Bone homeostasis regulating and controlling metaplasm prepared from lactobacillus paracasei CCFM1394 and application of bone homeostasis regulating and controlling metaplasm

Abstract

The invention discloses a Lactobacillus paracasei strain capable of relieving bone resorption and having an anti-inflammatory effect and a metagen thereof, and belongs to the technical field of microorganisms. The lactobacillus paracasei CCFM1394 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) is improved in vitro, and meanwhile, the expression of an RUNX gene of MC3T3-E1 is promoted; therefore, the lactobacillus paracasei 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 Lactobacillus paracasei CCFM1394 capable of alleviating bone resorption and having anti-inflammatory effects and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] Bone homeostasis refers to the dynamic balance between osteoblasts that maintain bone reconstruction and osteoclasts that dominate bone resorption in the skeletal system. This balance is essential for maintaining bone health. Bone homeostasis is mainly achieved through periodic bone reconstruction. The destruction of bone homeostasis will lead to the occurrence of diseases such as osteosclerosis and osteoporosis. Imbalance in oral bone homeostasis is closely related to the occurrence and development of oral destructive diseases such as periodontitis. Periodontitis is a chronic infectious disease of the periodontal supporting tissue caused by an imbalance of microbial flora in dental plaque, manifested by inflammatory cell infiltration, rupture of connective tissue attachment and alveolar bone resorption, which may increase the risk of loose teeth, increased tooth sensitivity, retrograde pulpitis and cardiovascular disease.

[0003] Macrophages play an important role in resisting periodontal pathogens and preventing and treating periodontal tissue damage. In periodontitis, the ratio of macrophages M1 / M2 increases, and there is a transition from M2 phenotype to M1 phenotype in macrophages in periodontal ligament and serum, and the M1 / M2 ratio is positively correlated with the severity of periodontitis. Periodontitis is often accompanied by alveolar bone resorption with abnormally enhanced osteoclast activity. The RANK / RANKL / OPG signaling pathway is closely related to bone resorption activity by participating in the differentiation and activation of osteoclasts. Inhibition of related signal transduction pathways can reduce alveolar bone resorption. Inflammatory factors such as IL-1, IL-6 and IL-8 induce periodontitis in the body by inducing PGE2 synthesis and activating osteoclasts. OPG can protect cementum and reduce root resorption, and is an important control factor in maintaining alveolar bone homeostasis.

[0004] At present, there are many patents involving the use of biological agents to treat periodontitis. These patents generally focus on the use of microorganisms or cell metabolites to regulate the proportion of gingival tissue macrophages or locally to promote the activity of osteoblasts and inhibit the function of osteoclasts, thereby alleviating periodontitis. Patent CN118021782A confirms that α-ketoglutaric acid can inhibit periodontitis and periodontal tissue inflammation caused by systemic diseases such as periodontitis by inhibiting osteoclast differentiation, inhibiting the activation of M1 macrophages, promoting the activation of M2 macrophages, and regulating the proportion of macrophages. Similarly, patent CN114569634B confirms that antler mesenchymal stem cell conditioned medium can significantly reduce alveolar bone resorption and gingival tissue inflammation by reducing the activity of osteoclasts and changing the ratio of M1 and M2. However, considering the complex environment of the oral cavity, the application of these substances in alleviating periodontitis still needs further verification. Therefore, it is urgent to develop a biological bacterial agent that can be used to treat periodontitis caused by bone homeostasis imbalance. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention provides a strain of Lactobacillus paracasei and its postbiotics which can alleviate bone resorption and have anti-inflammatory effects, aiming to solve the technical problem that the prior art lacks a biological bacterial agent that can be used to treat periodontitis caused by bone homeostasis imbalance.

[0006] The first technical solution provided by the present invention is a strain of Lactobacillus paracasei CCFM1394, the taxonomic name of the Lactobacillus paracasei CCFM1394 is Lactobacillus paracasei, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with a deposit number of GDMCC No.64758, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The Lacticaseibacillus paracasei CCFM1394 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 Lacticaseibacillus paracasei, and is named Lacticaseibacillus paracasei CCFM1394.

