Lactobacillus salivarius combination CCFM1397 capable of improving osteoblast activity and relieving bone resorption and periodontal tissue injury and metagen of lactobacillus salivarius combination CCFM1397

Abstract

The invention discloses a combined lactobacillus salivarius CCFM1397 for improving osteoblast activity and relieving bone resorption and periodontal tissue damage and a metagen thereof, and belongs to the technical field of microorganisms. The lactobacillus salivarius combination CCFM1397 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: the expression of MC3T3-E1 cell osteoprotegerin (OPG) protein and gene level 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 IL-1beta, IL-6 and TNF-alpha and myeloperoxidase MPO of the orthodontic bone absorption rats 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 CCFM1397 capable of improving osteoblast activity and alleviating bone absorption and periodontal tissue damage and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] Orthodontics is an important treatment method in the field of dentistry, which improves oral function and facial aesthetics by correcting abnormal tooth and jaw alignment. During orthodontic treatment, tooth movement involves the remodeling process of the alveolar bone, which is mainly achieved through bone resorption by osteoclasts and bone formation by osteoblasts. Osteoclasts can break down bone tissue during bone resorption to create space for tooth movement. Orthodontic force can stimulate the activity of osteoclasts and promote the resorption of alveolar bone. However, excessive orthodontic force and improper oral hygiene may induce periodontal problems such as gingival damage and alveolar bone damage, followed by excessive bone resorption of the alveolar bone. After osteoclasts absorb bone, osteoblasts calcify to form new bone tissue and stabilize the new position of the teeth.

[0003] The OPG / RANKL / RANK pathway is a key pathway that regulates the balance between bone resorption and bone formation. OPG (osteoprotegerin) is secreted by osteoblasts and can competitively bind to RANKL (receptor activator of nuclear factor κB ligand) on the surface of osteoclasts RANK receptors (receptor activator of nuclear factor κB), thereby inhibiting the differentiation and activity of osteoclasts. 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 involving the use of biological agents to treat alveolar bone resorption. These patents usually focus on the use of some Chinese herbal plant extracts and specific protease preparations to regulate gingival tissue inflammation, local metalloproteinase activity in gingival tissue, or local to promote osteoblast activity, inhibit osteoclast function, and thus reduce alveolar bone resorption. Patent CN116327762A found that coptisine hydrochloride can inhibit the pyroptosis pathway mediated by macrophage NLRP3 inflammasome, reduce the recognition of pathogen-associated molecular patterns (PAMPs), etc., and thus inhibit the occurrence of periodontal inflammation. However, this also indirectly reduces the individual's immunity and increases the risk of uncertainty in inaccurate dosage. In addition, in the application of microbial fields, some patented strains regulate femoral tissue bone metabolism by affecting the activity of osteoblasts and osteoclasts for the OPG / RANKL / RANK pathway and the Wnt signaling pathway, thereby slowing down the progression of osteoporosis. However, considering the complex environment of the oral cavity, the application of these strains in orthodontically induced bone resorption still needs further verification. At present, no treatment method that can effectively alleviate orthodontic-induced bone resorption has been found in clinical practice, nor has any probiotic product that can effectively alleviate orthodontic-induced bone resorption or relieve oral inflammation been found. Summary of the invention

[0005] Compared with the prior art, the present invention provides a new method for treating orthodontically induced excessive bone resorption using saliva combined with Lactobacillus salivarius CCFM1397. The Lactobacillus paracasei of the present invention can regulate the secretion of osteoprotegerin (OPG) by osteoblasts and increase the expression of RUNX genes related to osteoblast activity. 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.

[0006] The present invention provides a saliva-associated lactobacillus and its postbiotics that can improve osteoblast activity, alleviate bone resorption and have the effect of alleviating periodontal tissue damage. The taxonomic name of the saliva-associated lactobacillus (Ligilactobacillus salivarius) CCFM1397 is Ligilactobacillus salivarius, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with a deposit number of GDMCC No: 64761, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In one embodiment of the present invention, the salivary Lactobacillus (Ligilactobacillus salivarius) CCFM1397 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 salivary Lactobacillus, and is named as salivary Lactobacillus (Ligilactobacillus salivarius) CCFM1397.

