Bone homeostasis regulating and controlling metaplasm prepared from streptococcus salivarius CCFM1395 and application of bone homeostasis regulating and controlling metaplasm

Abstract

The invention discloses a bone homeostasis regulating metagen prepared from streptococcus salivarius CCFM1395 and application of the bone homeostasis regulating metagen, and belongs to the technical field of microorganisms. The streptococcus salivarius CCFM1395 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. Therefore, the streptococcus 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 postbiotic for regulating bone homeostasis prepared by a strain of Streptococcus salivarius CCFM1395 and application 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] Currently, 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 the systemic disease 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. Compared with biological preparations, probiotics have the advantages of high safety and non-dependence. The use of probiotics to prevent and / or treat periodontal inflammation or bone resorption has broad application prospects. However, no probiotics have been found that can effectively inhibit the activity and differentiation of osteoclasts. Summary of the invention

[0005] At present, no treatment method has been found in clinical practice that can effectively alleviate periodontitis-induced bone resorption. The present invention provides a new method for treating periodontitis caused by bone homeostasis imbalance using Streptococcus thermophilus CCFM1395, which has mild conditions and few side effects. The Streptococcus thermophilus of the present invention can regulate osteoblasts to secrete osteoprotegerin (OPG).

[0006] The present invention provides a thermophilic subspecies of Streptococcus salivarius and a postbiotic thereof that has the effects of relieving bone resorption and having anti-inflammatory effects. The thermophilic subspecies of Streptococcus salivarius (Streptococcus thermophilus) CCFM1395 is taxonomically named Streptococcus thermophilus, and has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with a deposit number of GDMCC No: 64759, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In one embodiment of the present invention, the thermophilic Streptococcus salivarius subspecies (Streptococcusthermophilus) CCFM1395 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 thermophilic Streptococcus salivarius subspecies (Streptococcusthermophilus) CCFM1395.

[0008] In one embodiment of the present invention, the colonies of the Streptococcus thermophilus CCFM1395 on the MRS culture medium are small translucent white circles.

[0009] The invention also provides a microbial preparation containing the thermophilic Streptococcus salivarius subspecies CCFM1395.

[0010] In one embodiment of the present invention, the number of cells of Streptococcus thermophilus CCFM1395 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 by the thermophilic Streptococcus salivarius subspecies CCFM1395.

[0012] In one embodiment of the present invention, the postbiotics contain the lysate and / or supernatant of the Streptococcus salivarius thermophilus subsp. CCFM1399.

[0013] The present invention also provides a method for preparing the postbiotics, comprising (a) or (b):

[0014] (a) culturing Streptococcus salivarius thermophilus subspecies CCFM1395 to a logarithmic growth phase, and then crushing the culture medium by high-pressure homogenization to obtain postbiotics;

[0015] (b) Cultivating Streptococcus salivarius thermophilus subspecies CCFM1395 to the logarithmic growth phase, collecting the fermentation supernatant, and obtaining postbiotics.

[0016] The present invention also provides products containing the thermophilic Streptococcus salivarius subspecies CCFM1395, or the microbial preparation, or the postbiotics, and the products include food, medicine, health care products or daily chemical products.

[0017] In one embodiment of the present invention, the food comprises fruit and vegetable products, bean products or dairy products.

[0018] In one embodiment of the present invention, the medicine includes biological medicine, chemical medicine or traditional Chinese medicine.

[0019] In one embodiment of the present invention, the components of the drug include Streptococcus thermophilus CCFM1395 and a carrier thereof.

[0020] In one embodiment of the present invention, the carrier is a pharmaceutically acceptable carrier.

[0021] 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.

[0022] 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.

[0023] The present invention also provides a method for producing osteoprotegerin, which comprises adding the lysate and / or supernatant of the thermophilic Streptococcus salivarius subspecies CCFM1395 into a culture environment of osteoblasts for culture.

[0024] The present invention also provides the use of the thermophilic Streptococcus salivarius subspecies CCFM1395, or the microbial preparation, or the postbiotic in producing osteoprotegerin.

