A strain of Limosilactobacillus salivarius CCFM1397 and its postbiotics for enhancing osteoblast activity and alleviating bone resorption and periodontal tissue damage

Abstract

The present invention discloses a Lactobacillus salivarius CCFM1397 and its postbiotics for improving the activity of osteoblasts and alleviating bone resorption and periodontal tissue damage, belonging to the field of microbial technology. The Lactobacillus salivarius CCFM1397 provided by the present invention is isolated from the oral dental plaque of healthy adolescents, and it has the effects of alleviating bone resorption and anti-inflammation, specifically manifested as: increasing the expression levels of osteoprotegerin (OPG) protein and gene in MC3T3-E1 cells in vitro; reducing the alveolar bone resorption amount of orthodontic bone resorption rats, reducing the number of osteoclasts in the alveolar bone, and increasing the level of OPG in the serum of rats; reducing the secretion of inflammatory factors IL-1β, IL-6, TNF-α and myeloperoxidase MPO in orthodontic bone resorption rats; improving the alveolar bone tissue pathological condition of orthodontic bone resorption rats. Therefore, this Lactobacillus salivarius has great application prospects in products for alleviating bone resorption and assisting in inhibiting periodontal tissue inflammation.

Description

Technical Field

[0001] The present invention relates to a Lactobacillus salivarius CCFM1397 and its postbiotics that can improve the activity of osteoblasts and alleviate bone resorption and periodontal tissue damage, belonging to the field of microbial technology. Background Art

[0002] Orthodontics is an important treatment method in the dental field, which can improve oral function and facial aesthetics by correcting abnormal tooth and jaw alignment. During orthodontic treatment, tooth movement involves the remodeling process of alveolar bone, which is mainly achieved through osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Osteoclasts can decompose bone tissue during bone resorption to create space for tooth movement. Orthodontic force can stimulate the activity of osteoclasts and promote alveolar bone resorption. However, excessive orthodontic force and poor oral hygiene status may induce periodontal problems such as gingival injury and alveolar bone injury, subsequently leading to excessive bone resorption of the alveolar bone. After osteoclasts absorb bone mass, osteoblasts calcify to form new bone tissue and stabilize the new position of the teeth.

[0003] The OPG / RANKL / RANK pathway is the key pathway regulating the balance between bone resorption and bone formation. Osteoprotegerin (OPG) is secreted by osteoblasts and can competitively bind to the receptor activator of nuclear factor-κB ligand (RANKL) on the surface of osteoclasts to inhibit the differentiation and activity of osteoclasts. During orthodontic treatment, by regulating this pathway, the metabolism of alveolar bone can be affected, and thus the speed and effect of tooth movement can be influenced. Increasing the level of OPG or inhibiting the activity of RANKL can reduce bone resorption, contribute to tooth stability, and thus achieve the bone homeostasis balance of the alveolar bone in orthodontic patients.

[0004] At present, there are already many patents related to the treatment of alveolar bone resorption using biological agents. These patents usually focus on using some traditional Chinese medicine plant extracts and specific protease preparations to regulate gingival tissue inflammation, the activity of matrix metalloproteinases in the local gingival tissue, or locally to promote the activity of osteoblasts and inhibit the function of osteoclasts, thereby reducing 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 uncertain risk of inaccurate dosage. In addition, in the application in the field of microorganisms, some patented strains target the OPG / RANKL / RANK pathway and the Wnt signaling pathway to regulate bone metabolism in femoral tissue by affecting the activity of osteoblasts and osteoclasts, thereby slowing down the process of osteoporosis. However, considering the complex oral environment, the application of these strains in orthodontic-induced bone resorption still needs to be further verified. At present, in clinical practice, no treatment method that can effectively relieve orthodontic-induced bone resorption has been found, nor has a probiotic product that can effectively relieve 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 using saliva combined with Ligilactobacillus salivarius CCFM1397 to treat orthodontic-induced excessive bone resorption. The paracasei paracasei of the present invention can regulate osteoblasts to secrete osteoprotegerin (OPG) 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 relieve bone resorption, inhibit the level of inflammatory factors in gingival tissue, and protect the stability of periodontal tissue.