[0008] In one embodiment of the present invention, the colonies of the salivary Lactobacillus salivarius CCFM1397 on the MRS culture medium are small translucent white circles.

[0009] The invention also provides a microbial preparation containing the saliva-combined lactobacillus CCFM1397.

[0010] In one embodiment of the present invention, the number of bacteria in the microbial preparation 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 CCFM1397, wherein the postbiotic comprises dead cells, lysate and / or supernatant of the saliva combined with Lactobacillus CCFM1397.

[0012] The present invention also provides products containing the saliva-combined lactobacillus CCFM1397, and / or the microbial preparation, and / or the postbiotics, and the products include food, medicine, health care products or daily chemical products.

[0013] In one embodiment of the present invention, the food comprises fruit and vegetable products, bean products or dairy products.

[0014] In one embodiment of the present invention, the ingredients of the medicine include Ligilactobacillus salivarius CCFM1397 and conventional pharmaceutical excipients and / or carriers.

[0015] In one embodiment of the present invention, the carrier is a pharmaceutically acceptable carrier.

[0016] 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.

[0017] 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.

[0018] The present invention also provides a method for producing osteoprotegerin, wherein the lysate and / or fermentation metabolites of the saliva combined with Lactobacillus CCFM1397 are added to a culture system containing osteoblasts for culture.

[0019] The present invention also provides the use of the saliva combined with Lactobacillus CCFM1397 and / or its postbiotics in the preparation of a medicine for inhibiting the colonization of Prevotella intermedia.

[0020] 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 combined with Lactobacillus salivarius CCFM1397.

[0021] The present invention also provides the use of the saliva combined with Lactobacillus CCFM1397 in preparing medicines for regulating oral inflammation function or in preparing medicines for alleviating bone absorption.

[0022] In one embodiment of the present invention, the oral inflammation includes periodontal inflammation and gingival inflammation.

[0023] In one embodiment of the present invention, the dosage form of the drug includes oral spray, tablet, capsule, paste, oral solution or lyophilized powder.

[0024] The present invention also provides the use of the saliva-combined Lactobacillus CCFM1397 or the microbial preparation in preparing care products for preventing and / or treating oral inflammation.

[0025] In one embodiment of the present invention, the oral inflammation comprises periodontitis or gingivitis.

[0026] 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.

[0027] The invention also provides application of the saliva combined with Lactobacillus CCFM1397 in preparing fermented food.

[0028] Beneficial Effects

[0029] 1. The present invention provides a saliva-associated Lactobacillus (Ligilactobacillus salivarius) CCFM1397, which is isolated from dental plaque in the oral cavity of healthy adolescents. The saliva-associated Lactobacillus CCFM1397 can promote osteoblast activity in vitro, increase osteoblast OPG expression (the expression level reaches 366.83 ng / L), prevent the combination of RANKL and RANK in alveolar bone tissue, inhibit the activity and differentiation of osteoclasts; and increase the expression of RUNX gene in MC3T3-E1 cells (the relative expression level is 6.14).

[0030] 2. The saliva combined with Lactobacillus CCFM1397 provided by the present invention has the effect of alleviating orthodontic-induced bone resorption and inhibiting periodontal inflammation, which is specifically embodied in:

[0031] (1) Significantly reduce the level of alveolar bone resorption in rats induced by orthodontic model.

[0032] (2) Reduce the gingival index and probing depth of rats induced by the orthodontic model.

[0033] (3) Reduce the number of osteoclasts at the apical bifurcation of the alveolar bone.

[0034] (4) Reduce the expression of RANKL in rat serum.

[0035] (5) Reduce the expression levels of IL-1β, IL-6, TNF-α, and MPO in gingival tissue.

[0036] (6) Improve the pathological condition of alveolar bone tissue.

[0037] Therefore, this saliva-associated Lactobacillus (Ligilactobacillus salivarius) CCFM1397 has great application prospects in the preparation of products (such as medicines or daily chemical products, etc.) for preventing and / or treating bone resorption.

