 22 In this context, B stands for the Ctnnb1 gene; Figure 22 In this context, C represents the mRNA expression level of the Ccnd1 gene; # Compared to the CON group, ## P<0.01, #### P<0.0001; Compared to the OSF group, P<0.05, P<0.01, P<0.0001; one-way ANOVA.

[0110] Figure 23 This image shows the expression results of tight junction pathway-related genes in the oral tissue of OSF mice after Ss intervention. # Compared to the CON group, #### P<0.0001. Compared to the OSF group, P < 0.05. One-way ANOVA.

[0111] Figure 24 The results show the effects of *Streptococcus saliva* on the diversity of oral microbiota in ARE-induced OSF mice, and the sparse curve of the Shannon index at the OTU level.

[0112] Figure 25 The results show the effects of *Streptococcus saliva* on the oral microbiota diversity of ARE-induced OSF mice, and the sparse curve results of Simpson's curve at the OTU level.

[0113] Figure 26 Figure 1 shows the effect of *Streptococcus saliva* on the diversity of oral microbiota in ARE-induced OSF mice, and figure 2 shows the results of Shannon index α diversity analysis.

[0114] Figure 27 Figure 1 shows the effect of *Streptococcus saliva* on the diversity of oral microbiota in ARE-induced OSF mice, and figure 2 shows the results of Simpson index α diversity analysis.

[0115] Figure 28 The results of the effect of Streptococcus salivarius on the oral microbiota diversity of ARE-induced OSF mice are shown in the figure, and the results of principal coordinate analysis (PCoA) of OTU levels in each group are also shown in the figure.

[0116] Figure 29 The figure shows the effect of Streptococcus saliva on the oral microbiota diversity of ARE-induced OSF mice, and the non-metric multidimensional scaling (NMDS) results of OTU levels in each group.

[0117] Figure 30 Figure showing the effect of *Streptococcus salivarius* on the oral microbiota diversity of ARE-induced OSF mice; Figure 30 In the figure, A represents the results of the Microbial Disorder Index (MDI) for each group; Figure 30 B in the figure represents the Venn diagram analysis results of the OTU levels in each group.

[0118] Figure 31 This is a diagram showing the results of oral microbial composition analysis at the phylum level.

[0119] Figure 32 The results of the oral microbial composition analysis at the class level are shown in the figure.

[0120] Figure 33 The image shows the results of an analysis of the oral microbial composition at the target level.

[0121] Figure 34 The results of the oral microbial composition analysis at the scientific level are shown in the figure.

[0122] Figure 35 The figure shows the results of the oral microbial composition analysis at the genus level.

[0123] Figure 36 This image shows the results of an analysis of oral microbiota differences among different treatment groups, comparing the relative abundance of Streptococcus spp. # Compared to the CON group, # P<0.05, ## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0124] Figure 37 This image shows the results of an analysis of oral microbiota differences among different treatment groups, comparing the relative abundance of the genus *Lactobacillus*. # Compared to the CON group, # P<0.05, ## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0125] Figure 38 This image shows the results of oral microbiota difference analysis among different treatment groups, comparing the relative abundance of *Mammalian cocci*. # Compared to the CON group, # P<0.05, ## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0126] Figure 39 This image shows the results of the oral microbiota difference analysis between different treatment groups, comparing the relative abundance of the genus *Cyclocarya*. # Compared to the CON group, # P<0.05, ## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0127] Figure 40 This image shows the results of the oral microbiota difference analysis between different treatment groups, comparing the relative abundance of the genus *Massezia*. # Compared to the CON group, # P<0.05, ## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0128] Figure 41 This image shows the results of the oral microbiota difference analysis among different treatment groups, comparing the relative abundance of *Sphingosine monocytogenes*. # Compared to the CON group, # P<0.05,## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0129] Figure 42 This image shows the results of the oral microbiota difference analysis between different treatment groups, comparing the relative abundance of the genus *Aquaticbacterium*. # Compared to the CON group, # P<0.05, ## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0130] Figure 43 This image shows the results of the oral microbiota difference analysis between different treatment groups, comparing the relative abundance of Enterococcus spp. # Compared to the CON group, # P<0.05, ## P<0.01; Compared to the OSF group, P<0.05, P<0.01.

[0131] Figure 44 The graph shows the effect of *Streptococcus salivarius* on the gut microbiota diversity of ARE-induced OSF mice, and the sparse curve of the Shannon index at the OTU level. # Compared to the CON group, #### P<0.0001; Compared to the OSF group, P<0.05; one-way ANOVA.

[0132] Figure 45 The graph shows the effect of *Streptococcus salivarius* on the gut microbiota diversity of ARE-induced OSF mice, and the sparse curve of the Simpson index at the OTU level. # Compared to the CON group, #### P<0.0001; Compared to the OSF group, P<0.05; one-way ANOVA.

[0133] Figure 46 Figure 1 shows the effect of *Streptococcus salivarius* on the gut microbiota diversity of ARE-induced OSF mice, and Figure 2 shows the results of Shannon index α-diversity analysis. # Compared to the CON group, ####P<0.0001; Compared to the OSF group, P<0.05; one-way ANOVA.

[0134] Figure 47 Figure 1 shows the effect of *Streptococcus salivarius* on the gut microbiota diversity of ARE-induced OSF mice, and figure 2 shows the results of Simpson index α-diversity analysis. # Compared to the CON group, #### P<0.0001; Compared to the OSF group, P<0.05; one-way ANOVA.

[0135] Figure 48 The graph shows the effect of *Streptococcus salivarius* on the gut microbiota diversity of ARE-induced OSF mice, along with the principal coordinate analysis (PCoA) results for OTU levels in each group. # Compared to the CON group, #### P<0.0001; Compared to the OSF group, P<0.05; one-way ANOVA.

[0136] Figure 49 The graph shows the effect of *Streptococcus salivarius* on the gut microbiota diversity of ARE-induced OSF mice, and the non-metric multidimensional scaling (NMDS) analysis results of OTU levels in each group. # Compared to the CON group, #### P<0.0001; Compared to the OSF group, P<0.05; one-way ANOVA.

[0137] Figure 50 This figure shows the effect of *Streptococcus salivarius* on the gut microbiota diversity of ARE-induced OSF mice. Figure 50 In the figure, A represents the results of the Microbial Disorder Index (MDI) for each group; Figure 50 In the diagram, B represents the Venn diagram analysis results of the OTU levels for each group. # Compared to the CON group, #### P<0.0001; Compared to the OSF group, P<0.05; one-way ANOVA.

[0138] Figure 51 The figure shows the results of the gut microbiota composition analysis at the phylum level.

[0139] Figure 52 The results of the gut microbiota composition analysis at the class level are shown in the figure.

[0140] Figure 53The figure shows the results of the intestinal microbial composition analysis at the target level.

[0141] Figure 54 The results of the intestinal microbiome composition analysis at the scientific level are shown in the figure.

[0142] Figure 55 The figure shows the results of the gut microbiota composition analysis at the genus level.

[0143] Figure 56 The results of LEfSe analysis of gut microbiota were presented in the figure (LDA>3) to analyze the differences in gut microbiota among the groups.

[0144] Figure 57 The LDA analysis results of gut microbiota were used to analyze the differences in gut microbiota among the groups (LDA>3).

[0145] Figure 58 A in the image is a transmission electron microscope (TEM) image of a salicylic streptococcal extracellular vesicle (SsEVs). Figure 58 B in the figure represents the nanoparticle tracking analysis (NTA) results of SsEVs.

[0146] Figure 59 Fluorescence microscopy image of oral squamous cell carcinoma cells taking up salivary streptococcal extracellular vesicles (SsEVs).

[0147] Figure 60 Figure showing the effects of SsEVs on the activity and proliferation of CAL-27 cells; Figure 60 In the figure, A represents the cell viability results after 24 hours of SsEV treatment. p<0.01, p<0.001; (analysis of variance) Figure 60 In the figure, B represents the cell viability results after 48 hours of SsEV treatment. p<0.001; (analysis of variance) Figure 60 The C in the text stands for real-time cell analysis (iCELLigence).

[0148] Figure 61 Figure A in the figure represents the effect of SsEVs on scratch healing of CAL-27 cells; Figure 61 B in the figure represents the quantitative analysis of the scratch test; Figure 61 In the image, C represents a representative image of CAL-27 cell migration and invasion after SsEVs treatment; Figure 61 In the figure, D represents the quantitative results of CAL-27 cells migrating and invading in each group. p<0.01, p<0.001; t-test).

[0149] Figure 62 Image A in the image represents the effect of SsEVs on the volume of CAL-27 xenograft tumors; Figure 62 In the figure, B represents the growth curve of the xenograft volume in nude mice; Figure 62 In the comparison, C represents the final tumor volume; Figure 62 In the figure, D represents the change in tumor volume between the control group and the SsEVs treatment group. p<0.001; t-test).

[0150] Figure 63 Volcano diagram of differential gene expression in CAL-27 cells after treatment with SsEVs.

[0151] Figure 64 KEGG bubble plot for downregulated gene enrichment.

[0152] Figure 65 The figure shows the GSEA enrichment analysis results for the Notch signaling pathway.

[0153] Figure 66 Figure showing the results of RT-qPCR validation of Notch signaling pathway-related gene expression in CAL-27 cells after SsEVs treatment; Figure 66 In this context, A represents the mRNA expression level of the NOTCH1 gene; Figure 66 In this context, B represents the mRNA expression level of the DLL1 gene; Figure 66 In this context, C represents the mRNA expression level of the DTX4 gene; Figure 66 In this context, D represents the mRNA expression level of the HEYL gene; Figure 66 In this context, E represents the mRNA expression level of the JAG1 gene; Figure 66 In this context, F represents the mRNA expression level of the MAML gene. p<0.01, p<0.001, p<0.0001; t-test).

[0154] Figure 67 In the figure, A represents the Western blotting changes of Notch1, Jagged1, and HEYL in CAL-27 cells after SsEVs treatment; Figure 67 B in the figure represents the quantitative analysis of the expression levels of Notch1, Jagged1, and HEYL proteins in the Notch signaling pathway in CAL-27 cells using Western blot analysis (SsEVs). p<0.01, p<0.001; t-test).

[0155] Figure 68 In Figure A, there are representative images and quantitative analysis of the immunohistochemical effects of SsEVs on Notch1 expression in the Notch signaling pathway in xenografts. Figure 68 In Figure B, there are representative images and quantitative analysis of the immunohistochemical effects of SsEVs on Jagged1 expression in the Notch signaling pathway in xenografts. Figure 68 C in the figure represents representative images and quantitative analysis of the immunohistochemical effect of SsEVs on HeyL expression in the Notch signaling pathway in xenografts. Staining intensity is expressed as mean optical density (IOD / Area). Data are expressed as mean ± standard deviation. p<0.01, p<0.001; t-test.

[0156] Figure 69 This is a comparison of the effects of S1-S7 and S8 of *Streptococcus salivarius* on oral cancer cells. p<0.01; t-test. Detailed Implementation

[0157] 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.

[0158] 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.

[0159] To ensure the reliability of the experimental results, each experiment was performed at least three times independently. The obtained data were statistically processed and plotted using GraphPad Prism 10.3.1 software. Independent samples t-tests were used to compare two groups, while differences among multiple groups were assessed using one-way ANOVA. A p-value < 0.05 was used as the criterion for statistical significance. The significance level was indicated as follows: ( P<0.05; P<0.01; P<0.001, p<0.0001).

[0160] Example 1: Obtaining experimental materials for the isolation and screening of oral streptococci. 1. Experimental strains The oral streptococci used in this invention were obtained from saliva samples of healthy volunteers at Harbin Medical University. All volunteers participated voluntarily with full informed consent, and the sample collection process strictly adhered to relevant ethical requirements and biosafety protocols. Fresh saliva samples underwent selective culture, isolation, purification, and preliminary screening to obtain candidate strains, which were further confirmed through morphological observation, mass spectrometry analysis, and 16S rDNA sequencing. Ultimately, oral streptococci were successfully isolated and identified for subsequent experimental research.

[0161] Streptococcus mutans ( Streptococcus mutans Sm)ATCC 35668 was derived from a standard strain preserved in the Department of Microbiology, Harbin Medical University, and was used as a control experiment.

[0162] 2. Animal source Six-week-old healthy male BALB / c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Production License No.: SCXK(Liaoning)2015-0001). The mice were housed in a barrier-type environment that complied with laboratory animal management regulations, and the housing conditions met the specific pathogen-free (SPF) level requirements. To minimize the potential impact of non-experimental factors on the research results, all mice underwent at least one week of acclimatization under laboratory conditions before the start of the experiment.

[0163] All animal experiments and related procedures strictly adhere to the ethical principles of laboratory animals and the standards for the use of laboratory animals.