[0006] The present invention provides a Ligilactobacillus salivarius and its postbiotics that can improve the activity of osteoblasts, relieve bone resorption and have the effect of relieving periodontal tissue damage at the same time. The taxonomic name of the Ligilactobacillus salivarius CCFM1397 is Ligilactobacillus salivarius, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on June 14, 2024, with the deposit number GDMCC No: 64761 and the deposit address being the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

[0007] In one embodiment of the present invention, the Ligilactobacillus salivarius CCFM1397 is derived from a dental plaque sample of a healthy volunteer oral cavity in Wuxi, Jiangsu. After sequencing analysis, its 16S rDNA sequence is shown in SEQ ID NO.1. The sequenced sequence is compared with nucleic acid sequences in NCBI, and the result shows that the strain is Ligilactobacillus salivarius, named Ligilactobacillus salivarius CCFM1397.

[0008] In one embodiment of the present invention, the colonies of the Ligilactobacillus salivarius CCFM1397 on MRS medium are small, semi-transparent, white and round.

[0009] The present invention also provides a microbial preparation containing the Ligilactobacillus salivarius 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 postbiotics prepared from the Ligilactobacillus salivarius CCFM1397, and the postbiotics include dead cells, lysates and / or supernatants of the Ligilactobacillus salivarius CCFM1397.

[0012] The present invention also provides products containing the Ligilactobacillus salivarius CCFM1397, and / or containing the microbial preparation, and / or containing the postbiotics, and the products include foods, drugs, health products or daily chemical products.

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

[0014] In one embodiment of the present invention, the components of the drug 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, wetting agent, disintegrant, binder, lubricant or flavoring agent.

[0017] In one embodiment of the present invention, the daily chemical products include mouthwash, mouthwash tablets, toothpaste, tooth powder, dental patches, periodontal gels, tooth gels or dental waxes.

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

[0019] The present invention also provides the use of Lactobacillus salivarius CCFM1397 and / or its postbiotics in the preparation of a medicament 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, which is to use the above-mentioned Lactobacillus salivarius CCFM1397.

[0021] The present invention also provides the use of Lactobacillus salivarius CCFM1397 in the preparation of a medicament for regulating oral inflammation or in the preparation of a medicament for relieving bone resorption.

[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 medicament includes oral sprays, tablets, capsules, pastes, oral liquids or lyophilized powders.

[0024] The present invention also provides the use of Lactobacillus salivarius CCFM1397 or the microbial preparation in the preparation of a nursing product for preventing and / or treating oral inflammation.

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

[0026] In one embodiment of the present invention, the nursing products include mouthwash, mouthwash tablets, toothpaste, tooth powder, dental patches, periodontal gels, tooth gels or dental waxes.

[0027] The present invention also provides the use of Lactobacillus salivarius CCFM1397 in the preparation of fermented foods.

[0028] Beneficial effects

[0029] 1. The present invention provides a strain of Ligilactobacillus salivarius CCFM1397, which is isolated from the oral dental plaque of healthy adolescents. The Ligilactobacillus salivarius CCFM1397 can promote the activity of osteoblasts in vitro, increase the expression of osteoprotegerin (OPG) in osteoblasts (the expression level reaches 366.83 ng / L), prevent the binding 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 Ligilactobacillus salivarius CCFM1397 provided by the present invention has the effect of alleviating orthodontic-induced bone resorption and simultaneously inhibiting periodontal inflammation, which is specifically manifested as follows:

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

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

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

[0034] (4) Reducing the expression of RANKL in the serum of rats.

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

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

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

[0038] 3. Ligilactobacillus salivarius is a kind of probiotic, which has currently been included in the List of Strains Allowed to be Used in Foods issued by the Ministry of Health. Therefore, the Ligilactobacillus salivarius CCFM1397 screened by the present invention will not bring any side effects to the human body, and has high safety when used in products (such as drugs or daily chemical products, etc.) for preventing and / or treating orthodontic-induced bone resorption. Description of the Drawings

[0039] Figure 1 : Diagram showing the effects of fermentation supernatants and lysates of different bacteria on osteoprotegerin (OPG) in MC3T3-E1 cells;

[0040] Figure 2 Effect of Lactobacillus salivarius CCFM1397 on the expression of RUNX gene in osteoblasts;

[0041] Figure 3 Effect of Lactobacillus salivarius CCFM1397 on the body weight (A) and OTM (B) of rats with orthodontic-induced bone resorption;

[0042] Figure 4 Effect of Lactobacillus salivarius CCFM1397 on the gingival index (A) and probing depth (B) of rats with orthodontic-induced bone resorption;

