Lactobacillus salivarius combination CCFM1398 for regulating and controlling bone homeostasis and relieving bone resorption and metagen of lactobacillus salivarius combination CCFM1398

Abstract

The invention discloses combined lactobacillus salivarius CCFM1398 for regulating and controlling bone homeostasis and relieving bone resorption and a metagen of the combined lactobacillus salivarius CCFM1398, and belongs to the technical field of microorganisms. The combined lactobacillus salivarius CCFM1398 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 rat serum OPG is improved; the secretion of inflammatory factors TNF-alpha, IL-6 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 combined lactobacillus salivarius has a huge application prospect in products for relieving bone resorption and assisting in inhibiting periodontal tissue inflammation.

Description

Technical Field

[0001] The invention relates to a saliva-associated lactobacillus CCFM1398 for regulating bone homeostasis and relieving bone absorption 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. At present, no simple, effective and side-effect-free method for alleviating orthodontic-induced bone resorption has been found, and there is a lack of probiotic products that can alleviate orthodontic-induced bone resorption or relieve oral inflammation. Summary of the invention

[0005] At present, no treatment method that can effectively alleviate orthodontic-induced bone resorption has been found in clinical practice. Compared with the prior art, the present invention provides a new method for treating orthodontically induced excessive bone resorption using saliva-combined lactobacillus (Ligilactobacillus salivarius) CCFM1398. The saliva-combined lactobacillus of the present invention can regulate osteoblasts to secrete osteoprotegerin (OPG) and increase the gene expression of osteoprotegerin (OPG). In a rat oral orthodontic model, the strain of the present invention can effectively slow down resorption, inhibit the level of inflammatory factors in gingival tissue, and protect the stability of periodontal tissue.

[0006] The present invention provides a saliva-associated lactobacillus and its postbiotics that relieve bone resorption and have anti-inflammatory effects. The taxonomic name of the saliva-associated lactobacillus (Ligilactobacillus salivarius) CCFM1398 is Ligilactobacillus salivarius, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with a deposit number of GDMCCNo: 64762, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The saliva-associated Lactobacillus (Ligilactobacillus salivarius) CCFM1398 is derived from oral 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 saliva-associated Lactobacillus, and is named saliva-associated Lactobacillus (Ligilactobacillus salivarius) CCFM1398.

[0008] The colonies of the Ligilactobacillus salivarius CCFM1398 on the MRS culture medium are small semi-transparent white circles.

[0009] The invention also provides a microbial preparation containing the saliva-combined lactobacillus CCFM1398.

[0010] In one embodiment of the present invention, in the microbial preparation, the number of cells of the saliva-combined Lactobacillus CCFM1398 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0011] The present invention also provides a postbiotic prepared by the saliva combined with Lactobacillus CCFM1398, wherein the postbiotic comprises dead cells, lysate and / or supernatant of the saliva combined with Lactobacillus CCFM1398.

[0012] The present invention also provides a product containing the saliva-combined Lactobacillus CCFM1398, and / or the microbial preparation and / or its postbiotics.

[0013] In one embodiment of the present invention, the product includes food, medicine, health care product or daily chemical product.

[0014] In one embodiment of the present invention, the food comprises fruit and vegetable products, bean products or dairy products.

[0015] In one embodiment of the present invention, the medicine further contains conventional pharmaceutical excipients or pharmaceutical carriers.

[0016] In one embodiment of the present invention, the carrier is a pharmaceutically acceptable carrier.

[0017] 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.

[0018] 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.

[0019] The present invention also provides a method for preparing a product for preventing and / or treating orthodontic-induced bone resorption, the method comprising using the saliva in combination with Lactobacillus CCFM1398.

[0020] The present invention also provides a method for producing osteoprotegerin, wherein the lysate and / or fermentation metabolites of the saliva combined with Lactobacillus CCFM1398 are added to a culture system containing osteoblasts for culture.

[0021] The present invention also provides the use of the saliva combined with Lactobacillus CCFM1398, and / or the microbial preparation and / or the postbiotic in preparing medicines for preventing and / or treating oral inflammation, or in preparing medicines for preventing and / or treating bone resorption.

[0022] In one embodiment of the present invention, the dosage form of the drug includes oral spray, tablet, capsule, paste, oral solution or lyophilized powder.