[0164] 3. Primers The specific primers are shown in Table 1: Table 1

[0165] 4. Main reagents The required bacterial culture reagents are shown in Table 2: Table 2

[0166] Reagents used for bacterial identification and screening are shown in Table 3: Table 3

[0167] The consumables and reagents used in animal experiments are shown in Table 4: Table 4

[0168] The reagents used for HE staining were the hematoxylin and eosin (HE) staining kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0169] The Masson staining reagents used were the Masson trichrome staining kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0170] Table 5 shows the reagents used for RNA extraction and RT-qPCR. Table 5

[0171] 5. Required instruments and equipment The required instruments and equipment are shown in Table 6: Table 6

[0172] Example 2 Preliminary identification and screening of oral streptococci 1. Sample collection and preprocessing (1) Volunteer recruitment and inclusion / exclusion criteria Twenty healthy volunteers were recruited from within the college to participate in this study, and all participants were informed and consented to participate. The entire research process strictly adhered to medical ethics principles and relevant personal information protection regulations, and the personal information and research data of the research subjects were kept strictly confidential throughout the entire process. All collected biological samples (such as saliva samples) were anonymized using a numbering system and did not involve names, ID numbers, or any other information that could identify an individual. The obtained samples and related data were used solely for statistical analysis and academic research purposes in this study and were not used for any other purpose.

[0173] The inclusion and exclusion criteria for volunteers are as follows: 1) Good oral health, with no obvious oral diseases such as tooth decay, periodontal disease, or halitosis; 2) No other major systemic diseases, such as diabetes, cardiovascular disease, immune system diseases, etc.; 3) Age between 20 and 40 years old; 4) Has not received antibiotic treatment in the past 6 months; 5) No history of smoking or alcohol abuse; 6) Non-pregnant women.

[0174] Those who do not meet any of the above conditions will not be included in this study.

[0175] (2) Collection, preservation and pretreatment of saliva Volunteers collected saliva samples upon waking in the morning or two hours after a meal. Prior to collection, participants were instructed to avoid brushing their teeth, eating, and drinking to minimize the impact of exogenous factors on the composition of the saliva microbiota. Saliva samples were collected using the natural drooling method, where participants naturally accumulated saliva in their mouths and spat it into sterile centrifuge tubes. Each volunteer collected approximately 2–5 mL of saliva. The entire sample collection process was conducted under sterile conditions to prevent contamination by exogenous microorganisms.

[0176] Saliva samples should be immediately placed in an icebox for cryogenic storage after collection and processed within 2-4 hours. Before processing, the saliva samples should be vortexed to mix thoroughly. Subsequently, the saliva samples should be serially diluted using sterile PBS at a ratio of 10:1. - ¹~10 -6 A series of dilutions were prepared by dilution factor. Each dilution was used in subsequent experiments related to the isolation and culture of Streptococcus salivarius to obtain an appropriate number of single colonies, thereby ensuring the accuracy and reproducibility of the isolation results.

[0177] 2. Isolation and purification of Streptococcus salivarius (1) Isolation of Streptococcus salivarius According to 10 - ¹~10 -6 Prepare saliva dilutions by dilution factor 1, and evenly spread 100 μL of each onto the surface of MSA selective medium. After inoculation, place the culture plates in a 37°C constant temperature aerobic incubator for 24–48 hours to promote the growth of Streptococcus salivarius.

[0178] After incubation, the colonies on the culture plates were observed, and preliminary screening was conducted based on their growth status and morphological characteristics. Special attention was paid to small, round colonies with regular edges, smooth surfaces, and predominantly white or milky white color, which were suspected to be Streptococcus salivarius. The distribution of colonies at different dilution gradients was also recorded to provide a basis for subsequent purification and identification of the strains.

[0179] (2) Purification of Streptococcus salivarius Single suspected colonies with morphological characteristics consistent with *Streptococcus salivarius* were picked from isolation culture plates using a sterile inoculation loop and streaked under the same culture conditions. Single colonies were obtained using the three-zone streak method, and the strain was purified. Each culture was conducted in a 37°C constant temperature aerobic incubator for 16–18 hours to allow the bacteria to grow to the logarithmic growth phase.

[0180] During the purification process, the consistency of colony morphology and the presence of contaminating bacteria on the surface of the culture medium were continuously observed. The above streak purification steps were repeated until a single colony with uniform morphology, stable growth, and no contamination was obtained, which was then determined to be a pure culture strain. The purified suspected Streptococcus salivarius strains (temporarily numbered from 1 to n) were used for subsequent morphological observation, biochemical identification, and molecular biological identification experiments.

[0181] 3. Preliminary identification of Streptococcus salivarius (1) Morphological observation 1) Prepare BHI agar plates and place them in a sterile environment.

[0182] 2) Remove the inoculation loop and flame it with an alcohol lamp. After cooling, take one loop of bacterial suspension and streak 3-5 lines parallel to each other in zone 1 of the plate. Rotate the plate about 60° and flame it again. Streak the inoculation loop twice from zone 1 to zone 2. Repeat the above steps to streak a total of 3 zones.

[0183] 3) After incubating the plates in a 37°C incubator for 16-18 hours, observe the colony morphology, including the size, shape, boundary clarity, surface condition, and color of the colonies.

[0184] (2) Gram staining 1) Draw a circle about 1 cm in diameter in the center of the slide with about 20 μL of sterile water. Pick a single colony from the plate and spread it evenly in the circle and mix it with water. Let the smear dry in the natural air.

[0185] 2) With the infected side facing up, quickly shake it over the flame of an alcohol lamp to fix it.

[0186] 3) Add crystal violet dye and stain for 1 minute. Slowly wash off the dye with distilled water and blot dry with absorbent paper.

[0187] 4) Add iodine solution as mordant for 1 minute, slowly wash off the dye with distilled water, and blot dry with absorbent paper.

[0188] 5) Add decolorizing solution, shake the slide, and decolorize for about 40 seconds depending on the thickness of the smear. When the effluent is no longer purple, immediately wash with distilled water to remove moisture.

[0189] 6) Finally, add safranin staining solution for counterstaining for 1 minute, rinse with distilled water, and air dry or blot dry.

[0190] 7) Observe the morphology of the strain under an oil immersion microscope and take photos for record-keeping.

[0191] (3) Catalase test To further clarify the biochemical characteristics of the strain, a catalase reaction was performed on the purified strain. A small amount of fresh culture solution was placed on a clean glass slide using a sterile inoculation loop, and 3% hydrogen peroxide (H2O2) solution was added. The presence of bubbles was immediately observed, and the test results were determined accordingly.

[0192] Strains that did not exhibit bubble formation were classified as catalase-negative, consistent with the typical biochemical characteristics of Streptococcus spp.; strains showing obvious bubble formation were classified as catalase-positive and excluded. The catalase test can effectively distinguish Streptococcus from other catalase-positive Gram-positive cocci, thereby improving the accuracy of preliminary identification.

[0193] After the above morphological observation and preliminary identification, strains that meet the basic characteristics of streptococci were screened out for further identification and analysis using subsequent molecular biological methods.

[0194] Fourteen morphologically stable purified bacterial strains were successfully obtained from oral saliva samples provided by 20 volunteers through selective culture and multiple streaking purification on BHI agar plates. These strains were inoculated onto BHI agar plates and incubated at 37°C for 24–48 hours, after which their colony morphology was observed.

[0195] 4. Experimental Results Most strains form small colonies on BHI agar plates, with colony color mainly grayish-white or milky-white. Figure 1 (A) has a smooth, moist surface, regular and neat edges, and some colonies are translucent. No obvious pigmentation was observed, and the overall characteristics are consistent with the typical colony morphology of *Streptococcus* spp. Gram staining showed ( Figure 1 (B in the sample) All strains showed a purple staining reaction, indicating they are Gram-positive bacteria. The bacteria were spherical in shape, relatively uniform in size, and mostly arranged in chains, with some cells appearing in pairs. None of the strains produced a significant bubble reaction after the addition of 3% hydrogen peroxide, and all were catalase-negative, consistent with the typical biochemical characteristics of Streptococcus.

[0196] In summary, the 14 isolated strains all possessed the basic characteristics of the genus Streptococcus in terms of morphology and biochemical tests, providing a reliable basis for further identification and functional studies.

[0197] Example 3 Functional characteristics of Streptococcus salivarius and its potential oral health benefits (a) Streptococcus salivarius has a stable ability to generate hydrogen peroxide. 1. Analysis of hydrogen peroxide production capacity The ability of *Streptococcus salivarius* to produce hydrogen peroxide (H₂O₂) was determined by colorimetry. *Streptococcus salivarius* (OD₆₀₀ = 0.5 or 1 × 10⁻⁶) was used as the sample. 7 The sample (containing CFU / mL) was inoculated into a suitable culture medium and cultured at 37°C until the logarithmic growth phase. The supernatant was then collected. The H2O2 content in the sample was determined by colorimetric reaction according to the instructions of the hydrogen peroxide detection kit.

[0198] Based on protein concentration, the H2O2 content (μmol / mg prot) = 1 × A Measurement ÷ A standard ÷ Cpr. Using H2O2 production as an evaluation index, the hydrogen peroxide production capacity of different Streptococcus salivarius strains was compared and analyzed, providing a reference for the study of their potential antibacterial mechanisms.

[0199] 2. Experimental Results Table 7 shows the H2O2 production levels of different *Streptococcus salivarius* strains. A certain amount of H2O2 was detected in all strains, but the production capacity varied among strains. Strains S1, S5, S7, and S8 produced relatively high amounts of H2O2, while strains S2 and S4 produced relatively low amounts. Overall, all tested strains exhibited some hydrogen peroxide production capacity. These results suggest that *Streptococcus salivarius* can potentially play an antagonistic role in the oral microecology by producing H2O2, helping to inhibit the growth of oral pathogens and potentially supporting the maintenance of oral flora homeostasis.

[0200] Table 7

[0201] (ii) Results of hemolysis test 1. Experimental Methods The Ss strain in its logarithmic growth phase was inoculated onto the surface of Columbia blood agar medium containing 5% sheep blood using the three-zone streak method and incubated at 37°C for 48 hours. After incubation, the hemolysis of the medium surrounding the colonies was observed. The type of hemolysis was determined based on the appearance of the hemolytic zone around the colonies, categorized as α-hemolysis, β-hemolysis, or γ-hemolysis. This hemolytic characteristic analysis assessed the potential pathogenicity of the strain, providing a reference for subsequent experiments.

[0202] 2. Experimental Results After culturing all tested strains on blood agar for 48 hours, no transparent hemolytic ring or green discoloration ring appeared on the blood agar surrounding the colonies. Figure 2The blood agar samples showed no significant changes in appearance, all exhibiting γ-hemolysis. These results indicate that the *Streptococcus salivarius* strains isolated in this study did not exhibit hemolytic activity and demonstrated good biosafety.

[0203] (iii) Streptococcus salivarius exhibits high sensitivity to a variety of commonly used antibiotics. 1. Experimental Methods The Kirby-Bauer disk diffusion method was used to determine the antibiotic susceptibility of screened *Streptococcus salivarius* strains. Standardized bacterial suspensions were evenly spread on BHI agar plates, and then antimicrobial susceptibility discs containing different antibiotics were attached to the plate surface. The plates were incubated at 37°C for 48 hours. After incubation, the diameter of the inhibition zone around each antibiotic disc was measured to determine the strain's susceptibility or resistance to different antibiotics. These results were used to assess the safety of the strains and their potential application feasibility.

[0204] 2. Experimental Results Table 8 shows the standards for antibiotic susceptibility testing of different antibacterial tablets. Table 9 shows the antibiotic susceptibility of different Streptococcus salivarius strains, indicating that all strains are generally sensitive (S) to commonly used antibiotics such as vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, ampicillin, and clindamycin; resistant (R) to streptomycin; and, with the exception of a few strains, most are resistant (R) to kanamycin. There are some differences in antibiotic susceptibility profiles among different strains, but no multidrug resistance was observed overall. These results indicate that the screened Streptococcus salivarius strains generally have good antibiotic susceptibility and safety, providing experimental evidence for their further development and application as potential oral probiotics.

[0205] Table 8

[0206] Table 9

[0207] (iv) *Streptococcus salivarius* has a good biofilm-forming ability and can effectively inhibit the formation of biofilms of *Streptococcus mutans*. 1. Experimental Methods The biofilm-forming ability of *Streptococcus salivarius* was determined using a 96-well plate crystal violet staining method. The biofilm-forming ability of *S. salivarius* bacterial suspension (OD200) was measured. 600 =0.5 or 1×10 7Add 200 μL of CFU / mL solution to each well of a sterile 96-well plate, with at least three replicates per sample. Use an equal volume of fresh culture medium as a negative control. Seal and incubate at 37°C for 24 hours. Discard the supernatant, gently wash twice with PBS, and air dry at room temperature. Then, fix each well with 200 μL of 99% methanol for 15 minutes, discard the methanol, and air dry at room temperature. Stain each well with 0.1% crystal violet solution for 45 minutes, discard the dye, wash twice with sterile water, and air dry at room temperature. Finally, elute with 200 μL of 95% ethanol for 45 minutes and measure the absorbance at 570 nm.

[0208] Simultaneously, the inhibitory effect of *Streptococcus salivarius* on biofilm formation by *Streptococcus mutans* was evaluated using a co-culture method. Two strains (OD...) were... 600 =0.5 or 1×10 7 CFU / mL) were co-inoculated at a certain ratio (1:1) and incubated statically at 37℃ under anaerobic conditions for 24 hours. Biofilm formation was then measured using the same method. The inhibitory effect of *Streptococcus salivarius* on biofilm formation by *Streptococcus mutans* was evaluated by comparing the biofilm formation levels under single-culture and co-culture conditions.