[0025] Beneficial Effects

[0026] 1. The present invention provides a strain of Streptococcus thermophilus CCFM1395, which is isolated from dental plaque in the oral cavity of healthy adolescents and has the effects of alleviating bone resorption and inhibiting periodontal inflammation, which is specifically embodied in:

[0027] (1) By promoting the high expression of OPG in MC3T3-E1 cells, the binding of RANKL and RANK in alveolar bone tissue is prevented, thereby inhibiting the activity and differentiation of osteoclasts.

[0028] Therefore, this Streptococcus thermophilus CCFM1395 has great application prospects in the preparation of products (such as medicines or daily chemical products, etc.) for preventing and / or treating bone resorption.

[0029] 2. Streptococcus thermophilus is a kind of probiotics and has been included in the "List of Bacteria that Can Be Used in Food". Therefore, the Streptococcus thermophilus CCFM1395 screened by the present invention will not cause any side effects to the human body, and is safer when used in products (such as medicines or daily chemical products) for preventing and / or treating orthodontic-induced periodontitis and bone resorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 :The effect of different bacterial fermentation supernatants and lysates on osteoprotegerin (OPG) in MC3T3-E1 cells;

[0031] (In the figure, * indicates significant difference, among which *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).

[0032] Biomaterial Deposit

[0033] A strain of Streptococcus thermophilus CCFM1395, taxonomically named Streptococcus thermophilus, was deposited in Guangdong Provincial Microbiological Culture Collection on June 14, 2024, with the deposit number GDMCC No: 64759, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0034] 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.

[0035] The culture medium involved in the following examples is as follows:

[0036] The MRS medium in Examples 1 and 2 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.

[0037] The complete cell culture medium in Examples 1 and 2 is: 92% (v / v) α-MEM culture medium + 7% (v / v) fetal bovine serum + 1% (v / v) 100× mixed solution of penicillin and streptomycin (the content of penicillin in the mixed solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL).

[0038] The osteogenic induction medium in Examples 1 and 2 includes complete cell culture medium + sodium β-glycerophosphate (10 mM / L), ascorbic acid (50 μg / mL), and dexamethasone (100 nM / L).

[0039] The strains and cells involved in the following examples are as follows:

[0040] In Examples 1 and 2, Streptococcus salivarius thermophilus subsp. CCFM1395 was obtained from the strain bank of the Biotechnology Center of Jiangnan University. Prevotella intermedia ATCC 25611 and Streptococcus salivarius thermophilus subsp. ATCC19258 were purchased from the American Type Culture Collection.

[0041] 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.

[0042] The preparation of the experimental samples involved in the following examples is as follows:

[0043] In Examples 1 and 2, the live bacterial suspension was prepared by inoculating Streptococcus salivarius thermophilus subspecies CCFM1395 and ATCC19258 in MRS liquid culture medium at a 2% (v / v) inoculation amount, culturing at 37°C for 24 h, and adjusting the bacterial concentration to 1×10 9 CFU / mL.

[0044] Preparation of supernatant: The concentration of 1×10 9 The live bacterial suspension of 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.

[0045] Preparation of lysis solution: The prepared live lactobacillus bacterial 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 was collected to obtain the bacterial lysis solution, which was then stored at -20°C.

[0046] Example 1 Isolation and identification of thermophilic Streptococcus salivarius subspecies CCFM1395

[0047] 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 -1Dilute 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.

[0048] 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.