[0023] The present invention also provides the use of the saliva combined with Lactobacillus CCFM1398, and / or the microbial preparation and / or the postbiotic in preparing care products for preventing and / or treating oral inflammation, wherein the oral inflammation includes periodontitis or gingivitis.

[0024] In one embodiment of the present invention, the care products include mouthwash, mouthwash granules, toothpaste, tooth cleaning powder, tooth stickers, periodontal gel, tooth gel or tooth wax.

[0025] The invention also provides application of the saliva combined with Lactobacillus CCFM1398 in preparing fermented food.

[0026] Beneficial Effects

[0027] 1. The saliva combined with Lactobacillus CCFM1398 provided by the present invention can promote the high expression of OPG in MC3T3-E1 cells in vitro (increased by 3.38 times compared with the control group), and increase the expression of the gene of osteoprotegerin (OPG) in MC3T3-E1 cells (increased by 1.53 times compared with the control group).

[0028] 2. The saliva combined with the living cells and dead cells of Lactobacillus CCFM1398 provided by the present invention can alleviate orthodontic-induced bone resorption and inhibit periodontal inflammation, which is specifically reflected in:

[0029] (1) Significantly reduce the level of alveolar bone resorption induced by orthodontic models.

[0030] (2) Reduce the gingival index and probing depth of rats induced by the orthodontic model.

[0031] (3) Reduce the number of osteoclasts at the apical bifurcation of the alveolar bone.

[0032] (4) Prevent the binding of RANKL and RANK in alveolar bone tissue, inhibit the activity and differentiation of osteoclasts, and reduce the expression of RANKL in rat serum.

[0033] (5) Reduce the expression levels of IL-6, TNF-α, and MPO in gingival tissue.

[0034] (6) Improve the pathological condition of alveolar bone tissue.

[0035] Therefore, the saliva-combined Lactobacillus salivarius CCFM1398 has great application prospects in the preparation of products (such as medicines or daily chemical products, etc.) for preventing and / or treating bone resorption.

[0036] Ligilactobacillus salivarius 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 Ligilactobacillus salivarius CCFM1398 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

[0037] Figure 1 :The effect of different bacterial fermentation supernatants and lysates on osteoprotegerin (OPG) in MC3T3-E1 cells;

[0038] Figure 2 :The effect of saliva combined with Lactobacillus CCFM1398 on osteoprotegerin (OPG) gene expression in osteoblasts;

[0039] Figure 3 :Effects of saliva combined with Lactobacillus CCFM1398 on body weight (A) and OTM (B) in rats with orthodontic-induced bone resorption;

[0040] Figure 4 :Effects of saliva combined with Lactobacillus CCFM1398 on gingival index (A) and probing depth (B) of rats with orthodontic-induced bone resorption;

[0041] Figure 5 :Effects of saliva combined with Lactobacillus CCFM1398 on the colonization of Prevotella intermedia in the oral cavity of rats with orthodontic-induced bone resorption;

[0042] Figure 6 :Effects of saliva combined with Lactobacillus CCFM1398 on gingival inflammatory factors in rats with orthodontic-induced bone resorption;

[0043] Figure 7 Effect of saliva combined with Lactobacillus CCFM1398 on the expression of OPG (A) and RANKL (B) in the serum of rats with orthodontic-induced bone resorption;

[0044] Figure 8 :Effects of saliva combined with Lactobacillus CCFM1398 on bone microstructure in rats with orthodontic-induced bone resorption (A) and the ratio of bone volume to total volume (B);

[0045] Fig. 9 :HE staining of saliva combined with Lactobacillus CCFM1398 on orthodontic-induced bone resorption in rats;

[0046] Fig.10:Trap staining of saliva combined with Lactobacillus CCFM1398 on orthodontic-induced bone resorption in rats (A) and analysis of osteoclast numbers (B).

[0047] (* in the figure indicates significant difference, among which *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)

[0048] Biomaterial Deposit

[0049] A strain of Ligilactobacillus salivarius CCFM1398, taxonomically named Ligilactobacillus salivarius, was deposited in the Guangdong Provincial Microbiological Culture Collection on June 14, 2024, with the deposit number GDMCC No: 64762, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0050] 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.

[0051] The culture medium involved in the following examples is as follows:

[0052] The MRS medium in Examples 1, 2, 3 and 4 includes: 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.