[0209] 2. Experimental Results All strains of *Streptococcus salivarius* possessed a certain biofilm formation ability, and their OD570 values ​​were significantly higher than those of the negative control group (P<0.05). Figure 3 (A) There are differences in biofilm formation ability among different strains, with some strains (such as S1, S3~S5, S7, S8) showing stronger biofilm formation ability.

[0210] The effect of *Streptococcus salivarius* on the biofilm formation ability of *Streptococcus mutans* was evaluated through co-culture experiments. The results showed ( Figure 3 Compared with the Sm culture group alone, the biofilm formation ability of *Streptococcus mutans* was significantly reduced under co-culture conditions with *Streptococcus salivarius*, with a significantly lower OD570 value (P<0.05). The inhibitory effect varied among different *Streptococcus salivarius* strains, with some strains (such as S3, S4, S7, and S8) showing more significant inhibitory effects. Ss effectively inhibited the formation of *Streptococcus mutans* biofilm, suggesting its potential application value in preventing dental caries and regulating oral pathogenic bacteria biofilms.

[0211] Example 4: Industrialization Advantages of Streptococcus salivarius S8 To further screen strains S1, S3, and S8, a small-scale expansion culture was conducted to evaluate the industrialization potential of each strain.

[0212] To clarify the industrialization capability of the strain, orthogonal experiments were conducted in the fermentation plant to determine the optimal culture medium and conditions, test the protective effects of different protectants, and optimize the freeze-drying process to obtain the maximum number of viable bacteria.

[0213] (a) Determination of culture medium After screening carbon and nitrogen sources and based on the previous experience of the fermentation plant, the final culture medium formula for the 15 L fermenter was determined to be: 7 g lactose, 3.5 g sucrose, 3.5 g glucose, 14 g tryptone, 7 g soybean peptone, 7 g yeast extract, 0.02 mol / L dipotassium hydrogen phosphate / potassium dihydrogen phosphate, 200 mg / L magnesium sulfate, 100 mg / L manganese sulfate, 60 mg / L calcium chloride, and 1 L distilled water.

[0214] (II) Test results of fermentation tank After determining the culture medium, fermentation tests were conducted in a 15 L fermenter. The temperature was controlled at 37 ℃, alkali was controlled with 20% sodium hydroxide solution, the pH was controlled at 5.4, and air pressure was maintained. During the fermentation process, samples were taken to observe changes in OD and pH, and the microscopic morphology of the bacterial cells was observed. After approximately 8-10 hours of cultivation, the OD reached around 10-12 and the growth slowed down. Fermentation was immediately stopped by cooling, and the bacterial sludge was collected by centrifugation and emulsified. The sludge was then obtained using a disc centrifuge.

[0215] (iii) Protectants and freeze-drying After optimization through experiments on the combination of protectant formulations, the protectant scheme is as follows: 20% trehalose, 2% sucrose, 3% maltodextrin, 1.5% sorbitol, 1.5% sodium vitamin C, and 72% water. The freeze-drying process was then carried out after optimizing the freeze-drying curve.

[0216] (iv) Results of the small-scale test After fermentation in a 15 L fermenter, centrifugation, and freeze-drying, the viable cell count was tested, and the viable cell count of strain S1 was 5.6 × 10⁻⁶. 10 CFU / g, the viable count of strain S3 is approximately 6.8 × 10⁻⁶. 10 CFU / g, 3.5×10⁻⁶ for S8 strain 11 CFU / g. The viable count of S8 is significantly higher than that of S1 and S3, making it more suitable for large-scale production and giving it a clear advantage in industrialization.

[0217] Based on the results of previous bacterial functional screening experiments and small-scale culture, Streptococcus salivarius S8 was finally selected as the strain for subsequent research and named Ss-08 (hereinafter referred to as Ss).

[0218] Example 5: The role of *Streptococcus salivarius* in the development and progression of OSF (I) Experimental Methods 1. Establishment of a mouse model of oral submucosal fibrosis induced by arecoline (1) Grouping and overall design Six-week-old male BALB / c rats were randomly divided into three groups of 10 each, and numbered using ear tagging. The grouping scheme is as follows: 1) CON group: Normal diet and water intake, no additional treatment given; 2) OSF group: 500 mg / L arecoline solution was given to the oral cavity to induce a submucosal fibrosis model. The arecoline solution should be prepared and used immediately to ensure its stability. 3) OSF+Ss group: In addition to free access to 500 mg / L arecoline solution in drinking water, a final concentration of 1×10⁻⁶ was added to the drinking water. 7 Ss live bacteria solution at CFU / mL.

[0219] The bacterial suspension of *Streptococcus salivarius* cultured to the logarithmic growth phase was centrifuged, washed, and resuspended. Under aseptic conditions, it was added to the arecoline drinking water in the OSF+Ss group, with a final concentration of 1×10⁻⁶. 7 CFU / mL. Change daily and prepare fresh for use to ensure strain activity and stability in mouse intake.

[0220] (2) General observation and measurement From the start of modeling, the general condition of the mice was observed daily, including their mental state, activity level, and food and water intake. Changes in the mice's weight and water intake were measured and recorded weekly, along with observations of local oral manifestations and mouth opening limitations. The experiment lasted for 8 weeks.

[0221] (3) Collection of materials Eight weeks later, feces were collected, and mice were euthanized under anesthesia. Tongue tissue and bilateral cheek tissue were harvested and divided into two parts: one part was fixed in 4% paraformaldehyde, and the other part was flash-frozen in liquid nitrogen and then transferred to... Store at 80℃ for subsequent experimental testing.

[0222] 2. Tissue paraffin embedding and sectioning (1) Paraffin embedding 1) Fixation: The tissue was fixed at room temperature with 4% paraformaldehyde.

[0223] 2) Place the fixed tissue into a disposable embedding box and mark it with a pencil.

[0224] 3) Dehydration: Soak in 75% ethanol for 2 hours, 85% ethanol for 2 hours, 95% ethanol for 1 hour, and anhydrous ethanol for 30 minutes, repeating twice.

[0225] 4) Transparency: Immerse the tissue in xylene solution for 10 minutes, repeat twice.

[0226] 5) Wax impregnation: After the tissue has become transparent, immerse it in pre-melted paraffin wax for 1-2 hours. Change the wax container and repeat once to ensure that the paraffin wax fully penetrates the tissue.

[0227] 6) Add an appropriate amount of dissolved paraffin wax to the embedding mold beforehand, use tweezers to pick up the tissue and place it in the mold with the cut side facing up, and place it on a freezing table to cool.

[0228] 7) After the paraffin has solidified, cover the embedding box with the lid and add more paraffin. Place it on the freezing table again until it is completely solidified.

[0229] 8) Store the embedded tissue in a -20℃ refrigerator.

[0230] (2) Paraffin sections 1) Before sectioning, the paraffin-embedded block is placed on a cold table for pre-cooling, and then the wax block is fixed on the paraffin microtome for rough trimming.

[0231] 2) After exposing the tissue, adjust the section thickness to 4~6μm, rotate the microtome at a constant speed, and cut continuous and complete paraffin strips.

[0232] 3) Gently spread the wax strip in warm water at 45℃ to allow the tissue to flatten naturally.

[0233] 4) Use a glass slide to pick up a flat slice, mark the glass slide with a pencil, and place it on a slide rack to dry for later use.

[0234] 3. HE staining and Masson staining (1) HE staining 1) Place the glass slide in a 60℃ oven and bake for 1 hour.

[0235] 2) Dewaxing and hydration: Placing paraffin sections in xylene for 10 minutes, repeating twice. Then, immersing the sections in anhydrous ethanol for 5 minutes (repeated twice), 95% ethanol for 2 minutes, 85% ethanol for 2 minutes, 75% ethanol for 2 minutes, and distilled water for 2 minutes.

[0236] 3) Stain with hematoxylin solution for 3-8 minutes, then rinse with distilled water for 30 seconds.

[0237] 4) Place the slices in 1% hydrochloric acid alcohol for 3-10 seconds to differentiate, then rinse with distilled water for 30 seconds.

[0238] 5) Blue with running water for 3-5 minutes, then rinse with distilled water for 30 seconds.

[0239] 6) Counterstain with eosin solution for 30 seconds to 2 minutes, then rinse with distilled water for 30 seconds.

[0240] 7) Dehydration: Place the slices in 75% ethanol for 10-20 seconds, 85% ethanol for 10-20 seconds, 95% ethanol for 20-30 seconds, and anhydrous ethanol for 1 minute in sequence, repeating twice.

[0241] 8) Transparency: Place the slice in xylene for 5 minutes, repeat twice.

[0242] 9) Observe the mounting with neutral resin.

[0243] (2) Masson staining 1) Place the glass slide in a 60℃ oven and bake for 1 hour.

[0244] 2) Dewaxing and hydration: Placing paraffin sections in xylene for 10 minutes, repeating twice. Then, immersing the sections in anhydrous ethanol for 5 minutes (repeated twice), 95% ethanol for 2 minutes, 85% ethanol for 2 minutes, 75% ethanol for 2 minutes, and distilled water for 2 minutes.

[0245] 3) Stain with Weigert iron hematoxylin for 5-10 minutes, then rinse with distilled water for 30 seconds.

[0246] 4) Add acidic differentiation solution for 5-15 seconds, then wash with distilled water for 30 seconds.

[0247] 5) Add Masson's blue solution for 3-5 minutes to restore blue color, then wash with distilled water for 30 seconds.

[0248] 6) Add poinsettia and fuchsin staining solution and stain for 5-10 minutes, then rinse with distilled water for 30 seconds.

[0249] 7) Add phosphomolybdic acid solution for 1-2 minutes, then rinse with distilled water for 30 seconds.

[0250] 8) Add aniline blue staining solution and stain for 1-2 minutes, then rinse with distilled water for 30 seconds.

[0251] 9) Dehydration: Place the slices in 75% ethanol for 10-20 seconds, 85% ethanol for 10-20 seconds, 95% ethanol for 20-30 seconds, and anhydrous ethanol for 1 minute in sequence, repeating twice.

[0252] 10) Transparent: Place the slice in xylene for 5 minutes, repeat twice.

[0253] 11) Observe the mounting with neutral resin.

[0254] (3) The criteria for judging Masson staining results are shown in Table 10: Table 10

[0255] (II) General condition and gross observation of the oral cavity of mice In the early stages of the experiment, mice in the OSF and OSF+Ss groups experienced a significant decrease in water intake due to the addition of drugs to their water. After gradually adapting, their water intake slowly recovered. Compared to the CON group, mice in the OSF and OSF+Ss groups experienced a decrease in body weight, which gradually recovered after week 3, while water intake slowly increased, stabilizing after week 5. The average body weight of both groups was significantly lower than that of the CON group. The trends in body weight and water intake in the OSF+Ss group were basically the same as those in the OSF group, but the changes were less pronounced. At the end of the experiment, the indicators were significantly higher in the OSF group than in the OSF group. Figure 4 (A and B in the text). Regarding general condition, CON group mice exhibited glossy fur, active behavior, and normal appetite; OSF group mice showed brittle fur, reduced activity, and decreased appetite in the later stages of the experiment; the OSF+Ss group also showed similar symptoms, but to a lesser degree. Gross oral examination revealed that the buccal mucosa of OSF group mice was paler, with no obvious fibrous strands, and the degree of passive mouth opening restriction gradually worsened with the extension of the experimental period; the pallor of the oral mucosa and the degree of mouth opening restriction in the OSF+Ss group were less severe than in the OSF group. In conclusion, Ss intervention can effectively slow down the occurrence and development of arecoline-induced OSF to a certain extent.

[0256] (III) Effects of Streptococcus salivarius treatment on oral mucosal tissue of OSF mice HE staining results showed that ( Figure 5 A- Figure 5 In the C and CON groups, the oral mucosal tissue structure was intact, with clear layers, uniform thickness, and a neatly arranged basal layer. The subepithelial connective tissue structure was loose, with fine and regularly arranged collagen fibers, and no obvious fibrosis was observed. Compared with the CON group, the OSF group showed obvious pathological changes, including abnormal epithelial structure, atrophy and thinning in some areas, changes in blood vessel diameter, thickening of the lamina propria, and significant fibrosis of the submucosal connective tissue. The collagen fibers were significantly thickened and arranged in dense bundles, replacing the normal loose connective tissue structure, which is consistent with the typical pathological features of oral submucosal fibrosis. After Ss intervention, the mucosal tissue structure of the OSF+Ss group was significantly improved compared with the OSF group, with enhanced epithelial integrity, more regular cell arrangement, and overall tissue morphology restored to that of the CON group.

[0257] The results of Masson trichrome staining further showed that ( Figure 5 D- Figure 5 In the OSF group (F), compared to the Con group, the epithelial area was atrophied and thinned, collagen fibers were significantly proliferated and densely arranged, and the blue-stained area was significantly increased, indicating that the degree of oral submucosal fibrosis was aggravated. In the OSF+Ss group, this situation was improved, the epithelial atrophy was reduced, and the degree of collagen deposition was alleviated, indicating that the intervention of saliva streptococci can effectively improve the changes in oral mucosal fibrosis in the OSF state.

