Streptococcus salivarius subsp. thermophilus YM1921, YM1921-TWK10 flora and metabolites and metachion for promoting bone development, improving bone density and reducing cartilage wear

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a Streptococcus salivarius subsp. thermophilus YM1921, a YM1921-TWK10 flora and metabolites and a metagenome for promoting bone development, improving bone density and reducing cartilage wear. The Streptococcus salivarius subsp. thermophilus YM1921 has been preserved in the China General Microbiological Culture Collection Center on September 10, 2024, and the preservation number is CGMCC No. 31927. Metabolites of the strain and metabolites of the YM1921-TWK10 flora containing the strain can promote bone cell proliferation and improve the ALP enzyme activity of C3H10T1 / 2 cells under the fermentation of metabolites in a simulated real human intestinal environment, thereby inducing osteogenic differentiation, and thus can promote bone development, improve bone density and reduce cartilage wear.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Streptococcus salivarius thermophilic subspecies YM1921, YM1921-TWK10, and their metabolites and metabiotics that promote bone development, increase bone density, and reduce cartilage wear. Background Technology

[0002] Osteoporosis and osteoarthritis are global health problems, and their incidence is increasing year by year with the aging population. Current clinical interventions mainly rely on drugs such as bisphosphonates and RANKL inhibitors, but long-term use can easily cause side effects such as gastrointestinal damage and osteonecrosis of the mandible, and cannot reverse cartilage degeneration.

[0003] In recent years, probiotics have become a hot topic in bone health research due to their safety. However, existing research focuses on Lactobacillus, and their mechanism of action is limited to promoting calcium ion absorption. They have a weak effect on regulating the dynamic balance of osteoblasts / osteoclasts and lack evidence of protection against bone metabolism. Summary of the Invention

[0004] To address the above-mentioned technical problems, this invention provides a strain of Streptococcus salivarius thermophilus subspecies YM1921, the YM1921-TWK10 bacterial community, along with their metabolites and metabiotics, which promote bone development, increase bone density, and reduce cartilage wear. This Streptococcus salivarius thermophilus subspecies YM1921 strain can simultaneously promote osteoblast proliferation and osteoblast differentiation, breaking through the single-mode of action of existing technologies, thus enabling it to promote bone development, increase bone density, and reduce cartilage wear.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a strain of Streptococcus salivarius thermophilus subsp. YM1921, whose taxonomic name is Streptococcus salivarius thermophilus subsp. YM1921. Streptococcus salivarius subsp. thermophilus It was deposited on September 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31927; the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] The *Streptococcus salivarius* subsp. *thermophilus* YM1921 strain provided in this invention was isolated from traditional dairy products. Experiments have shown that its fermentation metabolites in a simulated human intestinal environment can promote the proliferation of mesenchymal stem cells (C3H10T1 / 2) and increase the ALP enzyme activity of C3H10T1 / 2 cells. Alkaline phosphatase (ALP) is a hydrolytic enzyme widely found in human tissues such as bone, liver, intestines, kidneys, and placenta. In the skeletal system, the main function of ALP is to participate in bone formation and remodeling; increased ALP activity directly promotes bone formation and is a key indicator of osteogenic differentiation. Furthermore, ALP can directly promote hydroxyapatite deposition by hydrolyzing pyrophosphate (a mineralization inhibitor) and providing phosphate, which is the material basis for increased bone density. Studies have also shown that drugs that increase ALP activity can improve bone microstructure and bone mineral density in osteoporosis models. In addition, during mesenchymal stem cell differentiation, osteogenic tissue is formed not only in intramembranous osteoblasts but also in cartilage, thereby reducing [the risk of osteoporosis]. Therefore, the thermophilic subspecies of Streptococcus salivarius YM1921 can promote bone development, increase bone density, and reduce cartilage wear by promoting the proliferation of mesenchymal stem cells and increasing ALP enzyme activity.

[0007] The second aspect of the present invention provides metabolites of the above-mentioned Streptococcus salivarius thermophilic subspecies YM1921 strain.

[0008] Preferably, the metabolite is a metabolite produced by fermentation in an in vitro intestinal fermentation simulation system.

[0009] A third aspect of the present invention provides a YM1921-TWK10 bacterial group containing the above-mentioned Streptococcus salivarius thermophilic subsp. YM1921 strain and Lactobacillus plantarum TWK10; the Lactobacillus plantarum TWK10 was deposited at the China General Microbiological Culture Collection Center on June 27, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.25190.

[0010] Preferably, the YM1921-TWK10 bacterial group also contains *Bifidobacterium animalis* subsp. *lactodeum* BAL-28 and *Lactobacillus plantarum* TWK10; the *Bifidobacterium animalis* subsp. *lactodeum* BAL-28 strain was deposited at the China General Microbiological Culture Collection Center on June 26, 2022, with accession number CGMCC No. 7.462; the *Lactobacillus plantarum* TWK10 was deposited at the China General Microbiological Culture Collection Center on June 27, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 25190.