[0008] The colony morphology of the Lacticaseibacillus paracasei CCFM1394 on the MRS culture medium is characterized by small, translucent white circles.

[0009] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus paracasei CCFM1394 described in the first technical solution.

[0010] In certain embodiments, in the microbial preparation, the bacterial concentration of Lactobacillus paracasei CCFM1394 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0011] Furthermore, in the microbial preparation, the bacterial concentration of the Lactobacillus paracasei CCFM1394 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0012] The third technical solution provided by the present invention is a postbiotic, which includes one or more of the fermentation supernatant, bacterial lysate and fermentation broth of Lactobacillus paracasei 1394 described in the first technical solution.

[0013] In certain embodiments, the postbiotic is a fermentation supernatant obtained by centrifuging the Lactobacillus paracasei CCFM1394 after inoculating it into an MRS medium for culturing.

[0014] In certain embodiments, the postbiotic is obtained by high-pressure homogenization and membrane filtration of the fermentation broth after inoculating the Lactobacillus paracasei CCFM1394 in MRS medium to obtain a bacterial lysate.

[0015] In certain embodiments, the postbiotics can be dried into powder or used directly by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, fluidized bed drying, etc.

[0016] The fourth technical solution provided by the present invention is a product containing the Lactobacillus paracasei CCFM1396 described in the first technical solution, or containing the postbiotics described in the third technical solution.

[0017] In certain embodiments, the bacterial concentration of Lactobacillus paracasei CCFM1396 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0018] Furthermore, the bacterial concentration of Lactobacillus paracasei CCFM1396 in the product is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0019] In certain embodiments, the product is a medicine or a daily chemical product.

[0020] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0021] In certain embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0022] In certain embodiments, the pharmaceutical excipient comprises an excipient and an additive.

[0023] In certain embodiments, the pharmaceutical excipients include one or more of a stabilizer, a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a disintegrant, and a lubricant.

[0024] In certain embodiments, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0025] In certain embodiments, the daily chemical product is a mouthwash or toothpaste containing the Lactobacillus paracasei CCFM1394.

[0026] The present invention also provides the use of the Lactobacillus paracasei CCFM1394 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotics described in the third technical solution in preparing a product for preventing and / or treating bone homeostasis.

[0027] The present invention provides a fifth technical solution, which is the use of Lactobacillus paracasei CCFM1394 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotic described in the third technical solution in the preparation of a product for preventing and / or treating orthodontically induced bone homeostasis imbalance.

[0028] In certain embodiments, in the product, the viable count of Lactobacillus paracasei CCFM1394 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0029] Furthermore, in the product, the viable count of Lactobacillus paracasei CCFM1394 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0030] In certain embodiments, the product is a medicine or a daily chemical product.

[0031] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0032] In certain embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0033] In certain embodiments, the pharmaceutical excipient comprises an excipient and an additive.

[0034] In certain embodiments, the pharmaceutical excipients include one or more of a stabilizer, a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a disintegrant, and a lubricant.

[0035] In certain embodiments, the daily chemical product is a mouthwash or toothpaste containing the Lacticaseibacillus paracasei CCFM1394.

[0036] The present invention also provides application of the Lactobacillus paracasei CCFM1394 in preparing a medicine for preventing / alleviating bone homeostasis imbalance.

[0037] The present invention also provides the use of Lactobacillus paracasei CCFM1394 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of fermented food.

[0038] The sixth technical solution provided by the present invention is the use of Lactobacillus paracasei CCFM1394 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotic described in the third technical solution in the preparation of a product for promoting osteoblasts to express RUNX transcription factor.

[0039] The seventh technical solution provided by the present invention is a method for promoting osteoblasts to express RUNX transcription factor in vitro, wherein the method comprises co-culturing osteoblasts with the Lactobacillus paracasei CCFM1394 described in the first technical solution, or the microbial preparation described in the second technical solution, or the postbiotics described in the third technical solution.