[0258] Example 6 Transcriptome Sequencing Analysis Transcriptome sequencing and bioinformatics analysis were performed on mouse tongue tissue before and after arecoline induction and Ss intervention to systematically explore the changes in differential gene expression and related signaling pathways.

[0259] (a) Transcriptome sequencing 1. Sample preparation and sequencing On the day of mouse sacrifice, tongue tissue was collected, rapidly frozen in liquid nitrogen, and preserved. Store at 80℃. Tissue samples from at least three mice in each group were randomly selected for transcriptome sequencing analysis.

[0260] The prepared samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for further processing. The company uses the Trizol method for efficient extraction of total RNA from the samples. After extraction, the samples were analyzed using an Agilent 2100 Bioanalyzer.

[0261] 2. Bioinformatics Analysis This invention employs RNA sequencing (RNA-seq) technology to analyze the transcriptome expression profiles of mouse tissue samples from the CON control group, OSF model group, and OSF+Ss intervention group. After standardization, differentially expressed genes (DEGs) with statistical significance among the groups were screened using differential expression analysis, and the expression distribution and trends of DEGs were visualized using a volcano plot. Based on this, pathway enrichment analysis of DEGs was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to uncover key biological pathways and regulatory networks involved in the development of OSF. To further assess the dynamic changes in signaling pathways at the overall gene set level, Gene Set Enrichment Analysis (GSEA) was introduced to enrich the whole-gene expression matrix, revealing potential molecular regulatory patterns during OSF model establishment and Ss intervention. By combining the results of differential expression analysis, KEGG pathway enrichment, and GSEA, the mechanistic changes at the transcriptome level during OSF model formation and Ss intervention were systematically evaluated, providing important evidence for further in-depth mechanistic research.

[0262] (ii) RT-qPCR detection 1. RNA extraction Total RNA was extracted from cells / tissues according to the instructions of the rapid total RNA extraction kit for cells / tissues (NewSemi, Suzhou).

[0263] (1) To extract RNA from cells, take a 6-well plate as an example. Remove the old culture medium, wash twice with PBS, add 350 μL of lysis buffer (RLT Lysis Buffer) to each well, let stand for a while, scrape off the cells with a cell scraper and collect them in an enzyme-free 1.5 ml EP tube; take mouse tissue (15~30 mg) and add 600 μL of RLT Lysis Buffer. After thoroughly homogenizing the tissue, centrifuge at 12000 rpm for 5 minutes and take the supernatant.

[0264] (2) Add the lysis buffer to the DNA removal / RNA adsorption universal column and centrifuge at 12,000 rpm for 1 minute.

[0265] (3) Add an equal volume of 70% ethanol to the lysis solution and gently blow to mix evenly.

[0266] (4) Add the above mixture to a new DNA removal / RNA adsorption universal column, each volume less than 800 μL, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0267] (5) Add 500 μL of RW1 Buffer to the column, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0268] (6) Add 500 μL of RWB Wash Buffer to the column, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and repeat twice.

[0269] (7) Centrifuge at 12000 rpm for 2 minutes to remove as much rinsing solution as possible.

[0270] (8) Take out the universal column, put it into a 1.5 mL enzyme-free EP tube, add 25~50 μL of REB Elution Buffer, let it stand at room temperature for 1 minute, and centrifuge at 12000 rpm for 1 minute to obtain the RNA solution. Store the sample at -80℃ for a long time.

[0271] (9) The concentration and purity of RNA were detected using a NanoDrop 2000 spectrophotometer.

[0272] 2. Reverse transcription reaction (1) Prepare the reaction mixture according to the instructions in the kit.

[0273] (2) The specific reaction system is shown in Table 11: Table 11

[0274] (3) Vortex to mix, then briefly centrifuge. Reaction conditions: 42℃ for 15 minutes, 85℃ for 5 minutes. The reverse transcription product can be used directly for subsequent experiments or stored at -80℃ for a long time.

[0275] 3. Real-Time PCR (1) Prepare the reaction mixture on ice according to the kit instructions.

[0276] (2) The specific reaction system is shown in Table 12: Table 12

[0277] (3) Cover the 96-well PCR plate with a membrane and centrifuge briefly.

[0278] (4) The reaction conditions are shown in Table 13: Table 13

[0279] (5) Results Analysis: The average Ct value was taken for each sample in triplicate. ΔCt = Ct value of each gene - corresponding internal reference value. ΔΔCt = ΔCt value of experimental group - ΔCt of control group. A 2^ ΔΔCt is used to calculate the relative expression levels of each gene.

[0280] (III) Analysis of differential gene expression among different samples RNA-seq volcano plot results showed that, compared with the OSF group, a total of 218 significantly upregulated genes and 469 significantly downregulated genes were screened. Figure 6 In comparison between the OSF+Ss group and the OSF group, a total of 369 significantly upregulated genes and 48 significantly downregulated genes were screened. Figure 6 (B in the text). Compared with the OSF group, Ss intervention mainly upregulated differentially expressed genes, suggesting that Ss intervention can cause significant changes in the OSF-related transcriptional profile.

[0281] (iv) GO enrichment analysis of differentially expressed genes GO enrichment analysis was performed on differentially expressed genes in each comparison group, and the significantly enriched items were displayed in the form of bubble charts from three levels: biological process (BP), cellular component (CC), and molecular function (MF).

[0282] In the comparison between the OSF group and the CON group, differentially expressed genes at the BP level were mainly enriched in extracellular matrix organization, external encapsulating structure organization, and extracellular structure organization, which are related to extracellular matrix remodeling. At the same time, they were significantly enriched in inflammatory response, chemokine-mediated signaling pathway, and other processes related to inflammation and immune cell migration. Figure 7 At the CC level, differentially expressed genes were mainly enriched in structures such as the extracellular matrix and basolateral plasma membrane, suggesting that the structural composition of the extracellular matrix and cell membrane undergoes significant remodeling during OSF. Figure 8 Genes whose expression changes at the MF level are mainly involved in receptor ligand activity, chemokine receptor binding, and cytokine receptor binding, processes related to intercellular signal transduction and inflammation-related signal transduction. Figure 9 ).

[0283] In the comparison between the OSF+Ss group and the OSF group, differentially expressed genes at the BP level were mainly enriched in immune-regulatory processes such as immune response and immune system processes. Figure 10 At the CC level, differentially expressed genes were significantly enriched in extracellular space, extracellular region, apical plasma membrane, and immune synapse, pathways related to epithelial barrier structure and immune-related cell-to-cell interactions. Figure 11 At the MF level, it mainly involves pathways related to innate immune receptor signaling, such as Toll-like receptor binding and immunoglobulin receptor binding. Figure 12 ).

[0284] (V) GSEA-GO BP Analysis Based on Enrichment Map To further analyze the occurrence and development of OSF and the functional changes during Ss intervention from the perspective of overall transcriptional change trends, this invention uses GSEA and Cytoscape's enrichment map plugin to perform visual cluster analysis on GOBP.

[0285] The results showed that, compared with the CON group, the significantly enriched biological processes in the OSF group were mainly concentrated in keratinization, Wnt signaling pathway, cell cycle processes, ribosome biogenesis, chromosome organization, mRNA metabolism, and nucleotide excision repair, suggesting that OSF is accompanied by enhanced abnormal epithelial proliferation and differentiation, active protein synthesis, and remodeling of genome-related regulatory processes; at the same time, T cell proliferation-related processes showed a relatively negative enrichment, suggesting an alteration in the local immune regulatory state. Figure 13 (A) In the comparison between the OSF+Ss group and the OSF group, the biological processes enriched and changed mainly included keratinization, cell cycle processes, ribosome biogenesis, chromosome organization and separation, protein depolymerization, cGMP pathway and activin receptor signaling pathway, etc., and the overall trend was mainly downregulated, suggesting that Ss intervention can, to some extent, reverse the abnormal epithelial differentiation and proliferation activation state related to OSF, and regulate cell growth and signal transduction-related processes (A). Figure 13 (B in the middle).

[0286] (vi) KEGG pathway enrichment analysis of differentially expressed genes To further elucidate the key signaling pathways involved in differentially expressed genes, KEGG enrichment analysis was performed on differentially expressed genes in each comparison group.

[0287] The results showed that, compared with the CON group, differentially expressed genes were significantly enriched in cytokine-receptor interactions, calcium signaling pathways, estrogen signaling pathways, tight junctions, cell adhesion molecules, viral protein-cytokine and receptor interactions, IL-17 signaling pathways, and viral particle-hepatitis virus pathways, suggesting that OSF is accompanied by inflammatory microenvironment imbalance and abnormal changes in epithelial barrier structure and cell connectivity. Figure 14In the comparison between the OSF+Ss group and the OSF group, differentially expressed genes were mainly enriched in the cAMP signaling pathway, cell adhesion molecules, cGMP-PKG signaling pathway, tight junctions, cardiomyocyte adrenergic signaling pathway, leukocyte transendothelial migration, insulin secretion, cholinergic synapses, thyroid hormone synthesis, and viral particle-hepatitis virus pathways, suggesting that Ss intervention improves OSF-related pathological processes by regulating intercellular junction homeostasis, barrier function, and multiple signal transduction networks. Figure 15 ).

[0288] Further comparison of the KEGG enrichment results between the two groups revealed that cell adhesion molecules, tight junctions, and viral particles (hepatitis viruses) were common enrichment pathways in both groups. Among these, tight junctions, as key structures maintaining epithelial cell polarity and barrier integrity, can lead to impaired intercellular connections and altered mucosal permeability due to abnormalities, further affecting local microenvironment homeostasis. This process is closely related to impaired epithelial barrier function, abnormal intercellular connections, and the formation of a persistent inflammatory microenvironment in OSF, suggesting that tight junctions play a crucial role in the development and progression of OSF and in Ss intervention.

[0289] (vii) Tight junction pathway and related gene expression analysis KEGG pathway-gene association Sankey analysis showed that the tight junction pathway was among the top 10 significantly enriched pathways in both the OSF and CON groups and the OSF+Ss group. Given the important role of the oral mucosal barrier in the development of OSF, changes related to tight junctions should be given special attention. The results showed that tight junction-related genes, such as Cldn2, Cldn3, Cldn5, Cldn7, Cldn10, and Marveld3, were significantly downregulated in the OSF group. Figure 16 This suggests that tight junction-related molecules in OSF are affected, leading to alterations in mucosal barrier function. Compared to the OSF group, the OSF+Ss group showed a certain degree of reverse regulation of these tight junction-related genes. Figure 17 This suggests that intervention with *Streptococcus salivarius* can improve the abnormal expression of tight junction-related genes in the OSF state at the transcriptional level.

[0290] (viii) GSEA analysis showed changes in the Wnt signaling pathway during OSF and Ss interventions. The Wnt signaling pathway plays a crucial regulatory role in tissue development, cell proliferation, and differentiation. The development of OSF (osmotic fibrosis) is characterized by submucosal fibrosis and persistent inflammatory stimulation, and the Wnt signaling pathway plays a vital role in submucosal fibrosis by regulating fibroblast phenotypic transformation and collagen synthesis. Furthermore, Wnt signaling can further affect the stability of the epithelial barrier structure by regulating local cytokine expression, mechanical tension, and extracellular matrix composition. These changes can lead to abnormal expression and localization of tight junction proteins, thereby participating in the disruption or remodeling of intercellular connections, especially the abnormality of tight junction structures, ultimately resulting in impaired mucosal barrier function. The aforementioned GOBP analysis revealed a significant enrichment of Wnt-related biological processes in the OSF group compared to the CON group, suggesting a potentially important role for this pathway in the development of OSF. Therefore, based on the KEGG total enrichment results, this study further used GSEA to analyze the overall trend of Wnt signaling pathway changes to verify its dynamic changes during OSF and intervention.

[0291] In the comparison between the OSF group and the CON group, the Wnt signaling pathway showed significant positive enrichment ( Figure 18 The overall expression levels of multiple core genes in the Wnt pathway were increased in the OSF group. Figure 19 This suggests that the Wnt signaling pathway is abnormally activated under OSF conditions. In the comparison between the OSF+Ss group and the OSF group, the Wnt signaling pathway was significantly inhibited, showing a clear negative enrichment trend after intervention with *Streptococcus salivarius*. Figure 20 The overall expression levels of multiple core genes in the Wnt pathway were significantly reduced. Figure 21 This suggests that intervention with *Streptococcus salivarius* can effectively reverse the abnormal activation of the Wnt signaling pathway in OSF. In conclusion, the Wnt signaling pathway is one of the important molecular mechanisms by which *Streptococcus salivarius* improves the pathological state of OSF.

[0292] Example 7: RT-qPCR Validation of mRNA Expression of Genes Related to the Wnt Signaling Pathway To verify the changing trends of Wnt signaling pathway-related genes in the RNA-seq differential expression analysis results, RNA was extracted from tongue tissues of each group, and RT-qPCR analysis was performed on genes in the Wnt signaling pathway, namely Wnt3a, Ctnnb1, and Ccnd1.