[0011] Bifidobacterium animalis subsp. lactis BAL-28 strain has been disclosed in patent application number CN202310956580.0, entitled "Bifidobacterium animalis subsp. lactis BAL-28 and its fermentation products, bacterial flora CW and applications for preventing indigestion and bloating and promoting absorption". Lactobacillus plantarum TWK10 is a commercially available product and has been disclosed in patent application number CN202311653016.8, entitled "Use of Lactobacillus plantarum TWK10 in the preparation of anti-aging drugs, food and cosmetics".

[0012] More preferably, the ratio of viable counts of *Streptococcus salivarius* subsp. *thermophilus* YM1921, *Bifidobacterium animalis* subsp. *lactobacterium* BAL-28, and *Lactobacillus plantarum* TWK10 is 1:3:1.

[0013] The fourth aspect of the present invention provides metabolites of the above-mentioned YM1921-TWK10 bacterial community.

[0014] A fifth aspect of this invention provides a metabiotic prepared from the aforementioned *Streptococcus thermophilus* subsp. YM1921 strain or the aforementioned YM1921-TWK10 bacterial group. According to the well-known concept of "metabiotic" in the art, the metabiotic provided by this invention refers to the processed metabolites and bacterial cells of *Streptococcus thermophilus* subsp. YM1921 strain, including inactivated bacterial cells, bacterial cell fragments, cell lysates, and metabolites secreted by the strain in cell-free supernatant.

[0015] The sixth aspect of the present invention provides the use of the above-mentioned Streptococcus thermophilus subspecies YM1921 strain or its metabolites, the above-mentioned YM1921-TWK10 bacterial group or its metabolites in the preparation of products that promote the proliferation of mesenchymal stem cells and / or promote the differentiation of osteoblasts.

[0016] Preferably, the product is a food or medicine that promotes bone development.

[0017] Preferably, the product is a health food that helps improve bone density.

[0018] Preferably, the product is a medicine that reduces cartilage wear.

[0019] The seventh aspect of the present invention provides the application of the above-mentioned Streptococcus thermophilus subspecies YM1921 strain or its metabolites, the above-mentioned YM1921-TWK10 bacterial group or its metabolites in improving the alkaline phosphatase activity of mesenchymal stem cells (MSCs) in vitro.

[0020] Enhancing alkaline phosphatase activity in mesenchymal stem cells can promote osteogenic differentiation. In vitro osteogenic differentiation promotion is a core research method in bone biology and bone tissue engineering, and can be used to elucidate disease mechanisms, conduct high-throughput screening of drugs that regulate bone metabolism, and assist in the development of bone repair materials.

[0021] Preferably, the method of application specifically includes the following operations: after activating mesenchymal stem cells in DMEM medium, they are seeded into well plates. When the cells reach 70% to 80% confluence, the DMEM medium is replaced with fresh medium. The supernatant obtained from the fermentation of the above-mentioned Streptococcus thermophilus subsp. YM1921 strain in an intestinal in vitro fermentation simulation system or the supernatant obtained from the fermentation of the above-mentioned YM1921-TWK10 bacterial group in an intestinal in vitro fermentation simulation system is then inoculated in a 5% CO2 incubator at 37℃±1℃.

[0022] The beneficial effects of this invention are as follows: The fermentation metabolites of *Streptococcus salivarius* subsp. *thermophilus* provided in this invention can promote the proliferation of C3H10T1 / 2 cells and increase the ALP enzyme activity of C3H10T1 / 2 cells in a simulated human intestinal environment. This results in promoting bone development, increasing bone density, and reducing cartilage wear, breaking through the single-mode of action of existing technologies. These effects of the strain enable it not only to be used to produce products that promote mesenchymal stem cell proliferation and / or osteoblast differentiation, but also to promote osteogenic differentiation by increasing the alkaline phosphatase activity of mesenchymal stem cells in vitro, thus finding applications in bone biology and bone tissue engineering. Attached Figure Description

[0023] Figure 1 The cell morphology of Streptococcus salivarius subsp. thermophilus YM1921 in Example 1; Figure 2 The colony morphology of Streptococcus salivarius thermophilic subspecies YM1921 in Example 1 is shown. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0025] The present invention will be described below through specific embodiments.

[0026] The components and preparation methods of the culture medium used in the following examples are as follows: Modified MRS liquid culture medium: 10.0 g peptone, 5.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 1.0 mL Tween-80, 2.0 g dipotassium hydrogen phosphate, 5.0 g sodium acetate, 2.0 g triammonium citrate, 0.2 g magnesium sulfate (MgSO4·7H2O), 0.05 g manganese sulfate (MnSO4·4H2O), 1000 mL distilled water, pH 6.9±0.1.

[0027] MRS liquid medium (g / L): peptone, 10.0; beef extract, 10.0; yeast extract, 5.0; glucose, 20.0; triammonium citrate, 2.0; sodium acetate, 5.0; magnesium sulfate (MgSO4·7H2O), 0.1; manganese sulfate (MnSO4·4H2O), 0.05; dipotassium hydrogen phosphate, 2.0; Tween-80, 1.0; pH 6.5; solvent: water.