[0040] The beneficial effects of the present invention are as follows:

[0041] 1. The present invention provides a strain of Lactobacillus paracasei CCFM1394, which is isolated from dental plaque of healthy adolescents, and has the effects of relieving bone resorption and inhibiting periodontal inflammation, which is specifically embodied in: (1) promoting the high expression of OPG in MC3T3-E1 cells, preventing the combination of RANKL and RANK in alveolar bone tissue, and inhibiting the activity and differentiation of osteoclasts. (2) and increasing the expression of RUNX gene in MC3T3-E1 cells.

[0042] Therefore, this Lactobacillus paracasei CCFM1394 has great application prospects in the preparation of products (such as medicines or daily chemical products, etc.) for preventing and / or treating bone resorption.

[0043] 2. Lactobacillus paracasei 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 Lactobacillus paracasei CCFM1394 screened by the present invention will not cause any side effects to the human body, and when used in products (such as medicines or daily chemical products, etc.) for preventing and / or treating orthodontically induced bone resorption, it has higher safety.

[0044] Biomaterial Deposit

[0045] A strain of Lacticaseibacillus paracasei CCFM1394, taxonomically named Lacticaseibacillus paracasei, was deposited in the Guangdong Provincial Microbiological Culture Collection on June 14, 2024, with the deposit number GDMCC No.64758, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 :The effect of different bacterial fermentation supernatants and lysates on osteoprotegerin (OPG) in MC3T3-E1 cells.

[0047] Figure 2 :The effect of Lactobacillus paracasei on RUNX gene expression in osteoblasts.

[0048] In the figure, * indicates significant difference, where *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0050] The culture medium involved in the following examples is as follows:

[0051] MRS medium in Examples 1, 2 and 3: 5.0 g / L yeast powder, 10.0 g / L beef extract, 10.0 g / L peptone, 20.0 g / L glucose, 2.0 g / L anhydrous sodium acetate, 2.0 g / L diammonium hydrogen citrate, 2.6 g / L dipotassium hydrogen phosphate, 0.25 g / L manganese sulfate monohydrate, 0.5 g / L magnesium sulfate heptahydrate, 1 mL / L Tween-80, pH 6.2-6.4.

[0052] 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).

[0053] 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).

[0054] The strains and cells involved in the following examples are as follows:

[0055] In Examples 1, 2 and 3, Lactobacillus paracasei CCFM1394 was from the strain bank of the Biotechnology Center of Jiangnan University, and Lactobacillus paracasei ATCC334 was purchased from the American Type Culture Collection.

[0056] The mouse osteoblast precursor cells MC3T3-E1 in Examples 1 and 2 were deposited in the Biotechnology Center of the School of Food Science and Engineering, Jiangnan University.

[0057] The preparation of the experimental samples involved in the following examples is as follows:

[0058] In Examples 1, 2, and 3, the lactobacillus fermentation broth was prepared by inoculating Lactobacillus paracasei CCFM1394 and ATCC334 in MRS liquid medium at a 2% (v / v) inoculum amount, culturing at 37° C. for 24 h, and adjusting the bacterial concentration to 1×10 9 CFU / mL.

[0059] Preparation of supernatant: The concentration of 1×10 9 The lactobacillus fermentation broth with CFU / mL 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.

[0060] Lysis buffer preparation: the concentration is 1×10 9 The CFU / mL lactobacillus fermentation broth suspension 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., bacterial lysate, was collected and stored at -20°C.

[0061] Example 1 Isolation and identification of Lactobacillus paracasei CCFM1394

[0062] 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.

[0063] 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.

[0064] Table 1: Primer information

[0065]

[0066] The amplified 16S rDNA sequence was shown in SEQ ID NO: 1.

[0067] 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 paracasei. Then, 1.5 mL of the identified bacteria were taken into a 2 mL strain storage tube, centrifuged at 6000 r / min for 3 minutes, the supernatant was removed in a 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.

