Streptococcus salivarius CCFM1399 and postbiotic for alleviating bone resorption and periodontal tissue damage

Abstract

The application discloses a Streptococcus salivarius CCFM1399 and its postbiotic for relieving bone resorption and periodontal tissue damage, and belongs to the technical field of microorganisms. The Streptococcus salivarius CCFM1399 provided by the application is separated from oral plaque of healthy teenagers, has the effects of relieving bone resorption and resisting inflammation, and specifically has the following effects: improving the expression of osteoprotegerin (OPG) protein level and gene level of MC3T3-E1 cells in vitro; reducing the alveolar bone resorption amount of orthodontic bone resorption rats, reducing the number of osteoclasts in alveolar bone, and improving the OPG level in serum of rats; reducing the secretion of inflammatory factors IL-6, TNF-alpha and MPO of orthodontic bone resorption rats; and improving the pathological condition of alveolar bone tissue of orthodontic bone resorption rats. Therefore, the Streptococcus salivarius has great application prospect in products for relieving bone resorption and assisting in inhibiting periodontal tissue inflammation.

Description

Technical Field

[0001] This invention relates to a strain of Streptococcus salivarius CCFM1399 that alleviates bone resorption and periodontal tissue damage, and its postbiotics, belonging to the field of microbial technology. Background Technology

[0002] In a healthy skeletal system, osteoblasts and their bone-forming function, and osteoclasts and their bone-resorbing function, are normally in dynamic balance. However, periodontal diseases such as periodontitis, gingivitis, and peri-implantitis, as well as excessive orthodontic forces, can induce excessive bone resorption. Excessive oral bone resorption can lead to alveolar bone loss, tooth loosening and loss, worsening of periodontal disease, tooth displacement, and changes in occlusion, among other oral health problems, requiring timely prevention and treatment.

[0003] The OPG / RANKL / RANK signaling pathway plays a crucial role in maintaining bone resorption and bone formation homeostasis. Osteoporosis protectant (OPG), secreted by osteoblasts, competitively binds to RANK receptors on the surface of osteoclasts, thereby inhibiting osteoclast differentiation and bone resorption. Hormones such as estrogen and glucocorticoids can regulate bone resorption and bone formation by modulating this pathway. In orthodontic treatment, regulating this pathway can affect alveolar bone metabolism, thus influencing the speed and outcome of tooth movement. Increasing OPG levels or inhibiting RANKL activity can reduce bone resorption, contributing to tooth stability and achieving alveolar bone homeostasis in orthodontic patients.

[0004] Currently, several patents exist for alleviating excessive alveolar bone resorption, typically focusing on the use of bone resorption inhibitors and osteoclast agonists to regulate osteoclast differentiation. Patent CN116196424B uses protein transport inhibitors to reduce deubiquitinating enzyme activity, thereby achieving bone resorption or bone loss. Patent CN115770297A uses TGFβ or Wnt signaling agonists to activate osteocyte Wnt signaling, promoting increased TGFβ signaling and activating RANKL promoter activity, thus promoting osteoclast formation and providing a new research direction for the prevention and treatment of osteoporosis. However, for oral mucosal administration, the stability, absorption efficiency, and potential side effects of the drug in the oral cavity need to be considered. The use of protein transport inhibitors in the oral cavity may require specially designed drug delivery systems to ensure the efficacy and safety of the drug. Related agonists also need to be comprehensively evaluated for their mechanism of action under excessive bone resorption, potential adverse reactions, and impact on tumor risk. No safe, effective, and side-effect-free method for treating orthodontic-induced bone resorption has yet been discovered.

[0005] Food-grade probiotics, as safe products with no side effects, have broad application prospects. However, no edible probiotics have yet been found that can effectively treat orthodontic-induced bone resorption or alleviate oral inflammation. Summary of the Invention

[0006] Currently, no effective treatment for orthodontic-induced bone resorption has been found in clinical practice.

[0007] This invention provides a novel method for treating orthodontic-induced excessive bone resorption using *Streptococcus thermophilus* subsp. CCFM1399, which has a mild effect and few side effects. The *Streptococcus thermophilus* subsp. of this invention can regulate osteoblast secretion of osteoprotegerin (OPG) and increase the gene expression of bone morphogenetic protein (BMP). In a rat orthodontic model, the strain of this invention can effectively alleviate bone resorption, inhibit the level of inflammatory factors in gingival tissue, and protect periodontal tissue stability.

[0008] This invention provides a strain of Streptococcus thermophilus and its metabiotic that can alleviate bone resorption and periodontal tissue damage. The taxonomic name of the strain, Streptococcus thermophilus CCFM1399, is Streptococcus thermophilus, and it was deposited on June 14, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 64763), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] In one embodiment of the present invention, the Streptococcus thermophilus subsp. CCFM1399 was obtained from dental plaque samples from the oral cavity of healthy volunteers in Wuxi, Jiangsu Province. The 16S rDNA sequence of this strain is shown in SEQ ID No. 1. The sequenced sequence was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Streptococcus thermophilus subsp. CCFM1399.