[0038] 3. 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 CCFM1397 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

[0039] Figure 1 :The effect of different bacterial fermentation supernatants and lysates on osteoprotegerin (OPG) in MC3T3-E1 cells;

[0040] Figure 2 :The effect of saliva combined with Lactobacillus CCFM1397 on the expression of RUNX gene in osteoblasts;

[0041] Figure 3 :Effects of saliva combined with Lactobacillus CCFM1397 on body weight (A) and OTM (B) in rats with orthodontic-induced bone resorption;

[0042] Figure 4 :Effects of saliva combined with Lactobacillus CCFM1397 on gingival index (A) and probing depth (B) of rats with orthodontic-induced bone resorption;

[0043] 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;

[0044] Figure 6 :The effect of saliva combined with Lactobacillus CCFM1397 on the expression of inflammatory factors in gingival tissue of rats with orthodontic-induced bone resorption; (A) IL-1β, (B) IL-6, (C) TNF-α, (D) MPO;

[0045] Figure 7 :The effect of saliva combined with Lactobacillus CCFM1397 on the expression of OPG (A) and RANKL (B) in the serum of rats with orthodontic-induced bone resorption;

[0046] Figure 8 :The effect of saliva combined with Lactobacillus CCFM1397 on the bone microstructure of rats with orthodontic-induced bone resorption, (A) microstructure of rat model teeth, (B) ratio of the bone volume to the total volume of the rat first molar;

[0047] Fig. 9 :HE staining of saliva combined with Lactobacillus CCFM1397 on orthodontic-induced bone resorption in rats;

[0048] Fig.10 :Trap staining (A) and osteoclast count (B) of saliva combined with Lactobacillus CCFM1397 in rats with orthodontic-induced bone resorption;

[0049] (In the figure, * indicates significant difference, among which *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).

[0050] Biomaterial Deposit

[0051] A strain of salivary Lactobacillus (Ligilactobacillus salivarius) CCFM1397, taxonomically named Ligilactobacillus salivarius, was deposited in the Guangdong Provincial Microbiological Culture Collection on June 14, 2024, with the deposit number GDMCC No: 64761, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0052] 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.

[0053] The culture medium involved in the following examples is as follows:

[0054] 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.

[0055] 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).

[0056] 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).

[0057] BHI blood plate medium in Example 4: Qingdao Haibo BHI commercial medium 37.5g / L, agar powder 20g / L, sterile defibrinated sheep blood 100mL / L, 10% hemin-vitamin K 1mL / L

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

[0059] In Examples 1, 2, 3, and 4, Lactobacillus saliva CCFM1397 was from the strain bank of the Biotechnology Center of Jiangnan University, Lactobacillus saliva BNCC138618 was purchased from the Beina Biological Strain Collection, and Prevotella intermedia ATCC 25611 was purchased from the American Type Culture Collection.

[0060] 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.

[0061] 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).

[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 saliva with Lactobacillus CCFM1397 and BNCC138618 in MRS liquid medium at a 2% (v / v) inoculum, 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 saliva combined with lactobacillus live bacterial suspension was prepared by inoculating saliva combined with lactobacillus CCFM1397 and BNCC138618 in MRS liquid culture medium at a 2% (v / v) inoculation amount, culturing at 37°C for 24 hours, collecting the bacterial bodies by centrifugation at 8500g for 20 minutes, washing the bacterial bodies twice with sterile saline, resuspending the bacterial bodies with 10% skim milk aqueous solution, mixing well and then freeze-drying. Before the animal experiment, the freeze-dried lactobacillus powder was counted and then re-dissolved with PBS to a bacterial suspension concentration of 1×10 9 CFU / mL.

[0067] Preparation of saliva combined with dead Lactobacillus suspension: The preparation method and concentration of the dead bacteria group were the same as those of the live bacteria, except that after culturing at 37°C for 24 hours, the fermentation liquid was inactivated at 105°C for 10 minutes, and the inactivation effect was checked by plate coating. After heat treatment, the bacterial liquid was centrifuged at 8500g for 20 minutes, the bacteria were washed twice with sterile saline, and then resuspended with 10% skim milk solution, mixed thoroughly, and freeze-dried. Before intervention, it was resuspended with PBS to a concentration of 1×10 9CFU / mL.