[0068] Example 2 Ability of Lactobacillus paracasei to promote osteoblast osteoprotegerin (OPG) expression in vitro

[0069] 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.

[0070] 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 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, 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.

[0071] Table 2: Group information table

[0072]

[0073] 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 supernatant and lysate of Lactobacillus paracasei CCFM1394 significantly increased the OPG concentration in the cell culture supernatant, which was 236.8 ng / L and 347.24 ng respectively (p<0.0001). After the intervention of Lactobacillus paracasei ATCC334, the OPG content decreased significantly.

[0074] In summary, the screened Lactobacillus paracasei CCFM1394 can increase the expression of osteoprotegerin in osteoblasts in vitro.

[0075] Example 3 Ability of Lactobacillus paracasei to promote RUNX gene expression in osteoblasts in vitro

[0076] RUNX transcription factor is the most important transcription factor in the growth process of osteoblasts. It increases new bone formation and alleviates bone resorption by regulating the differentiation of downstream genes (including alkaline phosphatase, calcium regulatory protein), as well as the maturation of differentiated cells.

[0077] The MC3T3-E1 cell culture and induction test methods were the same as in Example 2. 4 After 21 days of differentiation induction with an initial concentration of 100 / mL, cellular RNA was extracted according to the detailed instructions provided by the Novozyme RNA extraction kit, and the operation was performed step by step. All experimental steps were performed in an RNase-free environment to avoid RNA degradation. The extracted RNA was measured for purity (A260 / A280 ratio) and concentration using a NanoDrop spectrophotometer.

[0078] The expression of RUNX gene in MC3T3-E1 cells after intervention with Lactobacillus paracasei CCFM1394 Figure 2 The Fold Change value of the RUNX gene in the lysate group was 5.91, and the expression of the RUNX gene in this group was significantly higher than that in the control group (p<0.001).

[0079] In summary, the screened Lactobacillus paracasei CCFM1394 can increase the expression of RUNX gene in osteoblasts in vitro.

[0080] Although the present invention has been disclosed as above in terms of 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 limited to the following.

Claims

1. A strain of Lactobacillus paracasei CCFM1394, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the deposit number GDMCC No.64758.

2. the microbial preparation of Lactobacillus paracasei CCFM1394 described in claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the bacterial concentration of Lactobacillus paracasei CCFM1394 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A postbiotic, characterized in that: The postbiotics include one or more of the fermentation supernatant, bacterial lysate and fermentation broth of the Lactobacillus paracasei CCFM1394 according to claim 1.

5. The postbiotic according to claim 4, characterized in that The postbiotics are fermentation supernatant obtained by centrifugation after inoculating the Lactobacillus paracasei CCFM1394 in MRS medium for cultivation; or the postbiotics are fermentation broth obtained by high-pressure homogenization and membrane filtration after inoculating the Lactobacillus paracasei CCFM1394 in MRS medium for cultivation to obtain bacterial lysate.

6. A product, characterized in that Contains the live strain and / or inactivated strain of Lactobacillus paracasei CCFM1394 according to claim 1, or contains the postbiotics according to claim 4 or 5.

7. The product according to claim 6, characterized in that The products are medicines or daily chemical products.

8. Use of the Lactobacillus paracasei CCFM1394 according to claim 1, or the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4 or 5 in preparing a medicament for regulating gingival tissue inflammation function and alleviating bone resorption.

9. Use of the Lactobacillus paracasei CCFM1394 according to claim 1, or the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4 or 5 in the preparation of a product for promoting the expression of RUNX transcription factor in osteoblasts.

10. A method for promoting osteoblasts to express RUNX transcription factor in vitro, characterized in that: The method comprises co-culturing the Lactobacillus paracasei CCFM1394 described in claim 1, or the microbial preparation described in claim 2 or 3, or the postbiotics described in claim 4 or 5 with osteoblasts.

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