Streptococcus salivarius CCFM1399 for relieving bone resorption and periodontal tissue injury and metaplasm of streptococcus salivarius CCFM1399

Abstract

The invention discloses streptococcus salivarius CCFM1399 for relieving bone resorption and periodontium injury and a metagen thereof, and belongs to the technical field of microorganisms. The streptococcus salivarius subsp. Thermophilus CCFM1399 provided by the invention is separated from oral dental plaque of healthy teenagers, has the effects of relieving bone resorption and resisting inflammation, and is specifically reflected in that expression of protein level and gene level of MC3T3-E1 cell osteoprotegerin (OPG) is improved in vitro; the bone absorption amount of orthodontic bone absorption rat alveolar bone is reduced, the number of alveolar bone osteoclasts is reduced, and the level of OPG in rat serum is improved; the secretion of inflammatory factors IL-6, TNF-alpha and MPO of the orthodontic bone absorption rat is reduced; and the pathological condition of orthodontic bone absorption rat alveolar bone tissue is improved. Therefore, the streptococcus salivarius subsp. Thermophilus has a huge application prospect in products with effects of relieving bone resorption and assisting in inhibiting periodontal tissue inflammation.

Description

Technical Field

[0001] The invention relates to a strain of Streptococcus salivarius CCFM1399 capable of alleviating bone resorption and periodontal tissue damage and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] In the skeletal system of a healthy body, osteoblasts and their bone-forming function and osteoclasts and their bone-resorbing function usually maintain a dynamic balance. However, periodontal diseases such as periodontitis, gingivitis, and peri-implantitis, as well as excessive orthodontic force, may induce excessive bone resorption. Excessive oral bone resorption can cause oral health problems such as alveolar bone loss, loose and falling teeth, worsening periodontal disease, tooth displacement, and changes in occlusal relationships, which require timely prevention and treatment.

[0003] The OPG / RANKL / RANK signaling pathway plays an important role in maintaining bone resorption and bone formation homeostasis. Osteoprotegerin (OPG) is secreted by osteoblasts and can compete with RANKL to bind to the RANK receptor 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 regulating this pathway. In orthodontic treatment, by regulating this pathway, the metabolism of the alveolar bone can be affected, thereby affecting the speed and effect of tooth movement. Increasing the level of OPG or inhibiting the activity of RANKL can reduce bone resorption and help stabilize teeth, thereby achieving alveolar bone homeostasis in orthodontic patients.

[0004] At present, there are many patents for alleviating excessive alveolar bone resorption, which usually focus on the use of bone resorption inhibitors and osteoclast agonists to regulate osteoclast differentiation. Patent CN116196424B reduces the activity of deubiquitinase through protein transport inhibitors to achieve bone resorption or bone loss. Patent CN115770297A activates the Wnt signal of osteocytes through TGFβ or Wnt signal agonists to promote the increase of its own TGFβ signal, activate the activity of RANKL promoter, and then promote osteoclast formation, providing a new research direction for the prevention and treatment of osteoporosis. However, for oral mucosal administration, the stability, absorption efficiency and possible 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 a specially designed drug delivery system to ensure the effectiveness and safety of the drug. Related agonists also need to comprehensively evaluate their mechanism of action under excessive bone resorption, potential adverse reactions and effects on tumor risk. No safe, effective and side-effect-free method for treating orthodontically induced bone resorption has yet to be found.

[0005] Probiotics that can be used in food have broad application prospects as a safe product with no side effects. However, no edible probiotics that can effectively treat orthodontic-induced bone resorption or relieve oral inflammation have been found. Summary of the invention

[0006] At present, no treatment method that can effectively alleviate orthodontic-induced bone resorption has been found in clinical practice.

[0007] The present invention provides a new method for treating orthodontically induced excessive bone resorption using Streptococcus thermophilus CCFM1399, which has a mild effect and few side effects. The Streptococcus thermophilus of the present invention can regulate osteoblasts to secrete osteoprotegerin (OPG) and increase the gene expression of bone morphogenetic protein (BMP). In a rat oral orthodontic model, the strain of the present invention can effectively alleviate bone resorption, inhibit the level of inflammatory factors in gingival tissue, and protect the stability of periodontal tissue.