[0010] In one embodiment of the present invention, the colonies of Streptococcus thermophilus CCFM1399 on MRS medium are small, translucent white circles.

[0011] The present invention also provides a microbial preparation containing the aforementioned Streptococcus thermophilus subsp. CCFM1399.

[0012] In one embodiment of the present invention, the number of *Streptococcus thermophilus* subsp. *salivarius* CCFM1399 cells in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6CFU / g.

[0013] The present invention also provides an epigenetic agent prepared from the thermophilic subspecies of Streptococcus salivarius CCFM1399, wherein the epigenetic agent contains dead cells, lysate and / or supernatant of the thermophilic subspecies of Streptococcus salivarius CCFM1399.

[0014] The present invention also provides a method for preparing a postbiotic of *Streptococcus salivarius* subsp. *thermophilus* CCFM1399, comprising (a), (b), or (c):

[0015] (a) The thermophilic subsp. salivator CCFM1399 was cultured to the logarithmic growth phase, and the cells were collected after heat treatment to obtain the metagener;

[0016] (b) The thermophilic subsp. salivarius CCFM1399 was cultured to the logarithmic growth phase, inactivated by heat treatment, and then homogenized under high pressure to obtain the metagener;

[0017] (c) The thermophilic subsp. salivary streptococcus CCFM1399 was cultured to the logarithmic growth phase, and the fermentation supernatant was collected to obtain the metagene.

[0018] The present invention also provides products containing the aforementioned Streptococcus thermophilus subsp. CCFM1399 and / or its metabiotics, said products being food, health products, pharmaceuticals or daily chemical products.

[0019] In one embodiment of the present invention, the food includes fruit and vegetable products, soy products, or dairy products.

[0020] In one embodiment of the present invention, the daily chemical products include mouthwash, mouthwash pellets, toothpaste, tooth powder, teeth whitening strips, periodontal gel, dental gel, or tooth wax.

[0021] In one embodiment of the present invention, the dosage form of the medicine includes oral spray, tablets, capsules, paste, oral liquid or lyophilized powder.

[0022] In one embodiment of the present invention, the pharmaceutical ingredient comprises Streptococcus sthermophilus CCFM1399 and conventional pharmaceutical excipients and / or carriers.

[0023] In one embodiment of the present invention, the carrier is a pharmaceutically acceptable carrier.

[0024] In one embodiment of the present invention, the carrier is one or more of a pharmaceutically acceptable filler, wetting agent, disintegrant, binder, lubricant or flavoring agent.

[0025] The present invention also provides the use of the aforementioned Streptococcus salivarius thermophilus subsp. CCFM1395, and / or the aforementioned microbial preparation, and / or the aforementioned metabiotic in the production of osteoprotegerin.

[0026] The present invention also provides the use of the aforementioned *Streptococcus salivarius* subsp. *thermophilus* CCFM1399, and / or the aforementioned microbial preparation, and / or the aforementioned metabiotic in the preparation of a medicament for regulating oral inflammatory function, wherein the oral inflammatory function includes periodontitis or gingivitis.

[0027] The present invention also provides the use of the aforementioned *Streptococcus salivarius* subsp. *thermophilus* CCFM1399, or the aforementioned microbial preparation, or the aforementioned metabiotic in the preparation of medicaments for the prevention and / or treatment of bone resorption.

[0028] The present invention also provides the use of the aforementioned *Streptococcus salivarius* subsp. *thermophilus* CCFM1399, and / or the aforementioned microbial preparation, and / or the aforementioned metabiotic in the preparation of care products for the prevention and / or treatment of oral inflammation, including periodontitis or gingivitis, and the care products including mouthwash, mouthwash pellets, toothpaste, dental powder, whitening strips, periodontal gel, dental gel, or dental wax.

[0029] Beneficial effects

[0030] 1. This invention provides a strain of Streptococcus thermophilus CCFM1399, isolated from dental plaque in the oral cavity of healthy adolescents. Cell lysates, live cells, and dead cells of the strain CCFM1399 all exhibit the effect of alleviating orthodontic-induced bone resorption and simultaneously inhibiting periodontal inflammation, specifically manifested in:

[0031] (1) By promoting osteoblast activity, it increases osteoblast OPG expression, prevents the binding of RANKL and RANK in alveolar bone tissue, and inhibits osteoclast activity and differentiation.

[0032] (2) Increase the expression of bone morphogenetic protein (BMP) gene in MC3T3-E1 cells.

[0033] (3) Significantly reduced the alveolar bone resorption level induced by orthodontic model in rats.

[0034] (4) Reduce the gingival index and probing depth induced by orthodontic model in rats.

[0035] (5) Reduce the number of osteoclasts at the root bifurcation of the alveolar bone.

[0036] (6) Reduce the expression of RANKL in rat serum.

[0037] (7) Reduce the expression levels of IL-6, TNF-α and MPO in gingival tissue.