[0293] The results show that ( Figure 22Compared with the CON group, the mRNA expression levels of the aforementioned genes in the OSF group were significantly increased (P<0.05). Further comparison between the OSF+Ss group and the OSF group revealed that the expression levels of the aforementioned related genes were significantly downregulated after Ss intervention (P<0.05), with the overall expression trend converging towards that of the CON group. These results are highly consistent with the significant positive enrichment and inhibition of the Wnt signaling pathway after intervention observed in RNA-seq and GSEA analyses, further confirming that *Streptococcus salivarius* can participate in the improvement of OSF pathological conditions by regulating the expression of key genes in the Wnt signaling pathway.

[0294] Example 8: RT-qPCR validation of mRNA expression of tight junction-related genes To further verify the expression changes of genes related to the tight junction pathway in the transcriptome analysis results, RT-qPCR was used to detect the mRNA expression changes of the Cldn3 tight junction protein gene.

[0295] The results show that ( Figure 23 In the OSF group, the mRNA expression was significantly lower than that in the CON group (P<0.05), while in the OSF+Ss group under Ss intervention, the mRNA expression of the Cldn3 gene was significantly higher (P<0.05).

[0296] Example 9: The effects of *Streptococcus salivarius* on the oral microbiota Based on the fact that *Streptococcus salivarius* can participate in the improvement of OSF pathological state by regulating oral microbiota structure, 16S rRNA gene sequencing analysis was performed on the oral microbiota of mice in the CON group, OSF group and OSF+Ss group to systematically evaluate the changes in oral microbiota under different experimental treatment conditions from the aspects of microbiota diversity, community structure and differential microbiota.

[0297] (I) The impact of Streptococcus salivarius on oral microbiota diversity Dilution curve analysis showed that at the OTU taxonomic level, the dilution curves of both the Shannon and Simpson indices gradually flattened with increasing sequencing depth, indicating that the sequencing data volume and depth of this invention can cover the vast majority of microorganisms in the sample, meeting the needs of subsequent analysis. Figure 24 , Figure 25 Compared with the CON group, there were no significant differences in α-diversity analysis, β-diversity analysis, and dysbiosis index (MDI) in the OSF group. Figures 26-29 , Figure 30 The A in the figure suggests that short-term ARE stimulation did not induce significant oral microbiota dysbiosis. After Ss intervention, α diversity analysis showed ( Figure 26 , Figure 27The Shannon index in the OSF+Ss group showed an increasing trend, while the Simpson index showed a decreasing trend, indicating a certain improvement in the richness and evenness of the bacterial community; PCoA / NMDS analysis showed that ( Figure 28 , Figure 29 The sample distribution in the OSF+Ss group differed significantly from that in the CON and OSF groups, indicating a significant shift in the oral microbiota community structure. As shown in Figure 16H, based on Venn diagram analysis at the OTU level, the number of OTU sets in the CON, OSF, and OSF+Ss groups were 123, 95, and 152, respectively, with a total of 61 OTUs (28.64%) across the three groups. The number of OTUs specific to the CON, OSF, and OSF+Ss groups were 41, 16, and 60, respectively, with the OSF group having the fewest OTUs, while the OSF+Ss group showed a significant increase in the number of OTUs specific to it. These results suggest that *Streptococcus salivarius* can actively reshape the composition of the oral microbiota community.

[0298] (II) The influence of Streptococcus salivarius on the composition of oral microbiota at various taxonomic levels To further investigate the effects of Ss intervention on the oral community composition of OSF mice, the oral microbiota composition of each group of mice was analyzed at five levels: phylum, class, order, family, and genus. Figures 31-35 Genus-level analysis showed that the dominant bacterial genera composition of the OSF group and the CON group was similar, with Diplococcus being the most prevalent. Gemella ) and Streptococcus spp. Streptococcus The main focus was on Ss intervention. After Ss intervention, the OSF+Ss group... Gemella Abundance decreased significantly, while Streptococcus and potentially beneficial bacteria Enterococcus spp. Enterococcus ), Neosphingomonas spp. Novosphingobium The abundance of Streptococcus salivarius was significantly increased, suggesting that Streptococcus salivarius can play a regulatory role in the oral microecology by optimizing the oral microbiota structure and enriching beneficial symbiotic bacteria. At other levels, Firmicutes (S. salivarius) were present in the OSF group. Bacillota Proteobacteria ( Pseudomonadota Staphylococci ( Staphylococcales ), Diplococcidae ( Gemellaceae Pasteuraceae ( Pasteurellaceae The relative abundance of ) increased, while unclassified_k__norank_d__Bacteria Phylum, Streptococcus ( Streptococcaceae The abundance of species such as *Rodentibacter* was significantly reduced. In the OSF+Ss group, the bacterial community structure underwent further adjustments, among which… unclassified_k__norank_d__Bacteria Phylum, Streptococcus ( Streptococcaceae The relative abundance of species such as Staphylococcus (Staphylococci) increased significantly, while the abundance of species such as Staphylococcus (Staphylococci) increased significantly. StaphylococcalesThe relative abundance of [groups such as] [[groups such as] ] decreased, and the overall trend was a recovery towards the CON group; in addition, Pasteurellaceae γ-Proteobacteria ( Gammaproteobacteria The abundance of Lactobacillus ( ) further increased; at the same time, the abundance of Lactobacillus ( ) further increased; Lactobacillales Streptococcal family ( Streptococcaceae The increased abundance of bacteria related to oral mucosal immune regulation, anti-inflammatory response, maintenance of microecological homeostasis and barrier function indicates that Ss improves OSF by reshaping the oral microbiota.

[0299] (III) Analysis of oral microbiota differences based on Genus level Multiple differential tests were used to analyze the relative abundance of individual genera in each group at the Genus level. The results are as follows: Figures 36-43 As shown. Streptococcus spp. as the intervening bacteria ( Streptococcus No significant differences were observed among the three groups, but a decreasing trend was observed in the OSF group, while a rebound was observed in the OSF+Ss group, suggesting that exogenous supplementation helps restore its ecological niche. Further analysis revealed that, compared with the CON group, the OSF group had a higher proportion of *Lactobacillus* spp. (…). Limosilactobacillus The relative abundance of *Mammalian cocci* was significantly reduced. Mammaliicoccus ) significantly increased; after Ss intervention, Limosilactobacillus The abundance was significantly higher than that of the OSF group, while Mammaliicoccus The significant decrease suggests that Ss intervention has a certain "reversal effect" on OSF-related bacterial imbalance. Furthermore, Ss intervention also had a significant impact on other bacterial genera. Compared with the CON group, the *Neosphomonas* genus in the OSF group (… Novosphingobium ) and genus Waterbacterium ( Aquabacterium The proportion of ) has increased significantly, while the proportion of genus Chlorella ( Chryseobacterium ), Masseilles ( Massilia ) and Enterococcus spp. Enterococcus The overall level was low or close to undetectable. After Ss intervention, Novosphingobium , Aquabacterium The increase was further amplified on top of the existing upward trend, and other genera also showed significant enrichment. Among them, Massilia and Enterococcus Significant abundance was detected only in the OSF+Ss group, suggesting that it may be a newly enriched bacterial genus related to the intervention.

[0300] Example 10: The effects of *Streptococcus salivarius* on the gut microbiota Based on the fact that *Streptococcus salivarius* can participate in the improvement of oral pathological conditions by regulating the structure of the gut microbiota, 16S rRNA gene sequencing analysis was performed on fecal samples from mice in the CON group, OSF group, and OSF+Ss group to systematically evaluate the changes in gut microbiota under different experimental treatments in terms of microbial diversity, community structure, and differential microbiota.

[0301] (I) The impact of Streptococcus salivarius on gut microbiota diversity Dilution curve analysis showed that at the OTU taxonomic level, the dilution curves of both the Shannon and Simpson indices gradually flattened with increasing sequencing depth, indicating that the sequencing depth in this study could adequately cover the main gut microbiota information in the samples, and the amount of data met the needs of subsequent diversity and community structure analysis. Figure 44 , Figure 45 Evaluation of the Shannon and Simpson indices in the alpha diversity index analysis revealed no statistically significant differences among the three groups. Figure 46 , Figure 47 This indicates that the interventions of OSF and *Streptococcus salivarius* had little impact on the overall richness and α-diversity of the gut microbiota. In the β-diversity analysis, the results of PCoA and NMDS analyses showed (…). Figure 48 , Figure 49 The three groups (CON, OSF, and OSF+Ss) were clearly separated in two-dimensional space, and the samples within each group aggregated well, indicating significant differences in the composition of the gut microbiota among the three groups. The results of the dysbiosis index (MDI) analysis showed... Figure 50 In Figure A), the MDI value of the OSF group was significantly higher than that of the CON group, indicating that ARE induced significant gut microbiota dysbiosis in the OSF group. The MDI value of the OSF+Ss group was significantly lower than that of the OSF group, suggesting that Ss intervention could partially alleviate the dysbiosis in OSF, but had not fully restored it to the control level. Venn plot analysis based on the OTU level of the samples showed (…). Figure 50 In the B group, the total number of OTUs in the CON, OSF, and OSF+Ss groups was 587 (47.41%), indicating the existence of a stable core microbial community composition among the samples. The number of OTUs specific to the CON, OSF, and OSF+Ss groups were 135, 86, and 144, respectively. The OSF+Ss group had the most specific OTUs, while the OSF group had the fewest OTUs, similar to the CON group. This suggests that Ss intervention may improve OSF by increasing microbial community richness and reshaping the microbial community structure.

[0302] (II) Effects of *Streptococcus salivarius* on the composition of intestinal flora at various taxonomic levels To investigate the effects of Ss intervention on the gut microbiota composition of OSF mice, the relative abundance of gut microbiota in each group of mice was assessed at five levels: phylum, class, order, family, and genus. Figures 51-55 At the phylum level, the gut microbiota of each group is mainly composed of Firmicutes (…). Bacillota ) and Bacteroidetes ( Bacteroidota The OSF group consisted of [a specific group of bacteria]. Compared to the CON group, the relative abundance ratio of the two dominant bacterial phyla shifted significantly in the OSF group, with [the specific phylum of bacteria] being [specifically, the percentage of bacteria that were present in the OSF group]. BacillotaThe relative abundance increased. Bacteroidota The relative abundance decreased, and the Firmicutes / Bacteroidetes (F / B) ratio increased significantly. After intervention with *Streptococcus salivarius*, the F / B ratio decreased, and the overall structure regressed to the CON group. At other levels, the Bacteroidetes class (…) was present in the OSF group. Bacteroidia Clostridium class ( Clostridia ), Lachnospirales ( Lachnospirales ), Lactobacillus genus ( Ligilactobacillus ), Prevotellaceae_UCG-001 When the abundance of the bacterial community decreases, Bacillus ( Bacilli Lactobacilli ( ) Lactobacillales ), family Ratbramycetes ( Muribaculaceae ), family Trichophyceae ( Lachnospiraceae ), Lactobacillus family ( Lactobacillaceae The proportions of various bacterial groups increased. After Ss intervention, the bacterial community structure changed, and the abundance of the aforementioned dominant bacteria regressed to that of the CON group. Among them, multiple bacterial groups, such as Bacteroides , Lachnospirales , Lactobacillales These bacteria are closely related to energy metabolism, short-chain fatty acid production, inflammation, and mucosal homeostasis. After Ss intervention, the relative abundance of these bacteria tended to approach that of the CON group.

[0303] (III) Differential analysis of gut microbiota based on LEfSe Building upon this, Lefse multilevel species differential discriminant analysis (LDA) was further employed to screen for characteristic bacterial groups with significantly different abundances among different groups at each level (from phylum to genus), with an LDA threshold of 3. The results showed that different groups exhibited different dominant bacterial groups (…). Figure 56 , Figure 57 In group CON, RIKEN's mycaceae family ( Rikenellaceae ), genus Wenken ( Rikenella ), genus Rocheria ( Roseburia Osmotherium spp. doribacter ) and Lactobacillus spp. Liglactobacillus The gut microbiota showed a significant enrichment of bacteria such as Lactobacillus and Lactobacillus, many of which are associated with maintaining intestinal homeostasis and metabolic balance. In contrast, OSF exhibited a clear microbiota imbalance, with an excessive enrichment of lactic acid bacteria, and its core microbiota mainly included Lactobacillus (Lactobacillus family). Lactobacillaceae ), Lactobacillus genus Corynebacterium ( Coriobacteriia ), Egeria ( Eggerthellaceae ) and Gordon's order ( Gordonibacteriales These bacteria, such as rumen bacteria, are mostly associated with inflammation, mucosal damage, and metabolic disorders. After Ss intervention, the differentially expressed bacterial composition in the OSF+Ss group changed significantly, with rumen bacteria (Rumenobacteria) becoming more prominent. Ruminococcaceae ), Ruminococcus , Oscillatorales ( Oscillospirales ), genus *Faecalibacterium* Faecalibaculum ), Dubosiella and Bifidobacterium spp. Bifidobacterium The gut microbiota was significantly enriched, and these microbiota typically play a beneficial role in maintaining gut homeostasis and health, including SCFA production (butyrate), intestinal barrier repair, and inflammatory immune regulation. The above results indicate that intervention with *Streptococcus salivarius* restores the gut microbiota to a normal level by reshaping the composition of key gut microbiota, and this trend is consistent with the improvement trend of OSF pathological state.