[0008] The present invention provides a thermophilic subspecies of Streptococcus salivarius and its postbiotics that can alleviate bone resorption and periodontal tissue damage. The thermophilic subspecies of Streptococcus salivarius (Streptococcus thermophilus) CCFM1399 is taxonomically named Streptococcus thermophilus, and has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with a deposit number of GDMCC No: 64763, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] In one embodiment of the present invention, the thermophilic Streptococcus salivarius subspecies (Streptococcusthermophilus) CCFM1399 is derived from dental plaque samples of healthy volunteers in Wuxi, Jiangsu Province. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID No. 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in NCBI, and the result shows that the strain is thermophilic Streptococcus salivarius subspecies (Streptococcusthermophilus) CCFM1399.

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

[0011] The invention also provides a microbial preparation containing the thermophilic Streptococcus salivarius subspecies CCFM1399.

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

[0013] The present invention also provides a postbiotic prepared by the thermophilic Streptococcus salivarius subspecies CCFM1399, wherein the postbiotic contains dead cells, lysate and / or supernatant of the thermophilic Streptococcus salivarius subspecies CCFM1399.

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

[0015] (a) culturing the thermophilic Streptococcus salivarius subspecies CCFM1399 to a logarithmic growth phase, collecting the cells after heat treatment, and obtaining postbiotics;

[0016] (b) culturing Streptococcus salivarius thermophilus subspecies CCFM1399 to the logarithmic growth phase, inactivating it by heat treatment, and then crushing it by high-pressure homogenization to obtain postbiotics;

[0017] (c) Cultivating Streptococcus salivarius thermophilus subspecies CCFM1399 to the logarithmic growth phase, collecting the fermentation supernatant, and obtaining postbiotics.

[0018] The present invention also provides a product containing the thermophilic Streptococcus salivarius subspecies CCFM1399 and / or its postbiotics, and the product is food, health care product, medicine or daily chemical product.

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

[0020] In one embodiment of the present invention, the daily chemical product includes mouthwash, mouthwash granules, toothpaste, tooth cleaning powder, tooth paste, periodontal gel, tooth gel or tooth wax.

[0021] In one embodiment of the present invention, the dosage form of the drug includes oral spray, tablet, capsule, paste, oral solution or lyophilized powder.

[0022] In one embodiment of the present invention, the ingredients of the drug include 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, a wetting agent, a disintegrant, a binder, a lubricant or a flavoring agent.

[0025] The present invention also provides the use of the thermophilic Streptococcus salivarius subspecies CCFM1395, and / or the microbial preparation, and / or the postbiotic in producing osteoprotegerin.

[0026] The present invention also provides the use of the thermophilic Streptococcus salivarius subspecies CCFM1399, and / or the microbial preparation, and / or the postbiotic in preparing a drug for regulating oral inflammation function, wherein the oral inflammation includes periodontitis or gingivitis.

[0027] The present invention also provides the use of the thermophilic Streptococcus salivarius subspecies CCFM1399, or the microbial preparation, or the postbiotic in preparing medicines for preventing and / or treating bone resorption.

[0028] The present invention also provides the use of the thermophilic subspecies CCFM1399 of Streptococcus salivarius, and / or the microbial preparation, and / or the postbiotic in the preparation of care products for preventing and / or treating oral inflammation, wherein the oral inflammation includes periodontitis or gingivitis, and the care products include mouthwash, mouthwash granules, toothpaste, tooth cleaning powder, tooth stickers, periodontal gel, tooth gel or tooth wax.

[0029] Beneficial Effects

[0030] 1. The present invention provides a strain of Streptococcus thermophilus CCFM1399, which is isolated from dental plaque in the oral cavity of healthy teenagers. The cell lysate, living cells and dead cells of the Streptococcus thermophilus CCFM1399 have the effects of alleviating orthodontic-induced bone resorption and inhibiting periodontal inflammation, which is specifically embodied in:

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

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

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

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

[0035] (5) Reduce the number of osteoclasts at the apical 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 CCFM1399 has great application prospects in the preparation of products (such as medicines or daily chemical products, etc.) for preventing and / or treating bone resorption.