[0053] The complete cell culture medium in Examples 1, 2 and 3 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).

[0054] The osteogenic induction medium in Examples 1, 2 and 3 includes complete cell culture medium + sodium β-glycerophosphate (10 mM / L), ascorbic acid (50 μg / mL) and dexamethasone (100 nM / L).

[0055] The BHI blood plate culture medium in Example 4: 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.

[0056] The strains, cells and animals involved in the following examples are as follows:

[0057] In Examples 1, 2, 3, and 4, Lactobacillus saliva CCFM1398 and Lactobacillus saliva FAHBZ8M2 were from the strain bank of the Biotechnology Center of Jiangnan University, and Prevotella intermedia ATCC 25611 was purchased from the American Type Culture Collection.

[0058] The rat osteoblast precursor cells MC3T3-E1 in Examples 1, 2, and 3 were deposited in the Biotechnology Center of the School of Food Science and Engineering, Jiangnan University.

[0059] In Example 4, 24 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).

[0060] The preparation of the experimental samples involved in the following examples is as follows:

[0061] In Examples 1, 2, and 3, the live lactobacillus suspension was prepared by inoculating saliva with Lactobacillus CCFM1398 and FAHBZ8M2 in MRS liquid 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.

[0062] 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.

[0063] 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.

[0064] In Example 4, the saliva-combined Lactobacillus live bacterial suspension was prepared by inoculating saliva-combined Lactobacillus CCFM1398 and FAHBZ8M2 in MRS liquid culture medium at a 2% (v / v) inoculum, culturing at 37°C for 24 h, collecting the bacterial bodies by centrifugation at 8500 g for 20 min, washing the bacterial bodies twice with sterile saline, resuspending the bacterial bodies with 10% (v / v) skim milk solution, mixing thoroughly, and then freeze-drying. Before the animal experiment, the freeze-dried Lactobacillus powder was counted and re-dissolved with PBS to a bacterial suspension concentration of 1×10 9 CFU / mL.

[0065] Preparation of saliva combined with Lactobacillus dead bacteria suspension: The preparation method and concentration of dead bacteria suspension are the same as those of live bacteria. The difference is that after culturing at 37°C for 24 hours, the fermentation liquid 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, the bacteria are washed twice with sterile saline, and then resuspended with 10% skim milk solution. After thorough mixing, freeze-drying is performed. Before intervention, it is resuspended with PBS to a concentration of 1×10 9 CFU / mL.

[0066] Preparation of live Prevotella intermedia ATCC 25611 suspension: 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, used for subsequent experiments. Example 1 Isolation and identification of saliva-associated Lactobacillus CCFM1398

[0067] 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.

[0068] 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.

[0069] Table 1: Primer information

[0070]

[0071] After sequencing, the 16S rDNA sequence information obtained by amplification is (SEQ ID No.1):

[0072] CTTAGACGGCTGGCTCCTTGCGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGG

[0073] TGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCG

[0074] ATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAAC

[0075] GGCTTTAAGAGATTAGCTAAACCTCGCGGTCTCGCGACTCGTTGTACCGTCCATTGTAGC

[0076] ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCC

[0077] GGTTTGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAACTGGCAACAAG

[0078] GGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCAT

[0079] GCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCCTAATCTCTTAGGTGGTCAAAGGAT

[0080] GTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTG

[0081] TGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAAT

[0082] GCTTATTGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATC

[0083] GTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGAACCTC

[0084] AGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACTGGTGTTCTTCCATATATCTACGC

[0085] ATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCC

[0086] AATGCACTACTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAGACCGCCTGCGT

[0087] TCCCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTG

[0088] GCACGTAGTTAGCCGTGACTTGCTGGTTAGATACCGTCATCGAATGAACAGTTACTCTCA

[0089] CTCGTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCACTCACGCGGC

[0090] GTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAG

[0091] TTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCAT

[0092] CACCTTGGTAGGCCGTTACCCCACCAACTAGTTAATACGCCGCGGGTCCATCTAAAAGC

[0093] GATAGCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTAGAGATATACGGTATTAGCA

[0094] CCTGTTTCCAAGTGTTATCCCCTTCTTTTAGGCAGGTTACCCACGTGTTACTCACCCGTCC

[0095] GCCACTCAACTTCTTACGGTGAATGCAAGCATTCGGTGTAAGAAAGTTTCGTACGACTGCATGTATAG.