[0304] Example 11: Acquisition of experimental materials for the role of Ss-08 in the progression of oral squamous cell carcinoma. 1. Experimental strains The *Streptococcus salivarius* used in this invention ( Streptococcus salivarius The *Streptococcus salivarius* strain numbered 08 (Ss-08) was used as the experimental strain in this study for subsequent related experimental research.

[0305] 2. Cell lines The human oral squamous cell carcinoma cell line CAL-27 used in this invention was preserved in the Department of Microbiology, Harbin Medical University. CAL-27 cells were isolated from tumor tissue of a patient with squamous cell carcinoma of the tongue. They are typical poorly differentiated oral squamous cell carcinoma cells with strong proliferative activity and invasive ability, and are widely used in research on the mechanisms of oral cancer and in evaluating the efficacy of antitumor drugs.

[0306] 3. Animal source This invention used 4–5 week old BALB / c-nu female nude mice for experiments. The experimental animals were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Production License No.: SCXK(Liaoning)2015-0001). All animal experiments were conducted after obtaining approval from the Ethics Committee of Harbin Medical University (Ethics No.: HMUIRB2025026). The nude mice used in the experiments were housed in a specific pathogen-free (SPF) level barrier system and were acclimatized in the animal room for at least one week before the formal start of the experiments to reduce the impact of environmental changes.

[0307] 4. Primers The required primers are shown in Table 14: Table 14

[0308] 5. Main reagents The required cell culture reagents are shown in Table 15: Table 15

[0309] The required reagents for SsEVs tracer are shown in Table 16: Table 16

[0310] The cell proliferation and viability assay kit was a CCK-8 assay kit, purchased from APExBIO Inc., USA.

[0311] The required reagents related to cell migration and invasion are shown in Table 17: Table 17

[0312] The reagents used for RNA extraction and RT-qPCR detection are shown in Table 18: Table 18

[0313] Reagents used for protein extraction and Western blot are shown in Table 19: Table 19

[0314] The required immunohistochemistry reagents are shown in Table 20: Table 20

[0315] 6. Required antibodies The required Western blot antibodies are shown in Table 21: Table 21

[0316] The required immunohistochemistry-related antibodies are shown in Table 22: Table 22

[0317] Example 12 Morphological characteristics and particle size distribution analysis of SsEVs 1. Experimental Methods (1) Resuscitation, activation and culture of Streptococcus salivarius: Formulate suitable for Streptococcus salivarius ( Streptococcus salivarius The BHI (Brain Heart Infusion) medium used for growing Ss was autoclaved at 121°C for 15 min before use.

[0318] The frozen suspension of Streptococcus salivarius was extracted from... Remove from the freezer at 80℃ and allow to thaw naturally at room temperature. Take 1.5 × 10 4CFU / mL bacterial culture was inoculated into 5 mL of sterile BHI liquid medium and incubated statically at 37°C for 16-18 h to allow the cells to grow to the logarithmic growth phase. Subsequently, 1% (v / v) of the logarithmic growth phase bacterial culture was inoculated into fresh BHI medium and cultured for two consecutive passages. The third generation activated bacterial culture was then selected as the strain for subsequent experiments.

[0319] (2) Extraction of Salicytic Streptococcus extracellular vesicles (SsEVs): Take 1.5 × 10 4 CFU / mL of Streptococcus salivarius culture was inoculated into eight sterile Erlenmeyer flasks containing 45 mL of BHI medium and incubated at 37°C for 16–18 h.

[0320] After cultivation, the bacterial suspension was centrifuged at 5000 rpm for 20 min at 4°C, the supernatant was discarded, and the precipitate was collected and resuspended. Subsequently, the resuspended suspension was transferred to eight culture flasks containing 400 mL of fresh BHI medium and incubated at 37°C for 16–18 h to obtain sufficient bacterial product.

[0321] The obtained bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged in batches at 4℃ and 5000 rpm for 20 min. The precipitate was discarded and the supernatant was retained.

[0322] The supernatant was centrifuged again under the same conditions (5000 rpm, 4 ℃, 20 min) to further remove bacterial residue and larger particulate impurities.

[0323] After centrifugation, the supernatant was aspirated using an electric pipette and filtered through a 0.45 μm PVDF microporous membrane. A portion of the filtrate was spread onto BHI agar plates and incubated at 37°C to detect bacterial growth. After confirming the filtrate was sterile, the remainder was collected for subsequent extracellular vesicle extraction.

[0324] Aliquot the supernatant after bacterial cell removal into ultracentrifuge tubes (each tube not exceeding 38 mL) and centrifuge at 120,000 × g and 4 °C for 60 min. Discard the supernatant and collect the precipitate obtained after centrifugation.

[0325] The precipitates obtained from multiple ultracentrifugations were collected in the same tube and subjected to ultracentrifugation again under the same conditions for further purification.

[0326] After centrifugation, discard the supernatant, add 2 mL of PBS to the tube to gently resuspend and rinse the precipitate, then add to a final volume of 38 mL and centrifuge at 120,000×g and 4℃ for 60 min.

[0327] Finally, the supernatant was discarded, and the precipitate was retained, which is the extracellular vesicle of *Streptococcus salivarius* (SsEVs). The obtained SsEVs were resuspended in 200 μL PBS, aliquoted, and placed in... Store in an 80°C refrigerator for later use.

[0328] (3) Culture and passage of CAL-27 cells: 1) Cell Culture CAL-27 cells were cultured routinely in a 37°C, 5% CO2 saturated humidity incubator. The culture medium was high-glucose DMEM (Dulbecco's Modified Eagle Medium), supplemented with 10% fetal bovine serum (FBS) to provide necessary nutrients. To prevent bacterial contamination, 100 U / mL penicillin and 0.1 mg / mL streptomycin were added to the culture system.

[0329] 2) Cell resuscitation Will CAL-27 cryopreserved cells stored at 80°C were rapidly removed and placed in a 37°C water bath for rapid thawing. After complete thawing, the cell suspension was transferred to a 15 mL sterile, enzyme-free centrifuge tube, and 9 mL of fresh complete culture medium was added and mixed thoroughly. The cells were then centrifuged at room temperature to remove the cryopreservation solution. After centrifugation, the supernatant was discarded, and 4 mL of complete culture medium was added to the cell pellet. The cells were gently resuspended by pipetting to form a homogeneous suspension. Finally, the cells were seeded into T25 cell culture flasks and cultured in an incubator at 37°C with 5% CO2.

[0330] 3) Cell passage When cell confluence reaches 80%-90% as observed under a microscope, discard the old culture medium and wash 2-3 times with sterile PBS to remove residual serum. Add 1 mL of 0.25% trypsin solution to the culture flask for digestion. Once the cells become round and begin to detach, gently tap the flask wall to promote cell dispersion. Then add 3 mL of complete culture medium to stop digestion. Collect the digested cell suspension and centrifuge at 800 rpm at room temperature for 5 min. Discard the supernatant and retain the cell pellet. Passage the cells at a ratio of 1:3.

[0331] 4) Cell cryopreservation Discard the culture medium and wash the cells 2–3 times with PBS. After trypsin digestion and centrifugation (800 rpm, room temperature, 5 min), discard the supernatant and resuspend the cell pellet in 1 mL of serum-free cell cryopreservation medium. Aliquot the cell suspension into sterile cryovials and immediately place them in a sterile cryopreservation container. Store in a refrigerator at 80°C.

[0332] (4) Transmission electron microscopy (TEM) detection Pre-purified *Streptococcus salivarius* extracellular vesicles (SsEVs) were taken and resuspended in PBS buffer to a final concentration of 800 μg / mL. An appropriate amount of the SsEV suspension was added to a 300-mesh carbon film copper grid for fixation. After negative staining, the ultrastructure of the sample was observed using a transmission electron microscope (TEM).

[0333] (5) Nanoparticle tracking analysis (NTA) The particle size and concentration of *Streptococcus salivarius* extracellular vesicles (SsEVs) were determined using a nanoparticle tracking analysis (NTA). An appropriate amount of SsEVs sample was diluted with sterile PBS to a suitable concentration range for detection. The sample was injected into the NTA detection system. The instrument captured the Brownian motion trajectory of the suspended particles by laser irradiation. Software performed real-time tracking analysis on each particle, calculating the hydrodynamic particle size distribution and concentration based on the Stokes–Einstein equation. All measurements were performed under isothermal conditions (25°C).

[0334] 2. Experimental Results SsEVs appear as elliptical particles of varying sizes under an electron microscope, with a clearly visible lipid bilayer membrane structure on the surface, exhibiting typical bacterial extracellular vesicle morphology characteristics, and their particle size is approximately 160 nm. Figure 58 In A). The diameter of SsEVs particles is mainly concentrated at around 160 nm, exhibiting a distinct concentration peak ( Figure 58 The results (B) are highly consistent with those observed by transmission electron microscopy.

[0335] The above results collectively indicate that the obtained SsEVs conform to the characteristics of typical bacterial extracellular vesicles in terms of both morphology and particle size distribution.

[0336] Example 13 Interaction between SsEVs and Cal-27 cells 1. Experimental Procedure (1) Marking of SsEVs membrane Cell slides were pre-placed in 24-well cell culture plates, and 1 mL of cell suspension (cell density 5.5 × 10⁶ cells / well) was seeded in each well. 5 (each cell / mL), and then incubated in a 37 ℃, 5% CO2 incubator for 12 h.

[0337] Fluorescent labeling of SsEVs membranes was performed using the ExoGlow™-Membrane EV Labeling Kit (following the kit instructions).

[0338] After labeling, discard the original culture medium in the 24-well plate, add fresh culture medium containing labeled SsEVs, and co-culture with the cells for 24 h (37 ℃, 5% CO2).

[0339] After co-culture, the cell nuclei were stained with Hoechst dye. After washing to remove excess dye, the cells were observed and fluorescent images were acquired under a confocal laser scanning microscope.

[0340] (2) Marking of SsEVs RNA Place the cell-coated slides into 24-well plates and add 1 mL of cell suspension (cell density 5 × 10⁶ cells / well) to each well. 5 (each cell / mL) was placed in a 37 ℃, 5% CO2 incubator and incubated overnight.

[0341] The RNA contained in SsEVs was fluorescently labeled using the ExoGlow™-RNA EV Labeling Kit (following the instructions). The reaction buffer was mixed with 100 μg of SsEVs, the RNA-labeling dye was added, and the mixture was incubated at 37 °C in the dark for 60 min.

[0342] After labeling, replace the original culture medium in the 24-well plate with the culture medium containing the labeled SsEVs, and co-culture with the cells for 24 h (37 ℃, 5% CO2).

[0343] After co-culture, the cell nuclei were stained with Hoechst dye. After washing to remove unbound dye, the cells were observed under a confocal microscope, and fluorescence images were acquired to analyze the distribution of SsEVs RNA in the cells.

[0344] 2. Experimental Results Confocal microscopy revealed numerous discrete fluorescent spots around the nuclei of Hoechst-labeled cells, primarily located in the cytoplasm and perinuclear region. This indicates that membrane-labeled SsEVs can effectively attach and be endocytosed by recipient cells, exhibiting typical cytoplasmic and perinuclear aggregation characteristics.

[0345] RNA-labeled fluorescent signals are also mainly distributed in the cytoplasm and show obvious perinuclear enrichment in some cells, indicating that recipient cells can take up SsEVs and internalize their RNA load.

[0346] This indicates that SsEVs can not only be effectively taken up by recipient cells, but also successfully deliver the RNA components they carry into the cell, laying the foundation for subsequent SsEVs-mediated intercellular communication. Figure 59 ).

[0347] Example 14 Effects of SsEVs on Oral Cancer Cell Phenotype (I) Effects of SsEVs on the Proliferative Capacity of Oral Cancer Cells 1. Experimental Procedure (1) Cell viability assay (CCK-8) CAL-27 cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well, at a cell density of 3 × 10⁶ cells / well. 4 Cells were cultured in 37°C overnight at 37°C. After discarding the culture medium, the cells were treated according to the experimental groups: the control and blank groups were treated with equal volumes of fresh complete culture medium, while the experimental groups were treated with culture medium containing 100, 200, 300, and 400 μg / mL SsEVs, respectively, and cultured for another 48 h. After the culture was completed, 10% of the original culture volume of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance of each well was then measured at 450 nm using a microplate reader to assess cell viability.

[0348] (2) OD values ​​were measured at a wavelength of 450 nm at 24 h and 48 h after treatment, respectively.

[0349] (3) Cell viability detection (iCELLigence real-time cell analysis system) Cells were seeded in E-Plate 16 electrode plates at a seeding density of 3 × 10⁶ cells per well. 4 Cells were cultured in wells with 10% FBS in DMEM complete medium to a total volume of 200 μL. The culture plates were placed in an incubator connected to the system (37°C, 5% CO2). The instrument automatically recorded changes in cell resistance and generated a cell index curve in real time. After cell adhesion stabilized, different concentrations of SsEVs were added to the experimental wells, while an equal volume of PBS buffer was added to the control group. Cultures were continued for 72 hours, and monitoring was maintained. Experimental data were acquired and analyzed in real time using iCELLigence software to assess cell proliferation and growth dynamics.