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

[0069] Example 1 Isolation and identification of saliva-associated Lactobacillus CCFM1397

[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] CTTAGACGGCTGGCTCCTTGCGGTTACCCACCGGCTTTGGGTGTTACAAACTCTCATGG

[0076] TGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCG

[0077] ATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAAC

[0078] GGCTTTAAGAGATTAGCTAAACCTCGCGGTCTTGCGACTCGTTGTACCGTCCATTGTAGC

[0079] ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCC

[0080] GGTTTGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAACTGACAACAAG

[0081] GGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCAT

[0082] GCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCCTAATCTCTTAGGTGGTCAAAGGAT

[0083] GTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTG

[0084] TGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAAT

[0085] GCTTATTGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATC

[0086] GTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGAACCTC

[0087] AGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACTGGTGTTCTTCCATATATCTACGC

[0088] ATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCC

[0089] AATGCACTACTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAGACCGCCTGCGT

[0090] TCCCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTG

[0091] GCACGTAGTTAGCCGTGACTTGCTGGTTAGATACCGTCATCGAATGAACAGTTACTCTCA

[0092] CTCGTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCACTCACGCGGC

[0093] GTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAG

[0094] TTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCAT

[0095] CACCTTGGTAGGCCGTTACCCCACCAACTAGTTAATACGCCGCGGGTCCATCTAAAAGC

[0096] GATAGCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTAGAGATATACGGTATTAGCA

[0097] CCTGTTTCCAAGTGTTATCCCCTTCTTTTAGGCAGGTTACCCACGTGTTACTCACCCGTCC

[0098] GCCACTCAACTTCTTACGGTGAATGCAAGCATTCGGTGTAAGAAAGTTTCGTACGACTGCATGTATAG.

[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 Lactobacillus salivarius. Then, 1.5 mL of the identified bacteria were taken into a 2 mL strain preservation tube. After centrifugation 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 Ligilactobacillus salivarius CCFM1397, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No: 64761.

[0100] Example 2 Ability of saliva combined with lactobacillus 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 times the volume of complete medium. After centrifugation, the supernatant was discarded and the cells were resuspended in 10 mL of complete medium. The cells were cultured at 37°C and 5% CO2, and the medium was replaced every 48 hours. After counting the cells, 1×10 4 Cells 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, and cell morphology was observed regularly. MC3T3-E1 cells that differentiated into osteoblasts usually became 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, and the OPG concentration in the saliva combined with Lactobacillus BNCC138618 lysate group was significantly lower than that in the control group (p < 0.01). The OPG content in the saliva combined with Lactobacillus CCFM1397 lysate group was 366.83 ng / L, which was significantly higher than that in the control group (p < 0.0001). The above results show that the saliva combined with Lactobacillus CCFM1397 bacterial lysate can significantly increase the expression of OPG in the culture supernatant of induced MC3T3-E1 cells.

[0106] In summary, the screened saliva-combined Lactobacillus CCFM1397 can increase the expression of OPG in osteoblasts in vitro.

[0107] Example 3 Ability of saliva combined with Lactobacillus to promote the expression of RUNX gene in osteoblasts in vitro

[0108] 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.

[0109] The expression of RUNX gene in MC3T3-E1 cells after intervention in each group Figure 2 The Fold Change value of RUNX gene in the saliva combined with Lactobacillus CCFM1397 lysate group was 6.14, which was significantly higher than that in the control group (p<0.01).

[0110] In summary, the screened saliva combined with Lactobacillus CCFM1397 increased the expression of RUNX gene in osteoblasts in vitro and promoted the activity of osteoblasts.

[0111] Example 4 Application of saliva combined with Lactobacillus CCFM1397 in alleviating orthodontic-induced bone resorption in rats

[0112] 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).

[0113] The experimental groups are shown in Table 3.

[0114] Table 3: Animal experimental plan and grouping

[0115]

[0116] 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.

[0117] 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).

[0118] For the saliva combined with live and dead bacteria groups of Lactobacillus CCFM1397, 6 hours after infection with Prevotella intermedia, the rats' oral cavity was irrigated with 1 mL of saliva combined with live and dead bacteria suspensions of Lactobacillus CCFM1397, respectively. The concentration of the bacterial suspension was 1×10 9 CFU / mL. The concentration of dead bacteria group was the same as that of live bacteria, and they were inactivated by high pressure steam at 105℃ for 10min, and the inactivation effect was checked by plate coating.

[0119] 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.