[0096] 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 Lactobacillus salivarius. Subsequently, 1.5 mL of the identified bacteria were taken into a 2 mL strain preservation tube, centrifuged at 6000 r / min for 3 minutes, and 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 resulting strain was named Ligilactobacillus salivarius CCFM1398, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No: 64762.

[0097] Example 2 Ability of saliva combined with lactobacillus to promote osteoblast osteoprotegerin (OPG) expression in vitro

[0098] 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.

[0099] 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 times the volume of complete 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 4Cells were seeded on 6-well plates 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. The medium was replaced every 48 hours. Cell morphology was observed regularly. 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.

[0100] Table 2: Group information table

[0101]

[0102]

[0103] 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, and that in the saliva combined with Lactobacillus BNCC138618 lysate group was 46.02 ng / L, which was significantly lower than that in the control group (p<0.01). The OPG content in the saliva combined with Lactobacillus CCFM1398 lysate group was 396.07 ng / L, which was significantly higher than that in the control group (p<0.0001). The above results indicate that the saliva combined with Lactobacillus CCFM1398 bacterial lysate can significantly increase the expression of osteoprotegerin in the induced MC3T3-E1 cell culture supernatant.

[0104] In summary, saliva combined with Lactobacillus CCFM1398 can increase the expression of OPG in osteoblasts in vitro.

[0105] Example 3 Ability of saliva combined with lactobacillus to promote osteoblast osteoprotegerin (OPG) gene expression in vitro

[0106] 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 μg / 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.

[0107] The expression of OPG gene in MC3T3-E1 cells was intervened by saliva combined with Lactobacillus CCFM1398 lysate. Figure 2 The Fold Change value of OPG gene in the saliva combined with Lactobacillus CCFM1398 lysate group was 1.53, which was significantly higher than that in the control group (p<0.05).

[0108] In summary, saliva combined with Lactobacillus CCFM1398 can increase the expression of OPG gene in osteoblasts in vitro.

[0109] Example 4 Application of saliva combined with Lactobacillus CCFM1398 in alleviating orthodontic-induced bone resorption in rats

[0110] 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).

[0111] The experimental groups are shown in Table 3.

[0112] Table 3: Animal experimental plan and grouping

[0113]

[0114] 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.

[0115] Except for the control group, the other groups underwent orthodontic modeling on day 0 (specific operation: 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 fix the ligature wire, and to ensure that the wire fracture did not damage the epithelial barrier. Except for the left first molar, the occlusal surfaces of the remaining maxillary 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 infected with Prevotella intermedia for a total of 12 days (days 1-6 and 8-13) (specific operation method of infection: 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).

[0116] For the saliva combined with live and dead bacteria groups of Lactobacillus CCFM1398, 1 mL of saliva combined with live and dead bacteria suspensions of Lactobacillus CCFM1398 were used to irrigate the rats’ oral cavity 6 hours after infection with Prevotella intermedia. The concentration of the bacterial suspensions was 1×10 9 CFU / mL.

[0117] 1. Weight of rats, distance between first molar and central incisor (OTM), gingival index and probing depth

[0118] The gingival index and probing depth of the periodontal tissue of all rats were measured on days 0, 7, and 14. Sterile swabs were used to collect rat saliva, which was stored in a 2-mL centrifuge tube containing 1 mL of 30% glycerol to analyze the colonization of Prevotella intermedia ( Figure 5 ). The rats' body weight, the distance between the first molar and the central incisor (OTM), the gingival index and the probing depth were also recorded.

[0119] (1) Changes in rat body weight and tooth movement distance (OTM)

[0120] like Figure 3 As shown in (A), rats showed significant weight loss on the 7th day after orthodontic modeling. After 14 days of intervention with saliva combined with live bacteria of Lactobacillus CCFM1398, the weight of rats recovered significantly compared with the model group (increased by 12% compared with the model group), and the result was statistically significant (p<0.05). There was no significant difference between the OTM groups ( Figure 3 B) indicates that saliva combined with Lactobacillus CCFM1398 does not cause excessive movement and remodeling of teeth.