[0043] Figure 5 Effect of Lactobacillus salivarius CCFM1398 on the colonization of Prevotella intermedia in the oral cavity of rats with orthodontic-induced bone resorption;

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

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

[0046] Figure 8 Effect of Lactobacillus salivarius CCFM1397 on the bone microstructure of rats with orthodontic-induced bone resorption, (A) Microstructure diagram of the teeth of the rat model, (B) Ratio of bone volume to total volume of the first molar of the rat;

[0047] Figure 9 HE staining diagram of rats with orthodontic-induced bone resorption treated with Lactobacillus salivarius CCFM1397;

[0048] Figure 10 Trap staining diagram (A) and number of osteoclasts (B) of rats with orthodontic-induced bone resorption treated with Lactobacillus salivarius CCFM1397;

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

[0050] Biomaterial preservation

[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] In Examples 1, 2, and 3, rat osteoprogenitor cells MC3T3-E1 were preserved in the Biotechnology Center of the School of Food Science and Technology, Jiangnan University.

[0061] In Example 4, 24 SPF-grade Wistar rats, male, 7 weeks old, weighing 235 - 255 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK(Beijing)2012 - 0001).

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

[0063] In Examples 1, 2, and 3, preparation of Lactobacillus live bacteria suspension: Lactobacillus salivarius CCFM1397 and BNCC138618 were inoculated in MRS liquid medium at an inoculation amount of 2% (v / v), cultured at 37 °C for 24 h, and then the bacterial concentration was adjusted to 1×10 9 CFU / mL.

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

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

[0066] In Example 4, preparation of Lactobacillus salivarius live bacteria suspension: Lactobacillus salivarius CCFM1397 and BNCC138618 were inoculated in MRS liquid medium at an inoculation amount of 2% (v / v), cultured at 37 °C for 24 h, the cells were collected by centrifugation at 8500 g for 20 min, washed twice with sterile saline, resuspended in a 10% aqueous solution of skim milk, and thoroughly mixed and then freeze-dried. Before the animal experiment, the Lactobacillus freeze-dried powder was counted and reconstituted with PBS to a bacterial suspension concentration of 1×10 9 CFU / mL.

[0067] Preparation of Lactobacillus salivarius dead bacteria 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 h, the fermentation broth was inactivated at 105 °C for 10 min, and the inactivation effect was checked by plate coating. After heat treatment, the bacterial solution was centrifuged at 8500 g for 20 min, washed twice with sterile saline, resuspended in a 10% skim milk solution, thoroughly mixed and then freeze-dried, and resuspended with PBS to a concentration of 1×10 9CFU / mL.

[0068] Preparation of Prevotella intermedia live bacterial suspension: Prevotella intermedia ATCC 25611 was anaerobically 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 for subsequent experiments.

[0069] Example 1 Isolation and Identification of Lactobacillus salivarius CCFM1397

[0070] Oral swab samples of healthy adolescents were collected and placed in an EP tube containing 1 mL of sterile normal saline. 0.2 mL was aspirated into 1.8 mL of sterile normal saline to obtain 10 -1 dilution, and then 0.5 mL of 10 -1 dilution was aspirated into 4.5 mL of normal saline to obtain 10 -2 dilution. According to this operation, 10 -3 , 10 -4 , 10 -5 , 10 -6 gradient dilutions were obtained in turn. 1 mL of 10 -4 , 10 -5 , 10 -6 dilutions were taken and placed in petri dishes, and MRS solid medium was poured in and gently mixed. After the medium solidified, it was incubated at 37 °C in an inverted position for 48 h.

[0071] Colonies with different morphologies were selected on the MRS plate for streak purification. The purified single colonies were picked and inoculated into 10 mL of liquid medium and cultured at 37 °C for 48 h. 1.5 mL of the cultured bacterial solution was taken, centrifuged at 6000 r / min for 3 min, the supernatant was discarded, and it was washed 3 times with 1.5 mL of sterile water and then resuspended in 1.5 mL of sterile water to be used as a template for bacterial species identification. A PCR system with a volume of 20 μL was set up, in which 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 were added. The primer information is shown in Table 1.