[0040] 2. Streptococcus thermophilus is a kind of probiotics and has been included in the "List of Bacteria that Can Be Used in Food" issued by the Ministry of Health. Therefore, the Streptococcus thermophilus CCFM1399 screened by the present invention will not cause any side effects to the human body, and is highly safe when used in products (such as medicines or daily chemical products, etc.) for preventing and / or treating orthodontic-induced bone resorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 :The effect of different bacterial fermentation supernatants and lysates on osteoprotegerin (OPG) in MC3T3-E1 cells;

[0042] Figure 2 :The effect of Streptococcus salivarius thermophilus subspecies CCFM1399 on the expression of bone morphogenetic protein (BMP) gene in osteoblasts;

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

[0044] Figure 4 :Effects of Streptococcus salivarius thermophilus subspecies CCFM1399 on gingival index (A) and probing depth (B) of rats with orthodontic-induced bone resorption;

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

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

[0047] Figure 7 :The effect of Streptococcus salivarius thermophilus subspecies CCFM1399 on bone microstructure in rats with orthodontic-induced bone resorption (A) and the ratio of bone volume to total volume (B);

[0048] Figure 8 :HE staining of Streptococcus salivarius thermophilus subspecies CCFM1399 on orthodontic-induced bone resorption in rats;

[0049] Fig. 9 :Trap staining of Streptococcus salivarius thermophilus subspecies CCFM1399 on orthodontic-induced bone resorption in rats (A) and analysis of osteoclast numbers (B);

[0050] (In the figure, * indicates significant difference, among which *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).

[0051] Biomaterial Deposit

[0052] A strain of Streptococcus thermophilus CCFM1399, taxonomically named Streptococcus thermophilus, was deposited in Guangdong Provincial Microbiological Culture Collection on June 14, 2024, with the deposit number GDMCC No: 64763, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0053] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

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

[0055] The complete cell culture medium in Examples 1 and 2 is: 92% (v / v) α-MEM culture medium + 7% (v / v) fetal bovine serum + 1% (v / v) 100× mixed solution of penicillin and streptomycin (the content of penicillin in the mixed solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL).

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

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

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

[0059] Streptococcus salivarius thermophilus subsp. CCFM1399 was obtained from the strain bank of the Biotechnology Center of Jiangnan University, and Prevotella intermedia ATCC 25611 and Streptococcus salivarius thermophilus subsp. ATCC19258 were purchased from the American Type Culture Collection.

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

[0061] SPF Wistar rats, female, 7 weeks old, weighing 235-255 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

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

[0063] In Examples 1, 2, and 3, the live lactobacillus suspension was prepared by inoculating Streptococcus salivarius thermophilus subspecies CCFM1399 and ATCC19258 in MRS liquid culture medium at a 2% (v / v) inoculation amount, culturing at 37°C for 24 h, and adjusting the bacterial concentration to 1×10 9 CFU / mL.

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

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

[0066] In Example 4, the live bacterial suspension of thermophilic Streptococcus salivarius was prepared by inoculating thermophilic Streptococcus salivarius CCFM1399 and ATCC19258 in MRS liquid culture medium at a 2% (v / v) inoculation amount, culturing at 37°C for 24 hours, collecting the bacterial cells by centrifugation at 8500g for 20 minutes, washing the bacterial cells twice with sterile saline, resuspending the bacterial cells with a 10% (v / v) skim milk powder aqueous solution, mixing well, and then freeze-drying. Before the animal experiment, the freeze-dried bacterial powder of thermophilic Streptococcus salivarius was re-dissolved with PBS to a bacterial suspension concentration of 1×10 9 CFU / mL.

[0067] Preparation of dead bacterial suspension of thermophilic subspecies of Streptococcus salivarius: The preparation method and concentration of dead bacterial suspension are the same as those of live bacteria. The difference is that a heat treatment step is added, and the fermentation liquid after 24 hours of inactivation culture is inactivated at 105°C for 10 minutes, and the inactivation effect is checked by plate coating. After heat treatment, the bacterial liquid is centrifuged at 8500g for 20 minutes, and the bacteria are washed twice with sterile saline, and then resuspended with 10% skim milk solution. After thorough mixing, freeze-drying is performed, and resuspended with PBS to a concentration of 1×10 9 CFU / mL.

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

[0069] Example 1 Isolation and Identification of Thermococcus salivarius Subspecies CCFM1399

[0070] Collect oral swab samples from healthy adolescents and place them in EP tubes containing 1 mL of sterile saline. Pipette 0.2 mL into 1.8 mL of sterile saline to obtain 10 -1 Dilution, then draw 0.5mL 10 -1 Dilute in 4.5 mL of saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution. Take 10 -4 , 10 -5 , 10 -6 Place 1 mL of each dilution in a plate, pour into MRS solid culture medium, mix gently, and after the culture medium solidifies, invert and culture at 37°C for 48 h.