[0120] (1) Changes in rat body weight and tooth movement distance (OTM)

[0121] like Figure 3As shown in A, rats showed significant weight loss on the 7th day after orthodontic modeling (p<0.0001). After intervention with saliva combined with live and dead bacteria of Lactobacillus CCFM1397, the weight of rats recovered significantly compared with the model group. The weight of the live bacteria group and the dead bacteria group were 0.83 times and 0.94 times that of the model group, respectively. On the 14th day of modeling, the weight of rats still showed a significant decrease (p<0.0001). After intervention with saliva combined with live bacteria of Lactobacillus CCFM1397, the weight of rats recovered significantly compared with the model group. The weight of the live bacteria group and the dead bacteria group were 1.08 times and 1.15 times that of the model group, respectively. The results were statistically significant (p<0.01). There was no significant difference in OTM between the groups ( Figure 3 B), indicating that salivary lactobacilli combined with lactobacilli do not cause excessive tooth movement and remodeling.

[0122] (2) Changes in periodontal tissue gingival index and probing depth

[0123] 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.

[0124] The changes of gingival index and periodontal probing depth are as follows Figure 4 As shown. On the 7th day, the gingival index of the saliva combined with live Lactobacillus CCFM1397 group decreased from 3 to 1.6, and the probing depth decreased from 1.33mm to 0.2mm, which were significantly decreased compared with the model group, and remained at a low level on the 14th day. The effect of the saliva combined with dead Lactobacillus CCFM1397 group on the gingival index was similar to that of the live bacteria group, and was significantly reduced compared with the model group (p < 0.01). This shows that after oral irrigation with Lactobacillus paracasei CCFM1397, the level of gingival inflammation in rats was reduced, the periodontal bleeding problem was alleviated, and the depth of the periodontal pocket also decreased significantly.

[0125] In summary, saliva combined with Lactobacillus CCFM1397 can reduce the formation of periodontal pockets by reducing the incidence of gingival tissue bleeding and gingival inflammation.

[0126] (3) Colonization of Prevotella intermedia in the rat oral cavity

[0127] like Figure 5 As shown, on the 14th day after orthodontic modeling, saliva combined with live and dead Lactobacillus CCFM1397 could significantly reduce the colonization of Prevotella intermedia in the rat oral cavity.

[0128] (4) Secretion of inflammatory factors IL-1β, IL-6, TNF-α, and MPO in rat gingival tissue and expression of OPG and RANKL in serum

[0129] The experimental period was 13 days (days 0 to 13). 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 interleukins (IL-6, IL-1β) and tumor necrosis factor (TNF-α) in gingival tissue, myeloperoxidase (MPO), osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in serum 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. 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.

[0131] Then, the levels of RANKL and OPG in serum and IL-1β, TNF-α, IL-6, and MPO in gingival tissue were determined according to the kit instructions.

[0132] The massive secretion of IL-1β, IL-6, TNF-α, and MPO causes periodontal inflammation and damages the periodontal tissue itself, 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-1β, IL-6, TNF-α, and MPO in rat gingival tissue Figure 6As shown. The IL-1β level in the control group was 6.21 ng / L. After orthodontic and pathogenic bacteria stimulation, the IL-1β level in the model group increased significantly to 25.30 ng / L. After saliva combined with live Lactobacillus CCFM1397 intervention, the IL-1β secretion decreased to 15.01 ng / L (p<0.05). The IL-6 level in the control group was 433.7 ng / L. The IL-6 level in the model group increased significantly to 660.17 ng / L. After saliva combined with Lactobacillus CCFM1397 intervention, the IL-6 secretion was significantly reduced, and the concentration in the dead bacteria group was 469.26 ng / L, which was significantly lower than that in the model group (p<0.01). The TNF-α level in the control group was 92.00 ng / L, and the concentration in the model group was significantly increased to 324.35 ng / L. After intervention with live bacteria of saliva combined with Lactobacillus CCFM1397, the TNF-α level was significantly reduced compared with the model group, and the live bacteria group and the dead bacteria group were 211.32 ng / L and 147.63 ng / L, respectively. The MPO level in the control group was 80.75 ng / L, and the MPO level in the model group was 128.94 ng / L. The live bacteria group and the dead bacteria group of saliva combined with Lactobacillus CCFM1397 were 97.37 ng / L and 100.89 ng / L, respectively, which were significantly lower than the model group. The above results show that saliva combined with Lactobacillus CCFM1397 can effectively reduce the level of gingival tissue inflammation in rats.