[0072] Table 1: Primer Information Table

[0073]

[0074] After sequencing, the 16S rDNA sequence information obtained by amplification was (SEQ ID No.1):

[0075]

[0076] The obtained 16S rDNA sequences were subjected to species identification through BLAST of NCBI (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 alignment results, and considering the values, it is preferably identified as the species marked with Complete genome. According to the Blast results, this bacterium was identified as Ligilactobacillus salivarius. Subsequently, for the accurately identified bacterial strain, 1.5 mL of the bacterial liquid was taken into a 2 mL bacterial strain preservation tube. After centrifugation at 6000 r / min for 3 min, the supernatant was removed in a laminar flow hood. 1 mL of 30% sterile glycerol was added, and after thoroughly mixing 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 Culture Collection of Microorganisms on June 14, 2024, with the deposit number GDMCC No: 64761.

[0077] Example 2 Ability of Ligilactobacillus salivarius to Promote the Expression of Osteoprotegerin (OPG) in Osteoblasts In Vitro

[0078] 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.

[0079] MC3T3-E1 cells were taken out from liquid nitrogen and quickly thawed 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 and cultured under the conditions of 37°C and 5% CO 2 . The medium was changed every 48 h. After cell counting, the cells were seeded on a 6-well plate at a density of 1×10 4 cells / well. 2 mL of osteogenic induction medium containing 5% (v / v) bacterial supernatant or lysate was added to each well, and the morphological changes of the cells were observed regularly. MC3T3-E1 cells differentiated into osteoblasts usually became flatter. After 21 days of culture, the cell culture supernatants of each group were collected, and the expression of OPG was determined using the Senbeijia OPG Elisa Kit. The grouping information for the cell experiments is shown in Table 2.

[0080] Table 2: Grouping Information Table

[0081]

[0082] The expression of OPG in MC3T3-E1 cells after the intervention of each group was as follows Figure 1 shown. The concentration of OPG in the cell culture supernatant of the control group was 117.78 ng / L, and the OPG concentration in the Lactobacillus salivarius combined with Lactobacillus BNCC138618 lysate group was significantly lower than that of the control group (p < 0.01). The content of OPG in the Lactobacillus salivarius combined with Lactobacillus CCFM1397 lysate group was 366.83 ng / L, which was significantly higher than that of the control group (p < 0.0001). The above results indicate that the cell lysate of Lactobacillus salivarius combined with Lactobacillus CCFM1397 can significantly increase the expression of OPG in the cell culture supernatant of induced MC3T3-E1 cells.

[0083] In summary, the selected Lactobacillus salivarius combined with Lactobacillus CCFM1397 can increase the expression of osteoblast OPG in vitro.

[0084] Example 3 Ability of Lactobacillus salivarius combined with Lactobacillus to promote the expression of osteoblast bone RUNX gene in vitro

[0085] The culture and induction test method of MC3T3-E1 cells was the same as that in Example 2. After inducing differentiation for 21 days at an initial concentration of 1×10 4 cells / mL, the cell RNA was extracted according to the detailed instructions provided by the Novizan RNA extraction kit, and the operations were carried out step by step. All experimental steps were carried out in an RNase-free environment to avoid RNA degradation. The extracted RNA was measured for purity (A260 / A280 ratio) and concentration using a NanoDrop spectrophotometer.

[0086] The expression of RUNX gene in MC3T3-E1 cells after the intervention of each group was as follows Figure 2 shown. The Fold Change value of the RUNX gene in the Lactobacillus salivarius combined with Lactobacillus CCFM1397 lysate group was 6.14, which was significantly higher than that of the control group (p < 0.01).

[0087] In summary, the selected Lactobacillus salivarius combined with Lactobacillus CCFM1397 can increase the expression of osteoblast RUNX gene in vitro and promote the activity of osteoblasts.

[0088] Example 4 Application of Lactobacillus salivarius combined with Lactobacillus CCFM1397 in relieving orthodontic-induced bone resorption in rats

[0089] Twenty-four SPF-grade Wistar rats, male, 7 weeks old, weighing 235 - 255 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

[0090] The experimental grouping is shown in Table 3.

[0091] Table 3: Animal experiment plan and grouping

[0092]

[0093] Rats were randomly divided into 4 groups according to body weight, with 6 rats in each group. All rats were normally fed throughout the experiment.