[0121] (2) Changes in periodontal tissue gingival index and probing depth

[0122] Changes in the gingival index can reflect changes in the degree of gingival inflammation. A decrease in the gingival index means improvements in symptoms such as gingival redness, swelling, and bleeding. Changes in probing depth are usually associated with improvements in periodontal tissue. A shallow probing depth suggests healthy periodontal tissue and a lower risk of periodontal disease.

[0123] The changes of gingival index and periodontal probing depth are as follows Figure 4 As shown. The saliva combined with dead bacteria group of Lactobacillus CCFM1398 reduced the gingival index from 3 to 1.4 on the 7th day, which was significantly lower than that of the model group, and remained at a low level on the 14th day (p<0.001); the gingival index of the live bacteria group decreased from 3 to 2.25 on the 7th day, and the effect lasted until the 14th day. The probing depth of the live bacteria group decreased from 1.33mm to 0.25mm on the 7th day, and from 1.67mm to 0.5mm on the 14th day, both of which were significantly lower than those of the model group (p<0.05); the probing depth of the dead bacteria group decreased to 0.8mm. This shows that after oral irrigation with saliva combined with Lactobacillus CCFM1398, live or dead bacteria can reduce the level of gingival inflammation in rats, alleviate periodontal bleeding, and significantly reduce the depth of periodontal pockets.

[0124] In summary, saliva combined with Lactobacillus CCFM1398 can reduce the formation of periodontal pockets by reducing the incidence of gingival tissue bleeding and gingival inflammation.

[0125] (3) Colonization of Prevotella intermedia in the rat oral cavity

[0126] like Figure 5 As shown, from the 7th to 14th day after orthodontic modeling, saliva combined with live and dead Lactobacillus CCFM1398 could significantly reduce the colonization of Prevotella intermedia in the rat oral cavity.

[0127] 2. Secretion of inflammatory factors IL-6, TNF-α, and MPO in rat gingival tissue and expression of OPG and RANKL in serum

[0128] 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.

[0129] Determination method: On the 14th day, before the rats were killed, 10 mL of blood was taken from the abdominal aorta of the rats. After standing for 30 minutes, the blood was centrifuged at 3000 r / min for 15 minutes 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 minutes, the supernatant was collected, and stored at -20°C.

[0130] Then, the levels of RANKL and OPG in serum and TNF-α, IL-6, and MPO in gingival tissue were determined according to the kit instructions.

[0131] The large amount of secretion of IL-6, TNF-α, and MPO can cause inflammation of periodontal tissues and damage the periodontal tissues themselves, accelerate the entry of pathogenic bacteria and toxins, and promote 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 and prevent RANKL from activating its receptor RANK, thereby inhibiting the differentiation and activity of osteoclasts and reducing bone resorption.

[0132] The secretion of inflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) in rat gingival tissue Figure 6The IL-6 level in the control group was 433.14 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 saliva combined with Lactobacillus CCFM1398, the secretion of IL-6 decreased. The IL-6 levels in the live bacteria group and the dead bacteria group were 513.94 ng / L and 514.24 ng / L, respectively, which were significantly lower than those in the model group (p<0.001). Similarly, after orthodontic and pathogenic bacteria stimulation, the secretions of TNF-α and MPO in the model group 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 saliva combined with live and dead Lactobacillus CCFM1398, the secretions of TNF-α and MPO in the model group were significantly reduced, with TNF-α levels dropping to 246.93 ng / L and 219.65 ng / L, and MPO levels dropping to 103.28 ng / L and 112.75 ng / L, respectively.

[0133] The expression of OPG and RANKL in rat serum Figure 7 As shown. The OPG level in the serum of the control group was 657.00 ng / L, the OPG level in the serum of the model group was 1130.33 ng / L, and the saliva combined with Lactobacillus CCFM1398 dead bacteria group and live bacteria group were both around 800 ng / L, which was significantly lower than that of the model group (p<0.01). The RANKL level in the serum of the control group was 190.58 pmol / L, the RANKL level in the serum of the model group was 301.57 pmol / L, and the saliva combined with Lactobacillus CCFM1398 dead bacteria group and live bacteria group were 240.90 pmol / L and 241.187 pmol / L, respectively.