[0134] The expression of OPG and RANKL in rat serum Figure 7 As shown. The serum OPG level in the control group was 657.00 ng / L, and the serum OPG level in the model group increased significantly to 1130.33 ng / L. After intervention with saliva combined with dead bacteria of Lactobacillus CCFM1397, the serum OPG level was significantly reduced to 854.46 ng / L (p<0.05). The serum RANKL level in the control group was 190.58 pmol / L, and the serum RANKL level in the model group was 301.57 pmol / L. After intervention with saliva combined with Lactobacillus CCFM1397, both the live bacteria group and the dead bacteria group dropped to about 230 pmol / L, which was significantly lower than the model group (p<0.05).

[0135] In summary, saliva combined with Lactobacillus CCFM1397 can reduce periodontal tissue inflammation and alleviate bone resorption by reducing the secretion of inflammatory factors and MPO in gingival tissue. After the orthodontic model was established, the level of bone resorption in rats was significantly aggravated, and the use of saliva combined with Lactobacillus CCFM1397 can slow down the bone remodeling process, reduce the ratio of RANKL / OPG, and alleviate orthodontic-induced bone resorption.

[0136] (5) Microstructure of rat alveolar bone tissue

[0137] 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.

[0138] 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.

[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 < 0.001). After intervention with saliva combined with Lactobacillus CCFM1397, the BV / TV ratio in the live bacteria group increased to 35.326%, close to the control group, and significantly increased compared with the model group (p < 0.01), while the BV / TV ratio in the dead bacteria group was 24.90 ng / L%, which was not significantly different from the model group.

[0141] In summary, saliva combined with Lactobacillus CCFM1397 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] (6) 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 determine the acuteness of inflammation and also observe changes in tissue morphology. Fig. 9As 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 live and dead bacteria of Lactobacillus CCFM1397 showed that the tissue inflammatory infiltration was relieved after intervention, 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 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 Lactobacillus CCFM1397 staining showed that saliva combined with Lactobacillus CCFM1397 could alleviate the excessive bone resorption induced by orthodontics, so that osteoclasts no longer gathered at the alveolar root apex bifurcation. And under the 20x field of view, the number of osteoclasts in the live bacteria group and the dead bacteria group decreased from 10 to 5.2 and 4.5, respectively, 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 Ligilactobacillus salivarius CCFM1397, characterized in that: The saliva-associated Lactobacillus CCFM1397 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No: 64761.

2. A microbial preparation containing the saliva-combined Lactobacillus CCFM1397 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The number of bacteria 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 CCFM1397 according to claim 1, characterized in that: The postbiotics include dead cells, lysate and / or supernatant of the saliva-combined Lactobacillus CCFM1397.

5. A product containing the saliva-associated Lactobacillus CCFM1397 according to claim 1, and / or the microbial preparation according to any one of claims 2 or 3, and / or the postbiotic according to claim 4, characterized in that: The products include food, medicine, health care products or daily chemical products.

6. The product according to claim 5, characterized in that The food includes fruit and vegetable products, bean 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 CCFM1397 according to claim 1 are added to a culture system containing osteoblasts for culture.

8. Use of the saliva combined with Lactobacillus CCFM1397 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 CCFM1397 according to claim 1, and / or the microbial preparation according to any one of claims 2 or 3, and / or the postbiotic according to claim 4 in the preparation of a drug for preventing and / or treating oral inflammation or bone resorption, characterized in that: The dosage form of the drug includes oral spray, tablet, capsule, paste, oral solution or lyophilized powder.

10. Use of the saliva-combined Lactobacillus CCFM1397 of claim 1, and / or the microbial preparation of any one of claims 2 or 3, and / or the postbiotic of claim 4 in the preparation of care products for preventing and / or treating oral inflammation, characterized in that: The oral inflammation includes periodontitis or gingivitis; the care products include mouthwash, mouthwash particles, toothpaste, tooth cleaning powder, tooth stickers, periodontal gel, tooth gel or tooth wax.

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