[0094] Except for the control group, orthodontic models were established in the other groups on day 0 (specific operation: apply a traction force of 50 g between the first molar and the central incisor of the rat using a nickel-titanium spring. This step requires first using a grinding tool to grind a groove perpendicular to the dental arch midline on the maxillary central incisor for fixing the stainless steel ligature wire. Both the maxillary central incisor and the first molar need to be fixed with ligature silk using fluid resin, and ensure that the silk fracture does not damage the epithelial barrier. Except for the left first molar, the occlusal surfaces of the other upper molars are fixed with fluid resin and the occlusal surfaces are raised to eliminate the occlusal interference of the orthodontically moved maxillary first molar), and all need to receive an infection of Prevotella intermedia for a total of 12 days (days 1-6, days 8-13) (specific operation method of infection: use a needleless syringe to aspirate 1 mL of Prevotella intermedia suspension with a concentration of 1×10 9 CFU / mL, slowly irrigate it into the rat's oral cavity, and place it in the cage after staying for 1 minute).

[0095] For the live bacteria group and dead bacteria group of Lactobacillus salivarius CCFM1397, 6 hours after the infection of Prevotella intermedia, the oral cavities of the rats were respectively irrigated with 1 mL of live bacteria and dead bacteria suspensions of Lactobacillus salivarius CCFM1397, and the concentration of the suspension was 1×10 9 CFU / mL. The concentration of the dead bacteria group was the same as that of the live bacteria, and it was inactivated by high-pressure steam at 105 °C for 10 minutes, and the inactivation effect was checked by plate coating.

[0096] All rats were measured for gingival index and probing depth of the periodontal tissue on day 7 and day 14, and the saliva of the rats was collected with a sterile swab and stored in a 2 mL centrifuge tube containing 1 mL of 30% glycerol. At the same time, the body weight of the rats, the distance from the first molar to the central incisor (OTM), gingival index and probing depth were recorded.

[0097] (1) Changes in rat body weight and tooth movement distance (OTM)

[0098] As Figure 3As shown in Figure A, the rats showed a significant weight loss on the 7th day after orthodontic model establishment (p<0.0001). After intervention with live and heat-killed Lactobacillus salivarius CCFM1397, the body weights of the rats recovered significantly compared with the model group. The body weights of the live bacteria group and the heat-killed bacteria group were 0.83 times and 0.94 times that of the model group, respectively. On the 14th day after model establishment, the rats still showed a significant weight loss (p<0.0001). After intervention with live Lactobacillus salivarius CCFM1397, the body weights of the rats recovered significantly compared with the model group. The body weights of the live bacteria group and the heat-killed bacteria group were 1.08 times and 1.15 times that of the model group, respectively, and this result was statistically significant (p<0.01). There was no significant difference in OTM among groups ( Figure 3 B), indicating that Lactobacillus salivarius does not cause excessive tooth movement and remodeling.

[0099] (2) Changes in gingival index and probing depth of periodontal tissue

[0100] Changes in the gingival index can reflect changes in the degree of gingival inflammation. A decrease in the gingival index means an improvement in symptoms such as gingival swelling and bleeding. Changes in the probing depth are usually related to the improvement of periodontal tissue. A shallower probing depth implies healthy periodontal tissue and a lower risk of periodontal disease.

[0101] The changes in the gingival index and periodontal probing depth are as Figure 4 shown. On the 7th day, the gingival index of the live Lactobacillus salivarius CCFM1397 group decreased from 3 to 1.6, and the probing depth decreased from 1.33 mm to 0.2 mm, showing significant decreases compared with the model group and remaining at a low level on the 14th day. The effect of the heat-killed Lactobacillus salivarius CCFM1397 group on the gingival index was similar to that of the live bacteria group, with significant decreases compared with the model group (p<0.01). This indicates that after oral irrigation with Lactobacillus paracasei CCFM1397, the level of gingival inflammatory response in the rats decreased, the problem of periodontal bleeding was alleviated, and the periodontal pocket depth also decreased significantly.

[0102] In summary, Lactobacillus salivarius CCFM1397 can reduce the incidence of gingival tissue bleeding and gingival inflammation and reduce the formation of periodontal pockets.

[0103] (3) Colonization of Prevotella intermedia in the oral cavity of rats

[0104] As Figure 5 shown, on the 14th day after orthodontic model establishment in rats, both live and heat-killed Lactobacillus salivarius CCFM1397 could significantly reduce the colonization of Prevotella intermedia in the oral cavity of rats.