[0350] 2. Experimental Results Compared with the control group, at both 24 h and 48 h, treatment with different concentrations of SsEVs significantly inhibited the proliferation of CAL-27 cells. Figure 60The inhibitory effect of SsEVs on cell proliferation gradually increased with increasing SsEV concentration. The inhibitory effect of SsEVs was most pronounced when the treatment time was extended to 48 h. Furthermore, cell proliferation was significantly inhibited at an SsEV concentration of 400 μg / mL, with the highest inhibition rate. Figure 60 (B in the middle).

[0351] 400 μg / mL of SsEVs significantly reduced the growth rate of CAL-27 cells, and the increase in Cell Index was significantly slowed down. Figure 60 (C in the text). This result is consistent with the CCK-8 assay results, further validating the time- and dose-dependent inhibitory effect of SsEVs on the proliferation of oral cancer cells.

[0352] (II) Effects of SsEVs on the migration and invasion abilities of oral cancer cells 1. Experimental Procedure (1) Cell scratch test CAL-27 cells were seeded in 6-well plates and cultured at 37°C in a 5% CO2 incubator. When cell confluence reached 90%, a straight line was vertically drawn in the center of the cell layer using a sterile 200 μL pipette tip. After washing with PBS to remove detached cells, the control group was cultured in serum-free DMEM, while the SsEVs-treated group was cultured in serum-free medium containing 400 μg / mL SsEVs. Cells were cultured under the same conditions, and the scratched area was photographed at 0 h, 12 h, and 24 h. Changes in scratch width were measured using ImageJ software to assess cell migration ability.

[0353] (2) Cell Transwell migration experiment 1) Cell migration ability was assessed using Transwell chambers (8 μm pore size, Corning).

[0354] 2) Add 500 μL of DMEM medium containing 20% ​​FBS to the lower chamber as a source of chemokines.

[0355] 3) After washing CAL-27 cells twice with PBS, resuspend them at a density of 8×10⁶ cells / mL. 4 Cell suspension of 200 μL cells was added to the upper chamber. The experimental group received additional SmEVs at a final concentration of 400 μg / mL in the upper chamber, while the control group received an equal volume of PBS. The Transwell plates were incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0356] 4) After incubation, discard the culture medium in the chamber and fix with 4% paraformaldehyde solution at room temperature for 20 minutes.

[0357] 5) After fixation, immerse the chamber in crystal violet staining solution for 8 minutes, then wash thoroughly with PBS and gently wipe away any unmigrated cells from the upper chamber with a cotton swab.

[0358] 6) After the chamber is completely dry, randomly select an observation area through a microscope to image and record the cells, and then perform subsequent statistical analysis on the cell count.

[0359] (3) Cell invasion experiment 1) From The invasion chambers, taken from the 80°C freezer, were placed at room temperature for about 30 minutes to allow them to fully equilibrate and rewarm. Then, 500 μL of serum-free DMEM was added to each chamber and the accompanying well plate, and the chambers were incubated at 37°C for 2 hours to complete the hydration step.

[0360] 2) Add 500 μL of DMEM medium containing 10% FBS to the lower chamber of the Transwell as a source of chemokines.

[0361] 3) Cells washed with PBS were prepared into a density of 8×10⁶ cells / cm². 4 200 μL of suspension was added to the upper chamber. The experimental group had SmEVs added to the upper chamber at a final concentration of 400 μg / mL, while the control group had an equal volume of PBS added. The mixture was then incubated at 37°C for 72 h.

[0362] 4) After 72 hours of incubation, remove the culture medium from the chamber and place the chamber in 4% paraformaldehyde solution to fix at room temperature for 20 minutes.

[0363] 5) After washing the chamber with PBS, stain it in crystal violet solution for 8 min. After staining, rinse again with PBS and gently wipe away any uninvaded cells in the upper chamber with a cotton swab to avoid interfering with the results.

[0364] 6) After the chamber has dried, observe it under a microscope and take pictures.

[0365] 2. Experimental Results Compared with the control group, the scratch healing area of ​​CAL-27 cells in the SsEVs treatment group was significantly reduced at both 24h and 48h, and the migration rate of cells to the scratch area was significantly decreased. Figure 61 (A in the text). This result indicates that SsEVs can effectively inhibit the horizontal migration ability of oral cancer cells.

[0366] Compared to the untreated control group, the number of CAL-27 cells capable of penetrating the membrane pores was significantly reduced in the SsEVs-treated group. Cell migration ability was significantly decreased. Furthermore, in the matrix gel invasion assay, the number of cells in the SsEVs-treated group that penetrated the matrix gel and adhered to the lower membrane surface was also significantly lower than that in the control group. Figure 61The results (D) indicate that SsEVs not only inhibit the migration ability of CAL-27 cells, but also significantly weaken their invasive potential.

[0367] In summary, both experiments demonstrated that SsEVs can inhibit the migration ability of CAL-27 cells, indicating that SsEVs have an inhibitory effect on the invasion and metastasis of oral cancer cells.

[0368] Example 15 Effect of SsEVs on the growth of human oral cancer cell xenografts in nude mice 1. Experimental Procedure (1) Experimental grouping and inoculation When constructing the CAL-27 cell xenograft model in nude mice, the mice were randomly divided into two groups: a control group (CAL-27 cells) and an SsEVs treatment group (CAL-27 cells + SsEVs co-injection).

[0369] BALB / c nude immunodeficient mice were randomly divided into two groups of 10 mice each, and numbered using ear tagging. The groupings are as follows: Group A: CAL-27 cells (1×10⁻⁶) 6 (each 0.15 mL) Group b: CAL-27 cells (1×10⁻⁶) 6 A mixture of 0.15 mL of ssEVs (400 μg / mL) and SsEVs (total injection volume 0.15 mL).

[0370] The prepared cell suspension was placed in a sterile EP tube and temporarily stored on ice. Before injection, it was gently mixed with a pipette. After fixing the nude mouse by hand, the mouse was disinfected with povidone-iodine and deiodized with 75% alcohol. After the local skin was dry, the cell suspension was slowly aspirated and injected subcutaneously into the right axilla on the back.

[0371] (2) Tumor growth observation and measurement Observe the nude mice daily for their mental state, diet, and activity. Once the tumor is palpable, measure its long and short diameters every two days using calipers. Calculate the tumor volume using the following formula: Tumor volume (mm³) = 0.5 × longest diameter (mm) × shortest diameter² (mm²).

[0372] (3) Material selection and measurement Once the tumors reached the reference standard, nude mice were anesthetized with isoflurane, and their tumor growth was photographed for record-keeping. The animals were then sacrificed, and the tumor tissue was dissected to measure and record its maximum transverse and minimum longitudinal diameters. Simultaneously, the isolated tumor-bearing mice were photographed.

[0373] (4) The paraffin embedding and sectioning of the tissue were performed in the same manner as in Example 5.

[0374] 2. Experimental Results After 14 days of tumor formation, the length and short diameter of the tumor were measured every two days, and the tumor volume was calculated. Results showed that, compared to the control group, the tumors in the SsEVs-treated group grew slowly and steadily during follow-up, with significantly smaller tumor volume from the early stages of tumor formation compared to the control group, and exhibiting significantly smaller tumor volume and weight at the end of the experiment. Figure 62 The tumor weight measured after final sacrifice also showed that the SsEVs-treated group was significantly lower than the control group (P<0.05). These results indicate that SsEVs can exert a sustained inhibitory effect on the growth of oral cancer cells in nude mice, suggesting that SsEVs have certain in vivo anti-tumor potential.

[0375] Example 16: Transcriptome Sequencing Analysis of Oral Cancer Cells Treated with SsEVs To more systematically elucidate the specific molecular mechanisms by which SsEVs inhibit oral cancer development, transcriptome sequencing was performed on CAL-27 cells after SsEV treatment, and various bioinformatics analysis methods were combined to systematically study the changes in differential gene expression and related signaling pathways.

[0376] (I) Analysis of differential gene expression in SsEVs 1. Sample preparation and sequencing CAL-27 cells were seeded into 6-well plates and cultured until adherence was achieved, at which point the culture medium was discarded. The experimental group received 2 mL of medium containing 400 μg / mL SsEVs, while the control group received an equal volume of fresh DMEM medium. The 6-well plates were then incubated in a cell culture incubator (37 °C, 5% CO2) for 48 h. After incubation, the culture medium was discarded, and the cells were washed 2-3 times with PBS buffer. 600 μL of TRIzol reagent was added to each well for lysis, and the cells were collected. Total RNA samples were flash-frozen in liquid nitrogen and stored at –80 °C for later analysis at Megabio.

[0377] 2. Bioinformatics Analysis Total RNA was extracted from each group of cells, and its quality and integrity were assessed. After confirming compliance with sequencing requirements, sequencing libraries were constructed and high-throughput sequencing was performed. The obtained raw sequencing data were then processed using a standardized bioinformatics workflow, including data quality filtering, alignment of sequencing sequences with a reference genome, and quantitative analysis of gene expression levels. Based on the expression levels, differentially expressed genes (DEGs) with significant changes were further analyzed to screen for these genes. Pathway enrichment analysis was then performed on the selected DEGs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to elucidate the related biological processes and signal transduction pathways regulated by SsEVs. To further assess pathway change trends at the gene set level, Gene Set Enrichment Analysis (GSEA) was used to enrich the whole-gene expression matrix, exploring the overall regulatory patterns of SsEVs on the transcriptional program of CAL-27 cells. By integrating RNA-seq differential expression, KEGG and GSEA enrichment analysis results, we systematically evaluated the effects of SsEVs on CAL-27 cell biological pathways.

[0378] 3. Results Bioinformatics analysis showed that after 48 hours of treatment with SsEVs, the overall transcriptional expression profile of CAL-27 cells was significantly altered. In the volcano plot, the red dots on the right correspond to genes with significantly elevated expression levels, while the blue dots on the left represent genes with significantly decreased expression levels; these two groups form clear and distinct clusters on the coordinate axis. Figure 63 ).

[0379] Further statistical results showed that 337 genes were upregulated after SsEVs treatment, while the expression of 625 genes was significantly suppressed. Figure 63 This extensive gene expression remodeling suggests that SsEVs play a regulatory role in multiple biological processes and signaling pathways.

[0380] (II) Analysis based on RNA-seq and KEGG / GSEA reveals key targets of SsEVs in inhibiting CAL-27. RNA-seq differential expression analysis showed that the number of downregulated genes in CAL-27 cells was significantly greater than the number of upregulated genes after SsEVs treatment, indicating that SsEVs have a predominantly inhibitory regulatory effect on the overall transcriptional program of oral squamous cell carcinoma cells. Since this experiment focuses on the inhibitory effect of SsEVs on tumor cell growth, subsequent pathway enrichment analysis was mainly based on the set of downregulated genes.

[0381] KEGG enrichment results showed that the downregulated genes were significantly enriched in multiple signaling pathways closely related to epithelial cell growth and proliferation, including the cell cycle, Wnt, Hippo, Ras / Rap1, TGF-β, and Notch signaling pathways (see KEGG enrichment plot). Figure 64 These pathways are widely involved in the regulation of cell proliferation, differentiation, and survival in epithelial-derived tumors. The concentrated distribution of downregulated genes in these pathways indicates that SsEVs inhibit the growth of CAL-27 cells by broadly suppressing multiple pro-proliferative signaling pathways.

[0382] KEGG enrichment analysis showed that the Notch signaling pathway reached a significant enrichment level, and the differentially enriched genes within the pathway were predominantly downregulated, indicating that this pathway was systematically suppressed after SsEVs treatment. Furthermore, GSEA analysis yielded consistent results: when using the Notch signaling pathway as the gene set, GSEA showed a significant negative enrichment trend, with most core enrichment genes located on the downregulated side of the enrichment curve, indicating that the Notch pathway as a whole was uniformly downregulated. Figure 65 Combined with the results at the gene level, Notch1 expression levels decreased significantly after SsEVs treatment, which is consistent with the overall inhibition of Notch signaling at the pathway level.

[0383] The above results collectively demonstrate that SsEVs-induced Notch1 downregulation is one of the important molecular bases for inhibiting the growth of oral squamous cell carcinoma cells CAL-27, and also provides a clear target for subsequent functional verification experiments around Notch1.

[0384] Example 17 RT-qPCR Validation of Expression Changes in Notch Signaling Pathway-Related Genes 1. Experimental Procedure (1) RNA extraction: 1) Discard the culture medium from the 6-well plate and wash twice with PBS. Add 1 mL of Trizol reagent to each well and gently pipette to mix and lyse the cells.

[0385] 2) Transfer the lysis buffer to an EP tube and place it on ice for 10 minutes to allow it to fully lyse.

[0386] 3) Add 200 μL of chloroform at a volume ratio of 5:1, shake vigorously for 15–30 s to mix, and then let stand for 2–5 min to promote separation.

[0387] 4) Centrifuge the sample at 4 ℃ and 12000 rpm for 15 min, then carefully aspirate the upper aqueous phase and transfer it to a new EP tube.

[0388] 5) Add isopropanol at a volume ratio of 1:1, mix well, place on ice for 10 min, and then centrifuge at 4 ℃ and 12000 rpm for 15 min to obtain RNA precipitate.