[0071] Select colonies of different morphologies for streaking purification on MRS plates, pick and inoculate purified single colonies into 10 mL of liquid culture medium, and culture at 37 ° C for 48 h. Take 1.5 mL of cultured bacterial solution, centrifuge at 6000r / min for 3 min, discard the supernatant, add 1.5 mL of sterile water to wash 3 times, resuspend in 1.5 mL of sterile water, and use it as a template for bacterial identification. Set up a PCR system with a volume of 20 μL, add 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

[0073]

[0074] The 16S rDNA sequence information obtained by sequencing and amplification 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 confirmed by NCBI's BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST). The closer the Query Cover and Identification are to 100%, the more feasible it is. If there are multiple species in the comparison results, the Complete genome species will be prioritized while taking into account the numerical value. According to the Blast results, the bacteria were identified as Streptococcus salivarius thermophilus subspecies. For the correctly identified species, 1.5 mL of bacterial solution was taken into a 2 mL strain preservation tube, centrifuged at 6000 r / min for 3 minutes, the supernatant was removed in the clean bench, 1 mL of 30% sterile glycerol was added, and after being thoroughly mixed with a vortex oscillator, it was stored in a -80 ° C refrigerator. The obtained strain was named Streptococcus salivarius thermophilus subspecies CCFM1399, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No: 64763.

[0100] Example 2 Ability of Streptococcus salivarius subsp. thermophilus to promote osteoblast osteoprotegerin (OPG) expression in vitro

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

[0102] MC3T3-E1 cells were removed from liquid nitrogen and thawed quickly 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, and the culture medium was replaced every 48 hours. After counting the cells, 1×10 4 Cells were seeded on a 6-well plate at a density of 10 cells / well. 2 mL of osteogenic induction medium containing 5% (v / v) bacterial supernatant or lysate was added to each well. Cell morphology was observed regularly and the medium was replaced every 48 hours. MC3T3-E1 cells differentiated into osteoblasts usually become flatter. After 21 days of culture, the cell culture supernatant of each group was collected and the expression of OPG was determined using the Senbega OPG Elisa Kit. The grouping information of the cell experiment is shown in Table 2.

[0103] Table 2: Group information table

[0104]

[0105] The expression of OPG in MC3T3-E1 cells after intervention in each group Figure 1 As shown. The OPG concentration in the cell culture supernatant of the control group was 117.78 ng / L. After being treated with the supernatant of S. salivarius CCFM1399, the supernatant of S. salivarius ATCC19258 and the lysate, the expression of OPG in the cells was not significantly different from that in the control group (p>0.05). However, the OPG content in the lysate group of S. salivarius CCFM1399 was as high as 375.05 ng / L, which was significantly higher than that in the control group (p<0.0001). The above results indicate that the bacterial lysate of S. salivarius CCFM1399 can significantly increase the expression of OPG in the culture supernatant of MC3T3-E1 cells after induction.

[0106] In summary, Streptococcus salivarius thermophilus subspecies CCFM1399 can increase the expression of OPG in osteoblasts in vitro.

[0107] Example 3 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, which can produce factors that inhibit osteoclast activity, such as OPG.

[0109] The MC3T3-E1 cell culture and induction test methods were the same as in Example 2. 4 After 21 days of differentiation induction with an initial concentration of 100 / mL, cellular RNA was extracted according to the detailed instructions provided by the Novozyme RNA extraction kit, and the operation was performed step by step. All experimental steps were performed in an RNase-free environment to avoid RNA degradation. The extracted RNA was measured for purity (A260 / A280 ratio) and concentration using a NanoDrop spectrophotometer.

[0110] The expression of BMP gene in MC3T3-E1 cells before and after intervention Figure 2 After intervention with the thermophilic Streptococcus salivarius subsp. CCFM1399 lysate, 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 salivarius thermophilus subspecies CCFM1399 can increase the expression of BMP genes in osteoblasts in vitro.