[0105] (4) Secretion of inflammatory factors IL-1β, IL-6, TNF-α, MPO in gingival tissue of rats and expression of OPG and RANKL in serum

[0106] The experimental period was 13 days (days 0 - 13). All rats were sacrificed on the 14th day, and serum, gingival tissue, and alveolar bone tissue were taken for subsequent histopathological analysis of the experimental tissues and microscopic analysis of the alveolar bone structure. The levels of soluble interleukin (IL-6, IL-1β) and tumor necrosis factor (TNF-α), myeloperoxidase (MPO) in the gingival tissue, osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in the serum were detected.

[0107] Determination method: On the 14th day, before sacrificing the rats, 10 mL of abdominal aortic blood was taken from the rats. After standing for 30 min, it was centrifuged at 3000 r / min for 15 min at 4°C, and the supernatant was taken as the blood sample. After sacrificing the rats, the gingival tissues on the lingual and buccal sides of the first molar were dissected. 20 mg of gingival tissue was weighed and mixed with 180 μL of PBS buffer, placed in a 1.5 mL centrifuge tube, 3 grinding zirconium beads were added, and it was ground in a high-throughput tissue grinder. Subsequently, the tissue homogenate was centrifuged at 12000 g for 15 min at 4°C, the supernatant was collected, and stored at -20°C.

[0108] Subsequently, according to the kit instructions, the levels of RANKL, OPG in the serum and IL-1β, TNF-α, IL-6, MPO in the gingival tissue were determined.

[0109] The massive secretion of IL-1β, IL-6, TNF-α, and MPO causes periodontal tissue inflammation and destroys the periodontal tissue itself, accelerates the entry of pathogenic bacterial toxins, and promotes the occurrence of excessive bone resorption by activating multiple downstream signaling pathways including NF-κB. OPG is a soluble protein belonging to the tumor necrosis factor receptor superfamily, and 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, prevent RANKL from activating its receptor RANK, thereby inhibiting the differentiation and activity of osteoclasts and reducing bone resorption.

[0110] The secretion of inflammatory factors IL-1β, IL-6, TNF-α, and MPO in the gingival tissue of rats was as follows Figure 6As shown in the figure. The level of IL-1β in the control group was 6.21 ng / L. After orthodontic treatment and pathogenic bacteria stimulation, the level of IL-1β in the model group increased significantly to 25.30 ng / L. After intervention with saliva combined with live Lactobacillus CCFM1397, the secretion of IL-1β decreased to 15.01 ng / L (p<0.05). The level of IL-6 in the control group was 433.7 ng / L. The level of IL-6 in the model group increased significantly to 660.17 ng / L. After intervention with saliva combined with Lactobacillus CCFM1397, the secretion of IL-6 decreased significantly. 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 level of TNF-α in the control group was 92.00 ng / L. The concentration in the model group increased significantly to 324.35 ng / L. After intervention with saliva combined with live Lactobacillus CCFM1397, the level of TNF-α was significantly lower than that in the model group. The levels in the live bacteria group and the dead bacteria group were 211.32 ng / L and 147.63 ng / L respectively. The level of MPO in the control group was 80.75 ng / L. The level of MPO in the model group was 128.94 ng / L. The levels in 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, both of which were significantly lower than that in the model group. The above results indicate that saliva combined with Lactobacillus CCFM1397 can effectively reduce the inflammatory level of rat gingival tissue.

[0111] The expression of OPG and RANKL in rat serum was as Figure 7 shown. The level of OPG in the serum of the control group was 657.00 ng / L. The level of OPG in the serum of the model group increased significantly to 1130.33 ng / L. After intervention with dead Lactobacillus CCFM1397 combined with saliva, the serum OPG level decreased significantly to 854.46 ng / L (p<0.05). The level of RANKL in the serum of the control group was 190.58 pmol / L. The level of RANKL in the serum of the model group was 301.57 pmol / L. After intervention with Lactobacillus CCFM1397 combined with saliva, the levels in both the live bacteria group and the dead bacteria group decreased to about 230 pmol / L, which were significantly lower than that in the model group (p<0.05).

[0112] To sum up, saliva combined with Lactobacillus CCFM1397 can reduce the secretion of inflammatory factors and MPO in gingival tissue, thereby reducing periodontal tissue inflammation and alleviating bone resorption. After the establishment of the orthodontic model, the level of bone resorption in rats increased significantly. However, 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.