[0038] (8) Improve the pathological condition of alveolar bone tissue.

[0039] Therefore, this Streptococcus thermophilus subsp. CCFM1399 has great application potential in the preparation of products for the prevention and / or treatment of bone resorption (such as pharmaceuticals or daily chemical products).

[0040] 2. *Streptococcus thermophilus* is a type of probiotic and is currently included in the "List of Bacterial Strains that Can Be Used in Food" issued by the Ministry of Health. Therefore, the *Streptococcus thermophilus* CCFM1399 screened in this invention will not cause any side effects to the human body and has a high safety profile when used in products (such as pharmaceuticals or daily chemical products) for the prevention and / or treatment of orthodontic-induced bone resorption. Attached Figure Description

[0041] Figure 1 Effects of fermentation supernatants and lysates of different bacteria on osteoprotegerin (OPG) in MC3T3-E1 cells;

[0042] Figure 2 Effect of Streptococcus salivarius subsp. thermophilus CCFM1399 on bone morphogenetic protein (BMP) gene expression in osteoblasts;

[0043] Figure 3 Effects of Streptococcus salivarius subsp. thermophilus CCFM1399 on body weight (A) and OTM (B) in orthodontic-induced bone resorption rats;

[0044] Figure 4 Effects of Streptococcus thermophilus subsp. CCFM1399 on gingival index (A) and probing depth (B) in orthodontic-induced bone resorption rats;

[0045] Figure 5 The effect of Streptococcus thermophilus subsp. CCFM1399 on the expression of inflammatory factors in gingival tissue of orthodontic-induced bone resorption rats: (A) IL-6, (B) TNF-α, (C) MPO;

[0046] Figure 6 The effect of Streptococcus salivarius subsp. thermophilus CCFM1399 on the expression of OPG(A) and RANKL(B) in the serum of orthodontic-induced bone resorption rats;

[0047] Figure 7 Effects of Streptococcus salivarius subsp. thermophilus CCFM1399 on bone microstructure in orthodontic-induced bone resorption rats (A) and the ratio of bone volume to total volume (B);

[0048] Figure 8 HE staining of orthodontic-induced bone resorption rats with *Streptococcus thermophilus* subsp. *salivarius* CCFM1399;

[0049] Figure 9 Trap staining of orthodontically induced bone resorption rats with Streptococcus salivarius subsp. thermophilus CCFM1399 (A) and osteoclast count analysis (B);

[0050] (* in the figure indicates a significant difference, where *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).

[0051] Preservation of biological materials

[0052] A strain of Streptococcus thermophilus (CCFM1399), taxonomically named Streptococcus thermophilus, was deposited on June 14, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 64763). The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0054] The MRS culture medium used in Examples 1, 2, and 3 consisted of: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, and pH 6.2–6.4.

[0055] In Examples 1 and 2, the complete cell culture medium consisted of 92% (v / v) α-MEM medium + 7% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the mixed solution contained 10,000 U / mL of penicillin and 10 mg / mL of streptomycin).

[0056] Osteogenic induction culture medium in Examples 1 and 2: complete cell culture medium + sodium β-glycerophosphate (10 mM / L), ascorbic acid (50 μg / mL), and dexamethasone (100 nM / L).

[0057] BHI blood agar medium in Example 3: 37.5 g / L of Qingdao Haibo BHI commercial medium, 20 g / L of agar powder, 100 mL / L of sterile defibrinated sheep blood, and 1 mL / L of 10% hemin-vitamin K.

[0058] The strains, cells, and animals involved in the following examples are as follows:

[0059] Streptococcus salivarius subsp. thermophilus CCFM1399 is from the culture collection of the Biotechnology Center of Jiangnan University. Prevotella intermedia ATCC 25611 and Streptococcus salivarius subsp. thermophilus ATCC19258 are purchased from the American Type Culture Collection.

[0060] Rat osteoblast precursor cells MC3T3-E1 are from the Biotechnology Center of the School of Food Science and Technology, Jiangnan University.

[0061] SPF-grade Wistar rats, female, 7 weeks old, weighing 235 - 255 g, are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK(Beijing)2012-0001).

[0062] The preparation of experimental samples involved in the following examples is shown as follows:

[0063] In Examples 1, 2, and 3, the preparation of live Lactobacillus suspension: Streptococcus salivarius subsp. thermophilus CCFM1399 and ATCC19258 were inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), cultured at 37 °C for 24 h, and then the cell concentration was adjusted to 1×10 9 CFU / mL.

[0064] The preparation of supernatant: The live Lactobacillus suspension with a concentration of 1×10 9 CFU / mL was centrifuged at 12000 rpm for 10 min, the supernatant was taken, filtered through a 0.22 μm filter membrane, and stored at -20 °C.

[0065] The preparation of lysate: The live Lactobacillus suspension with a concentration of 1×10 9 CFU / mL was homogenized (800 - 1200 MPa) 10 times in a high-pressure homogenizer, filtered through a 0.22 μm filter membrane, and the filtrate, that is, the cell lysate, was collected and stored at -20 °C.