[0134] In summary, saliva combined with Lactobacillus CCFM1398 reduces periodontal tissue inflammation and alleviates bone resorption by reducing the secretion of inflammatory factors and MPO in gingival tissue and inhibiting the secretion of OPG and RANKL in alveolar bone.

[0135] 3. Microstructure and pathological analysis of rat alveolar bone tissue

[0136] Bone microstructure analysis: After 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.

[0137] Histopathological observation: Alveolar bone tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sliced ​​into 5 μm thick sections for hematoxylin and eosin (HE) and Trap staining. Alveolar bone tissue samples were observed under a pathological section scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

[0138] (1) Microstructure of rat alveolar bone tissue

[0139] 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.

[0140] The microstructure of rat alveolar bone tissue Figure 8 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% (p < 0.0001). After the intervention of saliva combined with Lactobacillus CCFM1398, the BV / TV ratio of the live bacteria group was 33.48%, close to the control group, and significantly increased compared with the model group (p < 0.05). The BV / TV ratio of the dead bacteria group was 27.80%, which was not significantly different from the model group.

[0141] In conclusion, saliva combined with Lactobacillus CCFM1398 can increase the BV / TV ratio at the root apex of the first molar in the alveolar bone, thereby alleviating orthodontic-induced bone resorption.

[0142] (2) Histopathological analysis of rat alveolar bone

[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 reflect the acute degree of inflammation and also observe changes in tissue morphology. Fig. 9 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 the saliva combined with Lactobacillus CCFM1398 live bacteria group and the dead bacteria group showed that the tissue inflammatory infiltration was alleviated after the intervention, the inflammatory cells no longer gathered at the alveolar bone root apex, and the damage to the periodontal tissue was also alleviated. This shows that saliva combined with Lactobacillus CCFM1398 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.10 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 results of saliva combined with the live and dead bacteria groups of Lactobacillus CCFM1398 showed that the bacteria could alleviate orthodontic-induced excessive bone resorption and prevent osteoclasts from gathering at the alveolar root apex bifurcation. Among them, the number of osteoclasts in the dead bacteria group decreased from 10 to 3.6 compared with the model group under a 20x field of view, and the results were significantly different (p < 0.01); the number of osteoclasts in the live bacteria group decreased from 10 to 7 compared with the model group.

[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 Ligilactobacillus salivarius CCFM1398, characterized in that: The saliva-associated Lactobacillus CCFM1398 was deposited at the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No: 64762.

2. A microbial preparation containing the saliva-combined Lactobacillus CCFM1398 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The number of saliva-combined Lactobacillus CCFM1398 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. The postbiotic prepared by combining saliva with Lactobacillus CCFM1398 according to claim 1, characterized in that: The postbiotics include dead cells, lysate and / or supernatant of the saliva-combined Lactobacillus CCFM1398.

5. A product containing the saliva-combined Lactobacillus CCFM1398 according to claim 1, and / or the microbial preparation according to claim 2 or 3, and / or the postbiotic according to claim 4.

6. The product according to claim 5, characterized in that The products include food, medicine, health care products or daily chemical products; the food includes fruit and vegetable products, soy products or dairy products; the medicine also contains conventional pharmaceutical excipients and / or pharmaceutical carriers; the daily chemical products include mouthwash, mouthwash granules, toothpaste, tooth cleaning powder, tooth stickers, periodontal gel, tooth gel or tooth wax.

7. A method for producing osteoprotegerin, characterized in that: The lysate and / or fermentation metabolites of the saliva-lactobacillus CCFM1398 of claim 1 are added to a culture system containing osteoblasts for culture.

8. Use of the saliva combined with Lactobacillus CCFM1398 and / or its postbiotics according to claim 1 in the preparation of a medicine for inhibiting the colonization of Prevotella intermedia.

9. Use of the saliva-lactobacillus CCFM1398 according to claim 1, and / or the microbial preparation according to claim 2 or 3, and / or the postbiotic according to claim 4 in the preparation of a product for preventing and / or treating oral inflammation or bone resorption.

10. The use according to claim 9, characterized in that: The products include medicines or care products; the dosage forms of the medicines include oral sprays, tablets, capsules, pastes, oral liquids or freeze-dried powders; the care products include mouthwashes, mouthwash granules, toothpaste, tooth cleaning powder, tooth stickers, periodontal gels, tooth gels or tooth waxes.

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