[0112] Example 4 Application of Streptococcus salivarius thermophilus subspecies CCFM1399 in alleviating orthodontic-induced bone resorption in rats

[0113] Twenty-four SPF Wistar rats, male, 7 weeks old, weighing 235-255 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

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

[0115] Table 3: Animal experimental plan and grouping

[0116]

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

[0118] Except for the control group, the other groups underwent orthodontic modeling on day 0 (the specific operation was: a nickel-titanium spring was used to apply a 50g traction force between the first molar and the central incisor of the rat. This step required first using a grinding tool to grind a groove perpendicular to the midline of the dental arch in the maxillary central incisor for fixing the stainless steel ligature. The maxillary central incisor and the first molar both needed to be fixed with fluid resin to tie the silk thread, and to ensure that the silk thread fracture did not damage the epithelial barrier. Except for the left first molar, the occlusal surfaces of the remaining upper molars were fixed with fluid resin to make the occlusal surfaces convex to eliminate the occlusal interference of the maxillary first molar in orthodontic movement), and were required to be infected with Prevotella intermedia for a total of 12 days (days 1-6 and 8-13) (the specific operation method of infection was: 1×10 9 1 mL of Prevotella intermedia suspension with a CFU / mL was slowly irrigated into the rat's oral cavity and placed in the cage after leaving it for 1 minute).

[0119] For the live and dead bacteria groups of S. salivarius thermophilus subspecies CCFM1399, 6 hours after the intervention of Prevotella intermedia, the rats' oral cavity was irrigated with 1 mL of live and dead bacterial suspensions of S. salivarius thermophilus subspecies CCFM1399, respectively, with a bacterial suspension concentration of 1×10 9 CFU / mL.

[0120] 1. Weight of rats, distance between first molar and central incisor (OTM), gingival index and probing depth

[0121] The gingival index and probing depth of the periodontal tissue of all rats were measured on the 7th and 14th days, and the rat saliva was collected with a sterile swab and stored in a 2mL centrifuge tube containing 1mL of 30% glycerol. The rat body weight, the 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 A, rats showed significant weight loss on the 7th day after orthodontic modeling (p < 0.05), and the weight of rats recovered significantly after 7 days of intervention with live and dead bacteria of Streptococcus salivarius thermophilus subspecies CCFM1399 compared with the model group, increasing by 1.23 times and 1.17 times, respectively, compared with the model group. On the 14th day after modeling, the weight of rats still showed a significant decrease (p < 0.0001), while the weight of the live bacteria group recovered significantly (increased by 1.17 times) compared with the model group (p < 0.01). There was no significant difference between the OTM groups ( Figure 3 B), indicating that S. salivarius subsp. thermophilus does not cause excessive tooth movement and 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. If the gingival index decreases, it may mean that symptoms such as gingival redness, swelling, and bleeding have improved. Changes in probing depth are usually associated with improvements in periodontal tissues. Shallow probing depths indicate healthy periodontal tissues and a lower risk of periodontal disease.

[0126] The changes of gingival index and periodontal probing depth are as follows Figure 4 As shown. Compared with the model group, the gingival index and probing depth of the live and dead bacteria groups of Streptococcus salivarius thermophilus subspecies CCFM1399 decreased significantly on the 7th day (p<0.05). The gingival index and probing depth of the rats in the live bacteria group decreased by 56% and 75% on the 7th day, respectively, and remained significantly different from the model group on the 14th day. Except for the probing depth on the 7th day, the other indicators of the dead bacteria group were significantly lower than those of the model group (p<0.05). This shows that after oral irrigation with live and dead bacteria of Lactobacillus paracasei CCFM1399, the gingival inflammation level of rats was reduced, the depth of oral periodontal pockets was reduced, and the risk of periodontal disease was reduced.

[0127] In conclusion, S. salivarius thermophilus subspecies CCFM1399 can reduce the incidence of gingival bleeding and gingival inflammation in rats and reduce 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 to 14). All rats were killed on the 14th day, and serum, gingival tissue and alveolar bone tissue were collected for subsequent experimental histopathology and alveolar bone microstructure analysis, and the levels of soluble interleukin (IL-6) and tumor necrosis factor (TNF-α) in gingival tissue, myeloperoxidase (MPO), and serum osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) were detected.

[0130] Determination method: On the 14th day, before the rats were killed, 10 mL of blood was taken from the abdominal aorta of the rats, centrifuged at 3000 r / min for 15 min at 4°C, and the supernatant was taken as the blood sample. After the rats were killed, the lingual and buccal gingival tissues of the first molars were peeled off. 20 mg of gingival tissue was weighed and mixed with 180 μL PBS buffer, placed in a 1.5 mL centrifuge tube, 3 grinding zirconium beads were added, and ground in a high-throughput tissue grinder. The tissue homogenate was then centrifuged at 12000g at 4°C for 15 min, the supernatant was collected, and stored at -20°C.