[0066] In Example 4, the preparation of live bacterial suspension of *Streptococcus thermophilus* subsp. *salivarius* was as follows: *Streptococcus thermophilus* subsp. *salivarius* CCFM1399 and ATCC19258 were inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured at 37°C for 24 h. The bacterial cells were collected by centrifugation at 8500 g for 20 min, washed twice with sterile physiological saline, and then resuspended in a 10% (v / v) skim milk powder aqueous solution. After thorough mixing, the suspension was lyophilized. Before animal experiments, the lyophilized *Streptococcus thermophilus* subsp. *salivarius* bacterial powder was reconstituted with PBS to a bacterial suspension concentration of 1×10⁻⁶. 9 CFU / mL.

[0067] Preparation of dead bacterial suspension of *Streptococcus salivarius* subsp. *thermophilus*: The preparation method and concentration of the dead bacterial suspension are the same as for live bacteria, except that a heat treatment step is added. Fermentation broth inactivated at 105℃ for 10 minutes for 24 hours is used, and the inactivation effect is checked by plate plating. After heat treatment, the bacterial suspension is centrifuged at 8500g for 20 minutes, washed twice with sterile physiological saline, and then resuspended in 10% skim milk solution. After thorough mixing, the suspension is lyophilized, and before intervention, it is resuspended in PBS to a concentration of 1×10⁻⁶. 9 CFU / mL.

[0068] Prevotella intermedius bacterial suspension: Prevotella intermedius ATCC 25611 was anaerobically cultured in BHI medium at 37°C for 24 h, and the bacterial cells were collected by centrifugation and resuspended in PBS to a concentration of 1×10⁻⁶. 9 CFU / mL, used for subsequent experiments.

[0069] Example 1: Isolation and Identification of Streptococcus salivarius subsp. thermophilus CCFM1399

[0070] Collect oral swab samples from healthy adolescents and place them in an EP tube containing 1 mL of sterile saline. Pipette 0.2 mL into 1.8 mL of sterile saline to obtain 10... -1 Diluent, then take another 0.5 mL of 10 -1 The diluent was added to 4.5 mL of physiological saline to obtain 10. -2 Diluent, follow these steps to obtain 10 -3 10 -4 10 -5 10 -6 Serial dilution buffer. Take 10... -4 10 -5 10 -6 Add 1 mL of each diluent to a Petri dish, pour in MRS solid medium, mix gently, and incubate at 37°C upside down for 48 h after the medium has solidified.

[0071] Different morphological colonies were streaked and purified on MRS plates. A single purified colony was picked and inoculated into 10 mL of liquid culture medium and incubated at 37°C for 48 h. 1.5 mL of the cultured bacterial suspension was centrifuged at 6000 rpm for 3 min, the supernatant was discarded, and the suspension was washed three times with 1.5 mL of sterile water and resuspended in 1.5 mL of sterile water for use as a template for bacterial identification. A 20 μL PCR system was prepared, containing 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 10 μL of 2×Taq Mixture, 0.5 μL of bacterial suspension, and 8.5 μL of double-distilled water. Primer information is shown in Table 1.

[0072] Table 1: Primer Information Table

[0073]

[0074] The 16S rDNA sequence obtained by sequencing is (SEQ ID No. 1):

[0075] GGCAGGCGGGGTGCTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGG

[0076] ATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTA

[0077] TTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGG

[0078] GCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCT

[0079] CACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAG

[0080] ACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAAC

[0081] CCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTA

[0082] AGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAG

[0083] GGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGA

[0084] TTTATTGGGCGTAAAGCGAGGCCAGGCGTTTGATAAGTCTGAAGTTAAAGGCTGTGGC

[0085] TCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATT

[0086] CCATGTGTAGCGGTGAAATGCGTAGATATATGGGAGGAACCCGGTGGCGAAAGCGGCTC

[0087] TCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGGATTAGATACC

[0088] CTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTG

[0089] CCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCA

[0090] AAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACG

[0091] CGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTC

[0092] GGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT

[0093] TAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTA

[0094] GCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCC

[0095] TTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGA

[0096] CGGCGAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACA

[0097] TGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGC

[0098] CTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGACAGAGG.

[0099] The obtained 16S rDNA sequence was used for species identification via NCBI BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST). Query Cover and Identification scores closer to 100% are preferred. If multiple species are identified in the alignment results, the species with the Complete genome label should be prioritized, taking into account both numerical values. Based on the BLAST results, the bacterium was identified as *Streptococcus salivarius* subsp. *thermophilus*. For confirmed strains, 1.5 mL of bacterial culture was transferred to a 2 mL culture tube, centrifuged at 6000 rpm for 3 min, the supernatant was removed in a clean bench, 1 mL of 30% sterile glycerol was added, and the mixture was thoroughly mixed using a vortex mixer and stored at -80°C. The resulting strain was named *Streptococcus salivarius* subsp. *thermophilus* CCFM1399 and deposited at the Guangdong Provincial Microbial Culture Collection Center on June 14, 2024, with accession number GDMCC No: 64763.