[0049] Table 1: Primer information

[0050]

[0051] The 16S rDNA sequence information obtained by sequencing and amplification is (SEQ ID No.1):

[0052] CTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAAC

[0053] GGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAG

[0054] CTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCAC

[0055] TACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGAC

[0056] GATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGA

[0057] CTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCA

[0058] ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACG

[0059] GGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAAC

[0060] TACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGT

[0061] AAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTT

[0062] CGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGC

[0063] GGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTA

[0064] ACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC

[0065] ACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCCGAAGCTA

[0066] ACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTG

[0067] ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACC

[0068] TTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCG

[0069] GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG

[0070] CAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTG

[0071] CCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCT

[0072] GGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCGAGCT

[0073] AATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGG

[0074] AATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACAC

[0075] ACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAG。

[0076] 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 the thermophilic subspecies of Streptococcus salivarius. Then, 1.5 mL of the bacterial solution was taken from the correctly identified bacteria and placed in a 2 mL bacterial culture storage tube. After centrifugation 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. The obtained strain was named Streptococcus thermophilus CCFM1395, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the collection number GDMCC No.64759.

[0077] Example 2 Ability of Streptococcus salivarius subsp. thermophilus to promote osteoblast osteoprotegerin (OPG) expression 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 removed from liquid nitrogen and thawed quickly in a 37°C water bath. The thawed cell suspension was added to a centrifuge tube containing 5 volumes of complete cell culture medium. After centrifugation, the supernatant was discarded and the cells were resuspended in 10 mL of complete culture medium. The cells were cultured at 37°C and 5% CO2, and the culture medium was replaced every 48 hours. After counting the cells, 1×10 4 The cells were seeded on a 6-well plate at a density of 10 cells / well. 2 mL of osteogenic induction medium containing 5% (v / v) bacterial supernatant or lysate was added to each well, 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 ElisaKit. The grouping information of the cell experiment is shown in Table 2.

[0080] Table 2: Group information table

[0081]

[0082] The expression of OPG in MC3T3-E1 cells after intervention in each group Figure 1The OPG concentration in the cell culture supernatant of the control group was 117.78 ng / L, and the OPG content of the cells significantly increased to 286.77 ng / L after intervention with the thermophilic Streptococcus salivarius subsp. CCFM1395 lysate, which was significantly increased compared with the control group (p < 0.0001).

[0083] In summary, the screened thermophilic Streptococcus salivarius subspecies CCFM1395 can increase the expression of OPG in osteoblasts in vitro.

[0084] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Streptococcus thermophilus CCFM1395, characterized in that: The thermophilic subspecies of Streptococcus salivarius CCFM1395 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024, with the collection number GDMCC No: 64759.

2. A microbial preparation containing the thermophilic Streptococcus salivarius subspecies CCFM1395 according to claim 1.

3. The postbiotic prepared by the thermophilic Streptococcus salivarius subspecies CCFM1395 according to claim 1, characterized in that: The postbiotics contain the lysate and / or supernatant of the thermophilic Streptococcus salivarius subspecies CCFM1395 according to claim 1.

4. A method for preparing the postbiotics according to claim 3, characterized in that: Include (a) or (b): (a) culturing Streptococcus salivarius thermophilus subspecies CCFM1395 to a logarithmic growth phase, and then crushing the culture medium by high-pressure homogenization to obtain postbiotics; (b) Cultivating Streptococcus salivarius thermophilus subspecies CCFM1395 to the logarithmic growth phase, collecting the fermentation supernatant, and obtaining postbiotics.

5. A product containing the thermophilic Streptococcus salivarius subspecies CCFM1395 according to claim 1, or the microbial preparation according to claim 2, or the postbiotic according to claim 3, 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.

7. The product according to claim 5, characterized in that The medicine also contains conventional pharmaceutical excipients and / or pharmaceutical carriers.

8. The product according to claim 5, characterized in that The daily chemical products include mouthwash, mouthwash granules, toothpaste, tooth cleaning powder, tooth paste, periodontal gel, tooth gel or tooth wax.

9. A method for producing osteoprotegerin, characterized in that: The lysate and / or supernatant of the thermophilic Streptococcus salivarius subspecies CCFM1395 according to claim 1 is added to the culture environment of osteoblasts for culture.

10. Use of the thermophilic Streptococcus salivarius subspecies CCFM1395 according to claim 1, or the microbial preparation according to claim 2, or the postbiotic according to claim 3 in the production of osteoprotegerin.

Images