[0113] (5) Microstructure of rat alveolar bone tissue

[0114] Bone microstructure analysis: After the rats were sacrificed, the right maxilla tissues of the rats were dissected and placed in 4% paraformaldehyde solution for fixation for 48 h. The fixed maxilla of the rats was washed with PBS and air-dried. A high-resolution Micro-CT imaging system was used to scan the maxilla samples. 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 bone volume (BV) to total alveolar bone volume (TV) (BV / TV) at the modeling site was measured and calculated.

[0115] Histopathological observation: The alveolar bone tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into 5-μm thick sections, stained with hematoxylin and eosin (HE) and Trap. The alveolar bone tissue section samples were observed under a pathological section scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

[0116] Through microCT technology, the root resorption of the first molar can be visually observed. By calculation, the ratio of the bone volume (BV) of the first molar in the alveolar bone to the total volume (TV) can be obtained. This ratio is related to the risk of alveolar bone resorption. The lower the ratio, the higher the risk of bone resorption.

[0117] The microstructure of the alveolar bone tissue of rats is as Figure 8 shown. The BV / TV ratio at the apex of the first molar in the control group was 43.38%, and the BV / TV ratio at the apex of the first molar in the model group was significantly reduced to 24.81% (p < 0.001). After intervention with Lactobacillus salivarius CCFM1397, the BV / TV ratio in the live bacteria group increased to 35.326%, approaching that of the control group, and was significantly increased compared with the model group (p < 0.01). The BV / TV ratio in the heat-killed bacteria group was 24.90 ng / L%, showing no significant difference from the model group.

[0118] In summary, Lactobacillus salivarius CCFM1397 can increase the BV / TV ratio at the apex of the first molar in the alveolar bone, thereby alleviating orthodontic-induced bone resorption.

[0119] (6) Histopathological analysis of rat alveolar bone tissue

[0120] By performing HE staining and Trap staining on the rat alveolar bone tissue, the integrity, inflammation status, and the number and localization of osteoclasts per unit area of the alveolar bone tissue of rats in each group can be effectively evaluated.

[0121] HE staining can show the acute degree of inflammation and also observe the changes in tissue morphology. Such as Figure 9As shown, the periodontal tissue structure of the alveolar bone in the control group of rats was intact. After the maxilla slices were stained with HE, a complete periodontal tissue structure was shown. The gingiva was closely attached to the tooth surface, no periodontal pocket was formed, and no inflammatory cell aggregation was seen. The structures of the periodontal tissue and alveolar bone were uniform and no abnormalities were found. The maxilla slices of the model group of rats showed damage to the integrity of the periodontal tissue. A gap appeared between the gingiva and the tooth surface, forming a periodontal pocket. A large number of inflammatory cells were observed to aggregate in the apical region, 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 induced an inflammatory response and bone resorption in the periodontal tissue.

[0122] The HE staining results of the live and dead bacteria groups of Lactobacillus salivarius CCFM1397 showed that after the intervention, the tissue inflammatory infiltration was alleviated, the inflammatory cells no longer aggregated at the alveolar bone apex, and the damage to the periodontal tissue was also alleviated. This indicates that Lactobacillus salivarius can significantly relieve the symptoms of alveolar bone resorption in orthodontic model rats and inhibit the occurrence of inflammation.

[0123] Trap staining can effectively evaluate the integrity, inflammatory conditions, the number and localization of osteoclasts per unit area of the alveolar bone tissue in each group of rats. As Figure 10 shown, the number of osteoclasts in the model group was significantly increased compared with the control group (p < 0.01), mainly distributed at the apical bifurcation edge of the alveolar bone. The staining results of Lactobacillus salivarius CCFM1397 showed the effect that Lactobacillus salivarius CCFM1397 can relieve orthodontic-induced excessive bone resorption, making the osteoclasts no longer aggregate at the apical bifurcation of the alveolar bone. And under the 20-fold 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).

[0124] Although the present invention has been disclosed above with preferred embodiments, 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 protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Lactobacillus salivarius ( 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 the saliva combined with dead cells and lysate of 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 of the saliva-combined Lactobacillus CCFM1397 according to claim 1 is 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 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 a drug for preventing and / or treating oral inflammation or alleviating bone resorption, characterized in that: The dosage form of the medicine includes oral spray, tablet, capsule, paste, oral liquid or lyophilized powder; the oral inflammation is periodontitis or gingivitis.

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 is periodontitis or gingivitis; the care products include mouthwash, mouthwash grains, toothpaste, tooth cleaning powder, tooth stickers, periodontal gel, tooth gel or tooth wax.

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