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

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

[0133] The secretion of inflammatory factors IL-6, TNF-α, and MPO in rat gingival tissue Figure 5 The IL-6 level in the control group was 433.7 ng / L. After orthodontic and pathogenic bacteria stimulation, the IL-6 secretion in the model group increased to 660.17 ng / L. After the intervention of Streptococcus salivarius thermophilus subspecies CCFM1399, the secretion of IL-6 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, which were significantly lower than those in the model group (p<0.01). Similarly, after orthodontic and pathogenic bacteria stimulation, the secretions of TNF-α and MPO in the model group were significantly increased 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 salivarius subspecies CCFM1399, the secretions of TNF-α and MPO in the model group were significantly decreased, with the TNF-α level dropping to about 200 ng / L (p<0.001), and the MPO level dropping to about 100 ng / L (p<0.01).

[0134] The expression of OPG and RANKL in rat serum Figure 6As shown. The OPG level in the serum of the control group was 657.00 ng / L, and the OPG level in the serum of the model group increased significantly to 1130.33 ng / L. After intervention with dead bacteria of Streptococcus salivarius CCFM1399, it decreased significantly to 822.70 ng / L (p<0.01). The RANKL level in the serum of the control group was 190.58 ng / L, and the RANKL level in the serum of the model group increased significantly to 301.57 pmol / L. After intervention with live and dead bacteria of Streptococcus salivarius CCFM1399, it decreased significantly to about 240 pmol / L (p<0.05). OPG, as a ligand receptor of RANKL, inhibits the activity of osteoclasts after binding.

[0135] In summary, S. salivarius thermophilus subspecies CCFM1399 can reduce periodontal tissue inflammation and alleviate bone resorption by reducing 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: When the rats were killed, the right maxillary tissue of the rats was peeled off and fixed in 4% paraformaldehyde solution for 48 hours. The fixed maxilla of the rats was washed with PBS and dried. The maxillary samples were scanned using a high-resolution Micro-CT imaging system. The specific parameters were: voltage, 100 kV; current, 88 μA; field of view, 18 μm, imaging rotation 360°; acquisition time, 4 min. The ratio of the bone volume (BV) at the modeling site to the total alveolar bone volume (TV) (BV / TV) was measured and calculated.

[0138] The root resorption of the first molar can be observed intuitively through microCT technology, and the ratio of the alveolar bone volume (BV) to the total volume (TV) of the first molar can be obtained by calculation. This ratio is related to the risk of alveolar bone resorption. The lower the ratio, the higher the risk of bone resorption.

[0139] The microstructure of rat alveolar bone tissue Figure 7 As shown. The BV / TV ratio at the root apex of the first molar in the control group was 43.38%, and the BV / TV ratio at the root apex of the first molar in the model group was significantly reduced to 24.81%. After intervention with the thermophilic subspecies of Streptococcus salivarius CCFM1399, the BV / TV ratio in the live bacteria group increased to 33.06%, while the BV / TV ratio in the dead bacteria group increased significantly to 37.60% (p<0.05).

[0140] In conclusion, S. salivarius thermophilus subspecies CCFM1399 can increase the BV / TV ratio at the root apex of the first molar in the alveolar bone, thereby alleviating orthodontic-induced bone resorption.

[0141] 4. Histopathological analysis of rat alveolar bone

[0142] Histopathological observation: Alveolar bone tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sliced ​​into 5 μm thick slices, and stained with hematoxylin and eosin (HE) and Trap. Alveolar bone tissue slice samples were observed under a pathological slice scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

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

[0144] HE staining can determine the acuteness of inflammation and also observe changes in tissue morphology. Figure 8 As shown in the figure, the alveolar periodontal tissue structure of rats in the control group was intact. After HE staining, the maxillary bone sections showed a complete periodontal tissue structure, the gingiva was tightly attached to the tooth surface, no periodontal pocket was formed, and no inflammatory cells were observed to gather. The structure of the periodontal tissue and alveolar bone was uniform, and no abnormalities were found. The maxillary bone sections of rats in the model group showed that the integrity of the periodontal tissue was impaired. Gaps appeared between the gingiva and the tooth surface, forming periodontal pockets. A large number of inflammatory cells were observed to gather in the apical area, the gap between the first molar and the second molar increased, and the thickness of the periodontal apical tissue was uneven, indicating that the orthodontic model triggered an inflammatory response in the periodontal tissue and bone resorption.