[0100] Example 2: The ability of *Streptococcus salivarius* subsp. *thermophilus* to promote osteoprotegerin (OPG) expression in osteoblasts in vitro.

[0101] OPG is a glycoprotein secreted by osteoblasts and belongs to the tumor necrosis factor receptor superfamily. It can competitively bind to RANK (nuclear factor κB receptor activator) with RANKL (nuclear factor κB receptor activator ligand), thereby inhibiting osteoclast differentiation and activity.

[0102] MC3T3-E1 cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. The thawed cell suspension was added to a centrifuge tube containing 5 volumes of complete cell culture medium. After centrifugation, the supernatant was discarded, and the cells were resuspended in 10 mL of complete cell culture medium. The cells were cultured at 37°C and 5% CO2, with the medium changed every 48 hours. After cell counting, cells were sputtered at 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of [number] cells / well in 6-well plates. 2 mL of osteogenic induction medium containing 5% (v / v) bacterial supernatant or lysis buffer was added to each well. Cell morphology was observed regularly, and the medium was changed every 48 hours. MC3T3-E1 cells differentiating into osteoblasts typically became more flattened. After 21 days of culture, cell culture supernatant was collected from each group, and OPG expression was measured using the Sempercapto OPG Elisa Kit. Grouping information for the cell experiments is shown in Table 2.

[0103] Table 2: Grouping Information Table

[0104]

[0105] The expression of OPG in MC3T3-E1 cells after intervention in each group is as follows: Figure 1 As shown in the figure, the OPG concentration in the cell culture supernatant of the control group was 117.78 ng / L. After treatment with supernatant of *Streptococcus thermophilus* subsp. CCFM1399, supernatant of *Streptococcus thermophilus* subsp. ATCC19258, and lysis buffer, the OPG expression level in the cells was not significantly different from that in the control group (p > 0.05). However, the OPG content in the *Streptococcus thermophilus* subsp. CCFM1399 lysis buffer group was as high as 375.05 ng / L, significantly higher than that in the control group (p < 0.0001). These results indicate that the cell lysis buffer of *Streptococcus thermophilus* subsp. CCFM1399 can significantly increase the OPG expression level in the culture supernatant of induced MC3T3-E1 cells.

[0106] In conclusion, *Streptococcus salivarius* subsp. *thermophilus* CCFM1399 can enhance the expression of OPG in osteoblasts in vitro.

[0107] Example 3: The ability of *Streptococcus salivarius* subsp. *thermophilus* to promote bone morphogenetic protein (BMP) gene expression in osteoblasts in vitro.

[0108] BMP is a member of the transforming growth factor-β (TGF-β) superfamily and promotes the differentiation and function of osteoblasts. BMP can indirectly reduce bone resorption by promoting the activity of osteoblasts, and osteoblasts can produce factors that inhibit the activity of osteoclasts, such as OPG.

[0109] The culture and induction test methods of MC3T3-E1 cells were the same as those in Example 2. After inducing differentiation for 21 days at an initial concentration of 1×10 4 cells / mL, the cell RNA was extracted according to the detailed instructions provided by the Novizan RNA extraction kit, and the operations were carried out step by step. All experimental steps were carried out in an RNase-free environment to avoid RNA degradation. The extracted RNA was used to measure the purity (A260 / A280 ratio) and concentration using a NanoDrop spectrophotometer.

[0110] The expression of the BMP gene in MC3T3-E1 cells before and after intervention was as Figure 2 shown. After intervention with the lysate of Streptococcus thermophilus subsp. CCFM1399, the Fold Change value of the BMP gene in the lysate group was 2.8, which was significantly higher than that in the control group.

[0111] In summary, Streptococcus thermophilus subsp. CCFM1399 can increase the expression level of the BMP gene in osteoblasts in vitro.

[0112] Example 4 Application of Streptococcus thermophilus subsp. CCFM1399 in relieving orthodontic-induced bone resorption in rats

[0113] Twenty-four SPF-grade Wistar rats, male, 7 weeks old, weighing 235 - 255 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Beijing) 2012-0001).

[0114] The experimental groups are shown in Table 3.

[0115] Table 3: Animal experiment protocol and grouping

[0116]

[0117] The rats were randomly divided into 4 groups according to their body weight, with 6 rats in each group. All groups of rats were normally fed throughout the experiment.