[0389] 6) Discard the supernatant after centrifugation, add 800 μL of 75% ethanol to gently wash the precipitate, and centrifuge at 4 ℃ and 7000 rpm for 5 min.

[0390] 7) Repeat the ethanol washing step once.

[0391] 8) Discard the supernatant, open the tube cap to air dry any remaining liquid on the tube wall, and allow the RNA precipitate to air dry naturally.

[0392] 9) Add an appropriate amount of DEPC water to dissolve the RNA.

[0393] 10) Use Nanodrop to detect RNA concentration and purity.

[0394] (2) Genomic DNA removal Prepare the reaction mixture on ice: Mix 5× gDNA Eraser Buffer and gDNA Eraser thoroughly according to the manufacturer's instructions to prepare a master mix. Each 10 μL reaction mixture contains 1 μg of RNA. Calculate the volume to be added for each sample based on the measured RNA concentration, using RNase-free pipette tips and tubing, and briefly disconnect the pipette before proceeding to the next step.

[0395] The genomic DNA removal reaction system is shown in Table 23: Table 23

[0396] (3) The reverse transcription reaction system is shown in Table 24: Table 24

[0397] The reverse transcription reaction was carried out under the following conditions: 37℃ for 15 minutes; 85℃ for 5 seconds; and 4℃ cycling.

[0398] (4) Real-Time PCR 1) Prepare primer and reagent mixture according to the reaction system: Mix 10 μmol / L upstream and downstream primers with TB GreenPremix Ex Taq II in proportion to prepare primer-TB Green premix solution for later use.

[0399] 2) According to the system requirements, mix the cDNA sample with RNase-free ddH2O, mix thoroughly and set aside.

[0400] 3) According to the experimental design, add the above primer-TB Green premix and cDNA-ddH2O mixture to the 96-well plate in sequence, and collect the liquid to the bottom of the well by gently shaking or briefly centrifuging.

[0401] 4) The reaction system composition is shown in Table 25: Table 25

[0402] 5) Cover the 96-well PCR plate with a membrane and centrifuge briefly.

[0403] 6) Perform the following tests on the computer as shown in Table 26: Table 26

[0404] 7) Results Analysis: The average Ct value of the triplet wells was taken. The ΔCT value was obtained by subtracting the corresponding internal reference value from the Ct value of each gene. The ΔCT value was obtained by subtracting the ΔCT value of the control group from the ΔCT value of the experimental group. To determine the relative expression levels of each gene after real-time fluorescence PCR between groups, 2-ΔΔCt was used for calculation.

[0405] 2. Experimental Results In SsEVs-treated CAL-27 cells, Notch1 mRNA expression was significantly downregulated, with the most pronounced downregulation. Simultaneously, the expression levels of Notch1 ligand JAG1 (Jagged1) and downstream transcription factor HEYL were significantly reduced; DLL1 expression also showed a downregulation trend, but the decrease was relatively moderate. In contrast, no statistically significant differences were observed in the expression of Notch signaling transcription co-activators MAML2 and E3 ubiquitin ligase DTX4 between the SsEVs-treated group and the control group. Figure 66 Overall, the RT-qPCR results were consistent with the trend of RNA-seq analysis, indicating that the Notch signaling pathway in CAL-27 cells was inhibited at the transcriptional level after SsEVs treatment, and that this inhibition was centered on Notch1, accompanied by the synergistic downregulation of its ligands and downstream effector molecules.

[0406] Example 18 Changes in the expression of key proteins in the Notch signaling pathway in CAL-27 cells after SsEVs treatment 1. The Western blot-related antibodies required for this invention are described in Example 1.

[0407] The theoretical molecular weight of Notch1 is approximately 271 kDa, Jagged1 is approximately 134 kDa, and HeyL is approximately 35 kDa.

[0408] 2. Experimental Results Compared with the control group, the expression levels of the above three proteins in CAL-27 cells treated with SsEVs were significantly reduced, with Jagged1 protein showing the most significant downregulation, and Notch1 and HeyL proteins also showing a clear and consistent decreasing trend. Figure 67 This result is highly consistent with the downregulation trend of Notch1, Jagged1, and HeyL at the transcriptional level in the aforementioned RNA-seq and qRT-PCR analyses, further indicating that SsEVs can systematically inhibit the Notch signaling pathway at multiple levels, including receptors, ligands, and downstream effector molecules, providing experimental protein-level evidence for their role in regulating the biological behavior of oral squamous cell carcinoma cells CAL-27.

[0409] Example 19: Validation of IHC inhibition of the Notch1 / Jagged1 / HeyL axis by SsEVs in in vivo xenograft tissue 1. Experimental Procedure (1) Immunohistochemistry 1) Place the slide in a 60°C oven for 40 minutes to ensure that the tissue adheres fully to the slide.

[0410] 2) Dewax the sections in xylene for 10 minutes each time, repeating twice; then hydrate them in anhydrous ethanol for 10 minutes twice, and in 95% ethanol for 10 minutes twice, and finally soak them in distilled water for 5 minutes.

[0411] 3) Place the tissue sections in EDTA (1×) antigen retrieval solution and heat them for approximately 4 minutes using a dedicated antigen retrieval instrument to complete antigen retrieval, then allow them to cool naturally to room temperature. After cooling, rinse twice with PBS buffer and dry thoroughly. The required antigen is shown in Example 1.

[0412] 4) Add 3% H2O2 to the slice, incubate at room temperature in the dark for 10 minutes, wash with distilled water 3 times (5 minutes each time), and wipe dry.

[0413] 5) Add 20% sealing solution, let stand at room temperature for 20 minutes, remove excess liquid and wipe the edges dry.

[0414] 6) Dilute the primary antibody according to the instructions (Notch1 1:500; Hey L 1:200; Jagged1 1:200), add it to the slide, and incubate overnight at 4°C in a humidified chamber.

[0415] 7) After removing the slices and letting them stand at room temperature for 30 minutes, wash them three times with PBS for 2 minutes each time.

[0416] 8) Dilute Bio-goat anti-rabbit IgG at a ratio of 1:1000, incubate at 37°C for 30 minutes, and then wash three times with PBS for 2 minutes each time.

[0417] 9) Dilute streptavidin-HRP at a ratio of 1:1000, incubate at 37°C for 30 minutes, and wash with PBS 4 times for 5 minutes each time.

[0418] 10) Add the prepared DAB staining solution to the surface of the tissue section for staining. After about 1 minute, observe the staining degree under a microscope. When the appropriate intensity is reached, immediately rinse with distilled water to end the reaction. Counterstain with hematoxylin for 2 minutes, then rinse with distilled water.

[0419] 11) After the slices are briefly rinsed with distilled water (1–2 s), they are placed in 85%, 95% and 100% ethanol for 5 min each for gradient dehydration, with the 100% ethanol step repeated twice.

[0420] 12) The tissue sections were cleared in xylene solution for 5 minutes each time, and the treatment was repeated twice.

[0421] 13) After the sections are mounted with neutral resin, they are left to air dry at room temperature.

[0422] 14) Use a Leica microscope system (Leica, Germany) to observe the staining effect of the sections and collect and record images.

[0423] 2. Experimental Results Compared with the control group, the positive staining intensity of Notch1, Jagged1, and HeyL in the xenograft tissue of the SsEVs-treated group was significantly reduced. Figure 68 Further quantitative analysis of the staining results using ImageJ software revealed a significant decrease in the average optical density (IOD / area) of the aforementioned proteins in the SsEVs-treated group, suggesting that their protein expression levels were significantly suppressed in tumor tissue.

[0424] The above results demonstrate that SsEVs not only inhibit the expression of Notch signaling pathway-related proteins under in vitro experimental conditions, but also exhibit significant inhibitory effects in the in vivo tumor microenvironment. These in vivo IHC analysis results are highly consistent with the aforementioned in vitro Western blot results, mutually reinforcing each other at the histological and protein expression levels, thus enhancing the reliability of the experimental conclusions. Based on the combined in vivo and in vitro experimental data, it can be concluded that SsEVs inhibit the activation of the Notch signaling pathway by downregulating the activity of the Notch1 / Jagged1 / HeyL axis, thereby participating in the regulation of the growth and progression of oral squamous cell carcinoma.

[0425] Comparative Example 1: A comparative study of the inhibitory effects of extracellular vesicles of Streptococcus salivarius Ss-08 and other Streptococcus salivarius extracellular vesicles on the proliferation of oral cancer cells. To better demonstrate the ability of the extracellular vesicles of Streptococcus salivarius Ss-08 to inhibit oral cancer cells, this invention uses other isolated Streptococcus salivarius strains (S1-S7) as controls.

[0426] The specific steps are as follows: (1) Completely digest the Cal-27 cells that have grown in the culture flask, add DMEM culture medium, repeatedly pipette the digested cells to detach them from the cell wall and make a cell suspension, and centrifuge at 800 r / min for 5 min.

[0427] (2) Discard the supernatant, add an appropriate amount of culture medium to resuspend the cells, and transfer 20 μL of cells to the counting area. Count the cells using a cell counter. Adjust the cell density to 1×10⁻⁶. 4 per mL.

[0428] (3) Add 100 μL of cell suspension with adjusted density to each well into the 96-well plate to ensure that the cells are evenly distributed in the well plate. Place the 96-well plate in a cell culture incubator containing 5% CO2 at 37°C for culture.

[0429] (4) After the cells adhered, the cell culture medium in the 96-well plate was aspirated. Extracellular vesicles at a concentration of 200 μg / mL were added to the experimental wells, and fresh culture medium was added back to the control wells. The 96-well plate was then placed in a cell culture incubator and cultured for 48 h.

[0430] (5) After the extracellular vesicle activity is complete, add 10 μL of WST-1 solution to each well and incubate in a cell culture incubator for 1-2 h. Measure the absorbance at 450 nm using a microplate reader.

[0431] result( Figure 69It was found that extracellular vesicles of *Streptococcus salivarius* Ss-08 (200 μg / mL) significantly inhibited cell proliferation and reduced the viability of Cal-27 oral cancer cells after 48 hours of treatment. Other *Streptococcus salivarius* (S1-S7) at the same concentration (200 μg / mL) did not significantly inhibit these oral cancer cells, and their effects were significantly worse than those of *Streptococcus salivarius* Ss-08.

[0432] 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 type of Streptococcus salivarius ( Streptococcus salivarius Ss-08, characterized in that, The preservation number of the aforementioned Streptococcus salivarius Ss-08 is CCTCC NO:M20261361.

2. A preparation of Streptococcus salivarius Ss-08, characterized in that, The preparations include: fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, and extracellular vesicles.

3. The preparation according to claim 2, characterized in that, The prepared product is extracellular vesicles of Streptococcus salivarius Ss-08.

4. The preparation according to claim 3, characterized in that, The method for preparing extracellular vesicles of Streptococcus salivarius Ss-08 includes the following steps: (1) Resuscitate Streptococcus salivarius Ss-08; (2) Culture of Streptococcus salivarius Ss-08; (3) Obtain the supernatant from the bacterial culture; (4) Obtain the precipitate from the supernatant.

5. A microbial agent, characterized in that, The bacterial agent includes the *Streptococcus salivarius* Ss-08 as described in claim 1 or the preparation as described in any one of claims 2-4.

6. The use of the *Streptococcus salivarius* Ss-08 of claim 1, or the preparation of any one of claims 2-4, or the bacterial agent of claim 5, in the preparation of medicaments for the prevention and / or treatment of oral diseases.

7. The application according to claim 6, characterized in that, The viable count of Streptococcus salivarius Ss-08 in the drug is 1×10⁻⁶. 6 -1×10 8 CFU / mL or 1×10 6 -1×10 8 CFU / mg; the content of extracellular vesicles of Streptococcus salivarius Ss-08 in the drug is 300-600 μg / mL.

8. The application according to claim 6, characterized in that, The drug includes a pharmaceutically acceptable carrier.

9. The application according to claim 6, characterized in that, The drug has at least one of the following effects: (1) Improve oral mucosal pathological damage caused by oral submucosal fibrosis; (2) Inhibit abnormal activation of gene signaling pathways in the tongue; (3) Promote the recovery of expression of molecules related to tight junctions in oral epithelium; (4) Reduce the progression of oral fibrosis; (5) Regulating oral and intestinal flora to participate in the regulation of oral submucosal fibrosis; (6) Inhibits transcription of oral squamous cell carcinoma cells; (7) Inhibits the progression of oral squamous cell carcinoma cells.

10. The application according to claim 6, characterized in that, The oral diseases mentioned are selected from one of the following: dental caries and pulp diseases, periodontal tissue diseases, oral mucosal diseases, oral and maxillofacial surgical and developmental diseases, and dentition and occlusion abnormalities.

11. A product for preventing and treating oral diseases, characterized in that, The product comprises the *Streptococcus salivarius* Ss-08 as described in claim 1, or the preparation as described in any one of claims 2-4, or the bacterial agent as described in claim 5.

12. The product according to claim 11, characterized in that, The product is a drug, and the viable count of Streptococcus salivarius Ss-08 in the drug is 1×10⁻⁶. 6 -1×10 8 CFU / mL or 1×10 6 -1×10 8 CFU / mg; the content of extracellular vesicles of Streptococcus salivarius Ss-08 in the drug is 300-600 μg / mL.

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