[0145] The HE staining results of live and dead bacteria groups of Streptococcus salivarius thermophilus subspecies CCFM1399 showed that under the intervention of the bacteria, inflammatory infiltration was alleviated, inflammatory cells no longer gathered at the alveolar bone root apex, and periodontal tissue damage was also alleviated. This indicates that Streptococcus salivarius thermophilus subspecies CCFM1399 can significantly alleviate the symptoms of alveolar bone resorption in orthodontic model rats and inhibit the occurrence of inflammation.

[0146] Trap staining can effectively evaluate the integrity of alveolar bone tissue, inflammation, and the number and location of osteoclasts per unit area in rats of each group. Fig. 9 As shown in the figure, the number of osteoclasts in the model group increased significantly compared with the control group (p < 0.01), and was mainly distributed at the edge of the alveolar root apex bifurcation. The staining results of the live and dead bacteria groups of Streptococcus salivarius thermophilus subspecies CCFM1399 showed that the bacteria could alleviate orthodontic-induced excessive bone resorption, and osteoclasts no longer gathered at the alveolar root apex bifurcation. Among them, the number of osteoclasts in the dead bacteria group decreased from 10 to 4.5 compared with the model group under a 20x field of view, and the results were significantly different (p < 0.05).

[0147] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Streptococcus thermophilus CCFM1399, characterized in that: The thermophilic subspecies of Streptococcus salivarius CCFM1399 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No: 64763.

2. A microbial preparation containing the thermophilic Streptococcus salivarius subspecies CCFM1399 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The number of Streptococcus salivarius thermophilic subspecies CCFM1399 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. The postbiotic prepared by Streptococcus salivarius thermophilus subspecies CCFM1399 according to claim 1, characterized in that: The postbiotics contain dead cells, lysate and / or supernatant of the thermophilic Streptococcus salivarius subspecies CCFM1399.

5. A method for preparing postbiotics of Streptococcus salivarius thermophilus subspecies CCFM1399, characterized in that: Includes (a), (b) or (c): (a) culturing the thermophilic Streptococcus salivarius subspecies CCFM1399 described in claim 1 to a logarithmic growth phase, collecting the cells after heat treatment, and obtaining postbiotics; (b) culturing Streptococcus salivarius thermophilus subspecies CCFM1399 to the logarithmic growth phase, inactivating it by heat treatment, and then crushing it by high-pressure homogenization to obtain postbiotics; (c) Cultivating Streptococcus salivarius thermophilus subspecies CCFM1399 to the logarithmic growth phase, collecting the fermentation supernatant, and obtaining postbiotics.

6. A product containing the thermophilic Streptococcus salivarius subspecies CCFM1399 and / or its postbiotics according to claim 1, characterized in that: The products are food, health products, medicines or daily chemical products.

7. The product according to claim 6, characterized in that The food includes fruit and vegetable products, bean products or dairy products; the daily chemical products include mouthwash, mouthwash grains, toothpaste, tooth cleaning powder, tooth stickers, periodontal gel, tooth gel or tooth wax; the dosage form of the medicine includes oral spray, tablets, capsules, paste, oral liquid or freeze-dried powder.

8. Use of the thermophilic Streptococcus salivarius subspecies CCFM1399 according to claim 1, and / or its microbial preparation, and / or its postbiotics in the production of osteoprotegerin.

9. Use of the thermophilic Streptococcus salivarius subspecies CCFM1399 according to claim 1, and / or its microbial preparation, and / or its postbiotics in the preparation of a drug for preventing and / or treating oral inflammation, or in the preparation of a drug for preventing and / or treating bone resorption, characterized in that: The oral inflammation includes periodontitis or gingivitis.

10. Use of the thermophilic Streptococcus salivarius subspecies CCFM1399, and / or its microbial preparation, and / or its postbiotics as claimed in claim 1 in the preparation of care products for preventing and / or treating oral inflammation, characterized in that: The oral inflammation includes periodontitis or gingivitis, and the care products include mouthwash, mouthwash particles, toothpaste, tooth cleaning powder, tooth stickers, periodontal gel, tooth gel or tooth wax.

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