[0118] Except for the control group, all other groups underwent orthodontic model creation on day 0 (specifically, a nickel-titanium spring was used to apply a 50g traction force between the rat's first molar and central incisor; this step required first using a grinding tool to create a groove perpendicular to the dental arch midline in the maxillary central incisor to fix the stainless steel ligature. Both the maxillary central incisor and first molar required the ligature to be fixed with fluid resin, ensuring that the broken ligature did not damage the epithelial barrier. Except for the left first molar, the occlusal surfaces of the other maxillary molars were fixed with fluid resin and made convex to eliminate occlusal interference from the orthodontic movement of the maxillary first molar). They also underwent a total of 12 days (days 1-6 and 8-13) of Prevotella intermediate infection (specifically, 1×10⁻⁶ oz. of bacteria was drawn using a needle-free syringe). 9 1 mL of a CFU / mL Prevotella intermediate suspension was slowly flushed into the oral cavity of the rats, left for 1 minute, and then placed in the cage.

[0119] For the live and dead bacteria groups of *Streptococcus thermophilus* subsp. CCFM1399, after 6 hours of intervention with *Prevotella intermedia*, the oral cavity of rats was rinsed with 1 mL of live and dead bacterial suspensions of *Streptococcus thermophilus* subsp. CCFM1399, respectively. The bacterial suspension concentration was 1×10⁻⁶. 9 CFU / mL.

[0120] 1. Rat body weight, distance from first molar to central incisor (OTM), gingival index, and probing depth.

[0121] On days 7 and 14, the gingival index and probing depth of the periodontal tissues of all rats were measured. Saliva was collected from the rats using sterile swabs and stored in 2 mL centrifuge tubes containing 1 mL of 30% glycerol. Rat body weight, distance from the first molar to the central incisor (OTM), gingival index, and probing depth were also recorded.

[0122] (1) Changes in rat body weight and tooth movement distance (OTM)

[0123] like Figure 3 As shown in Figure A, rats exhibited significant weight loss on day 7 after orthodontic model establishment (p < 0.05). However, after intervention with live and dead *Streptococcus thermophilus* subsp. CCFM1399 for 7 days, rat weight recovered significantly compared to the model group, increasing by 1.23 times and 1.17 times, respectively. On day 14 after model establishment, rat weight continued to show significant decline (p < 0.0001), while the live bacteria group showed significant weight recovery compared to the model group (increasing by 1.17 times) (p < 0.01). There was no significant difference between the OTM groups. Figure 3 B) indicates that *Streptococcus thermophilus* subsp. *salivarius* does not cause excessive tooth remodeling.

[0124] (2) Changes in periodontal tissue gingival index and probing depth

[0125] Changes in the gingival index can reflect changes in the degree of gingival inflammation. A decrease in the gingival index may indicate an improvement in symptoms such as gingival redness, swelling, and bleeding. Changes in probing depth are usually associated with improvements in periodontal tissues; shallower probing depth suggests healthy periodontal tissues and a lower risk of periodontal disease.

[0126] Changes in gingival index and periodontal probing depth are as follows: Figure 4 As shown in the figure, compared with the model group, both the live and dead *Streptococcus paracasei* subsp. *thermophilus* CCFM1399 groups showed significant decreases in gingival index and probing depth on day 7 (p<0.05). Specifically, the live bacteria group showed a 56% and 75% decrease in gingival index and probing depth, respectively, on day 7, and maintained a significant difference from the model group on day 14. The dead bacteria group showed no significant difference in probing depth compared to the model group on day 7, but all other indicators were significantly lower than the model group (p<0.05). This indicates that oral irrigation with live and dead *Lactobacillus paracasei* CCFM1399 reduced gingival inflammation, decreased periodontal pocket depth, and reduced the risk of periodontal disease in rats.

[0127] In conclusion, *Streptococcus thermophilus* subsp. CCFM1399 can reduce the incidence of gingival bleeding and gingival inflammation in rats and decrease the formation of periodontal pockets.

[0128] 2. Secretion of inflammatory factors IL-6, TNF-α, and MPO in rat gingival tissue, and expression of OPG and RANKL in serum.

[0129] The experimental period was 14 days (days 0-14). All rats were sacrificed on day 14, and serum, gingival tissue, and alveolar bone tissue were collected for subsequent experimental histopathology, alveolar bone microstructure analysis, and to detect the levels of soluble interleukin (IL-6) and tumor necrosis factor (TNF-α) in gingival tissue, myeloperoxidase (MPO), osteoprotegerin (OPG) in serum, and receptor activator of nuclear factor κB (RANKL) ligand.

[0130] Assay Method: On day 14, before sacrifice, 10 mL of blood was collected from the abdominal aorta of the rats and centrifuged at 3000 rpm for 15 min at 4°C. The supernatant was collected as the blood sample. After sacrifice, the lingual and buccal gingival tissues of the first molars were dissected. 20 mg of gingival tissue was weighed and mixed with 180 μL of PBS buffer, placed in a 1.5 mL centrifuge tube, and 3 zirconium beads were added. The mixture was then homogenized in a high-throughput tissue homogenizer. The homogenate was then centrifuged at 12000 g at 4°C for 15 min, and the supernatant was collected and stored at -20°C.

[0131] Subsequently, the levels of RANKL and OPG in serum and IL-6, TNF-α, and MPO in gingival tissue were measured according to the kit instructions.

[0132] The excessive secretion of IL-6, TNF-α, and MPO causes periodontal tissue inflammation and damages the periodontal tissue itself, accelerates the entry of pathogenic bacterial toxins, and promotes excessive bone resorption by activating multiple downstream signaling pathways, including NF-κB. OPG is a soluble protein belonging to the tumor necrosis factor receptor superfamily, while RANKL is a transmembrane protein mainly produced by osteoblasts and activated T cells. RANKL can bind to the RANK receptor on osteoclast precursor cells, promoting osteoclast differentiation and maturation and increasing bone resorption. OPG can bind to RANKL, preventing RANKL from activating its receptor RANK, thereby inhibiting osteoclast differentiation and activity and reducing bone resorption.

[0133] The secretion of inflammatory factors IL-6, TNF-α, and MPO in rat gingival tissue is as follows: Figure 5 As shown in the figure, the IL-6 level in the control group was 433.7 ng / L. After orthodontic treatment and stimulation by pathogenic bacteria, the IL-6 secretion in the model group increased to 660.17 ng / L. However, after intervention with *Streptococcus thermophilus* subsp. CCFM1399, the IL-6 secretion decreased. The IL-6 levels in the live bacteria group and the dead bacteria group were 485.16 ng / L and 529.82 ng / L, respectively, both significantly lower than those in the model group (p<0.01). Similarly, after orthodontic treatment and stimulation by pathogenic bacteria, the secretion levels of TNF-α and MPO in the model group increased significantly from 92.00 ng / L and 80.75 ng / L to 324.35 ng / L and 128.94 ng / L, respectively, compared with the control group. However, after intervention with live and dead bacteria of Streptococcus thermophilus subsp. CCFM1399, the secretion levels of TNF-α and MPO in the model group decreased significantly, with TNF-α levels dropping to around 200 ng / L (p<0.001) and MPO levels dropping to around 100 ng / L (p<0.01).

[0134] The expression levels of OPG and RANKL in rat serum are as follows: Figure 6As shown in the figure, the serum OPG level in the control group was 657.00 ng / L, while the serum OPG level in the model group significantly increased to 1130.33 ng / L. However, after intervention with live and dead *Streptococcus thermophilus* subsp. CCFM1399, the OPG level significantly decreased to 822.70 ng / L (p<0.01). The serum RANKL level in the control group was 190.58 ng / L, while the serum RANKL level in the model group significantly increased to 301.57 pmol / L. However, after intervention with live and dead *Streptococcus thermophilus* subsp. CCFM1399, the RANKL level significantly decreased to approximately 240 pmol / L (p<0.05). OPG, as a ligand receptor for RANKL, inhibits osteoclast activity upon binding.

[0135] In conclusion, *Streptococcus thermophilus* subsp. CCFM1399 can reduce periodontal inflammation and alleviate bone resorption by decreasing the secretion of inflammatory factors and MPO in the gingival tissue of rats with excessive bone resorption.

[0136] 3. Microstructural analysis of rat alveolar bone tissue

[0137] Bone microstructure analysis: At the time of rat sacrifice, the right maxilla tissue was dissected and fixed in 4% paraformaldehyde solution for 48 hours. The fixed maxilla was then washed with PBS and air-dried. The maxilla samples were scanned using a high-resolution Micro-CT imaging system. Specific parameters were: voltage, 100 kV; current, 88 μA; field of view, 18 μm; imaging rotation, 360°; acquisition time, 4 min. The ratio of bone volume (BV) to total alveolar bone volume (TV) at the model site (BV / TV) was measured and calculated.

[0138] MicroCT technology allows for direct observation of the resorption of the first molar root. The ratio of the alveolar bone volume (BV) of the first molar to the total volume (TV) can be calculated. This ratio is related to the risk of alveolar bone resorption; the lower the ratio, the higher the risk of bone resorption.

[0139] Microstructure of rat alveolar bone tissue, such as Figure 7 As shown in the figure, the BV / TV ratio at the root apex of the first molar in the control group was 43.38%, while in the model group it was significantly reduced to 24.81%. After intervention with *Streptococcus thermophilus* subsp. CCFM1399, the BV / TV ratio in the live bacteria group increased to 33.06%, while the BV / TV ratio in the dead bacteria group significantly increased to 37.60% (p<0.05).

[0140] In conclusion, *Streptococcus thermophilus* subsp. CCFM1399 can increase the bone volume (BV) to total volume (TV) ratio at the root apex of the first molar, thereby alleviating orthodontic-induced bone resorption.

[0141] 4. Histopathological analysis of rat alveolar bone tissue

[0142] Histopathological observation: Alveolar bone tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5μm thick sections. Hematoxylin and eosin (HE) staining and trap staining were performed. Alveolar bone tissue sections were observed at 20x magnification using a pathological slide scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary).

[0143] HE staining and Trap staining of rat alveolar bone tissue can effectively assess the integrity, inflammation status, and number and location of osteoclasts per unit area of ​​alveolar bone tissue in different groups of rats.

[0144] HE staining can reveal the acuteness of inflammation and also observe changes in tissue morphology. For example... Figure 8 As shown, the control group rats exhibited intact alveolar bone and periodontal tissue structure. After HE staining, their maxillary bone sections revealed a complete periodontal tissue structure, with the gingiva tightly adhering to the tooth surface, no periodontal pockets, and no inflammatory cell aggregation. The periodontal tissue and alveolar bone structures were homogeneous and showed no abnormalities. In contrast, maxillary bone sections from the model group rats showed impaired periodontal tissue integrity. Gaps appeared between the gingiva and the tooth surface, forming periodontal pockets. Numerous inflammatory cells were observed to accumulate in the apical region, the gap between the first and second molars widened, and the periodontal periapical tissue showed uneven thickness, indicating that the orthodontic model induced an inflammatory response and bone resorption in the periodontal tissues.

[0145] HE staining results of live and dead *Streptococcus salivarius* subsp. *thermophilus* CCFM1399 showed that intervention with this bacterium alleviated inflammatory infiltration, reduced inflammatory cell aggregation at the alveolar bone apex, and reduced periodontal tissue damage. This indicates that *Streptococcus salivarius* subsp. *thermophilus* CCFM1399 can significantly alleviate alveolar bone resorption symptoms and inhibit inflammation in orthodontic model rats.

[0146] Trap staining can effectively assess the integrity, inflammation status, and number and location of osteoclasts per unit area in the alveolar bone tissue of rats in different groups. Figure 9 As shown, the number of osteoclasts in the model group was significantly increased compared to the control group (p < 0.01), mainly distributed at the periapical bifurcation edge of the alveolar bone. Staining results of live and dead *Streptococcus thermophilus* subsp. CCFM1399 showed that this bacterium could alleviate orthodontic-induced excessive bone resorption, and osteoclasts no longer accumulated at the periapical bifurcation of the alveolar bone. Specifically, under 20x magnification, the number of osteoclasts in the dead bacterial group decreased from 10 to 4.5 compared to the model group, a significant difference (p < 0.05).

[0147] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A Streptococcus salivarius subsp. thermophilus (S. thermophilus) strain CCFM1399, characterized in that, Streptococcus thermophilus The Streptococcus thermophilus CCFM1399 has been deposited with the Guangdong Microbial Culture Collection Center on June 14, 2024, and the deposit number is GDMCC No: 64763. ​ 2. A microbial preparation containing the Streptococcus thermophilus CCFM1399 of claim 1.

3. The microbial preparation of claim 2, characterized in that, The number of bacteria of Streptococcus thermophilus CCFM1399 in the microbial preparation is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

4. The probiotic prepared from Streptococcus thermophilus CCFM 1399 according to claim 1, characterized in that, The postbiotic contains dead cells, lysate and / or supernatant of the Streptococcus thermophilus CCFM1399.

5. A method for preparing a S. thermophilus CCFM1399 postbiotic, characterized in that, comprising (a), (b) or (c): (a) culturing the Streptococcus thermophilus CCFM1399 of claim 1 to the logarithmic growth phase, collecting cells after heat treatment to obtain a postbiotic; (b) culturing the Streptococcus thermophilus CCFM1399 to the logarithmic growth phase, obtaining a postbiotic after inactivation by heat treatment and high-pressure homogenization; (c) culturing the Streptococcus thermophilus CCFM1399 to the logarithmic growth phase, collecting fermentation supernatant to obtain a postbiotic.

6. Product containing the Streptococcus thermophilus subsp. CCFM1399 and / or its postbiotic of claim 1, characterized in that, The product is a food, health product, pharmaceutical product or daily chemical product.

7. The product of claim 6, wherein, The food includes fruit and vegetable products, bean products or dairy products; the daily chemical product includes mouthwash, mouthwash granules, toothpaste, tooth powder, toothpaste, periodontal jelly, tooth gel or tooth wax; the dosage form of the pharmaceutical product includes oral spray, tablet, capsule, paste, oral liquid or lyophilized powder.

8. Use of S. thermophilus CCFM1399, and / or of a microbial preparation thereof, and / or of a postbiotic thereof, according to claim 1, for the preparation of a medicinal product for the prevention and / or treatment of oral inflammation, or for the preparation of a medicinal product for the prevention and / or treatment of bone resorption, characterized in that, The oral inflammation includes periodontitis or gingivitis.

9. Use of Streptococcus thermophilus CCFM1399, and / or of a microbial preparation thereof, and / or of a postbiotic thereof, according to claim 1, for the preparation of a care product for the prevention and / or treatment of oral inflammation, characterized in that, The oral inflammation includes periodontitis or gingivitis, and the care product includes mouthwash, mouthwash granules, toothpaste, tooth powder, toothpaste, periodontal jelly, tooth gel or tooth wax.

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