[0028] Intestinal model culture medium (CMGM) (g / L): Starch, 5.0; Pectin, 2.0; Guar gum, 1.0; Mucin (porcine gastric type III), 4.0; Xylan, 2.0; Arabinogalactan, 2.0; Inulin, 1.0; Casein, 3.0; Peptone water, 5.0; Tryptone, 5.0; Bile salts, 0.4; Yeast extract, 4.5; Ferrous sulfate (FeSO4·7H2O), 0.005; Sodium chloride, 4.5; Potassium chloride, 4.5; Potassium dihydrogen phosphate (KH2PO4), 0.5; Magnesium sulfate (MgSO4·7H2O), 1.25; Calcium chloride (CaCl2·6H2O), 0.15; Sodium bicarbonate (NaHCO3), 1.5; Cysteine, 0.8; Heme, 0.05; Tween 80, 1.0; Solvent: Water.

[0029] *Streptococcus salivarius* subsp. *thermophilus* YM131 and YM003 were isolated from traditional dairy yogurt from Tashkurgan Tajik Autonomous County (altitude 4000 meters, latitude 35°37′-38°40′N, longitude 71°20′-77°01′E) using conventional techniques by technicians from Hebei Yuanmin Biotechnology Co., Ltd. BLAST sequence alignment of the 16S rDNA sequences in the NCBI database confirmed that strains YM131 and YM003 are similar to *Streptococcus salivarius* subsp. *thermophilus* (…). Streptococcus salivarius subsp. thermophilus The Identity of this strain is 100.00%. Based on cell morphology observation, colony morphology observation, and Gram staining, this strain is consistent with the thermophilic subsp. salivarius.

[0030] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the materials and reagents used in the following embodiments are all commercially available.

[0031] Example 1 This invention provides the thermophilic subspecies of Streptococcus salivarius YM1921 strain, its acquisition process, and its identification process.

[0032] 1. Acquisition process Local traditional dairy curds were obtained in Tashkurgan Tajik Autonomous County (altitude 4000 meters, latitude 35°37′-38°40′N, longitude 71°20′-77°01′E). 1g of the curds was added to 10mL of physiological saline and serially diluted 10-fold six times. 1mL of each serial dilution was spread onto MRS plates and anaerobically incubated at 37°C for 48 hours. Single colonies of different morphologies were streaked three times until purified single colonies were obtained. Single colonies of the purified YM1921 strain were transferred to MRS liquid medium and incubated for 24 hours. The culture was then mixed with 30% glycerol and stored at -80°C.

[0033] 2. Identification 2.1 Observation of colony morphology Cell morphology: Oval, arranged in pairs or chains (e.g.) Figure 1 (As shown); Colony morphology observation: In MC agar medium, the colonies are medium to small, with neat and smooth edges, and are red (as shown). Figure 2 (As shown); Gram staining: positive.

[0034] 2.2 Identification of 16S rDNA of the strain The 16S rDNA sequence of this strain (as shown in SEQ ID No. 1):

[0035] BLAST sequence alignment in the NCBI database showed that strain YM1921 is related to *Streptococcus salivarius* subsp. *thermophilus*. Streptococcus salivarius subsp. thermophilus The Identity of ) is 100.00%.

[0036] Cell morphology observation, colony morphology observation, Gram staining, and 16S rDNA sequence comparison confirmed that this strain is identical to *Streptococcus salivarius* subsp. *thermophilus*. This *Streptococcus salivarius* subsp. *thermophilus* YM1921 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 31927.

[0037] Example 2 This embodiment provides a bacterial suspension of Streptococcus salivarius subsp. thermophilicus YM1921, and its preparation method is as follows: Seed culture of Streptococcus salivarius subsp. thermophilus YM1921 was inoculated into modified MRS liquid medium and activated for 3 generations. The resulting bacterial cells were then adjusted to a bacterial suspension using PBS.

[0038] Example 3 This embodiment provides a YM1921 probiotic powder, prepared as follows: *Streptococcus salivarius* subsp. *thermophilus* YM1921 is added to a culture medium at an inoculum rate of 3% for fermentation at 37°C for 12 hours. After fermentation, the mixture is centrifuged at 6000 r / min for 30 min to obtain bacterial sludge. The sludge is then mixed evenly with a freeze-drying protectant, freeze-dried, and finally pulverized to obtain YM1921 probiotic powder (live count 1.0 × 10⁻⁶). 10 CFU / g).

[0039] Preparation method of culture medium: Mix 40g glucose, 4g soybean peptone, 3g yeast extract, 2g diammonium citrate, 6g anhydrous sodium acetate, 3g dipotassium hydrogen phosphate, 1g isomaltooligosaccharide, 3g starch and other food-grade materials, then add 1000g pure water and mix until there are no visible particles. After the mixture is dissolved, adjust the pH to 5.0-6.8 and sterilize at 117.5-118.5℃ for 20min to obtain the culture medium.

[0040] Preparation method of freeze-drying protectant: Mix 100g starch, 20g glucose, 60g maltodextrin, 30g sucrose, 10g lactose, 50g skim milk powder and other food ingredients evenly, add 1000g pure water to dissolve until no visible particles are present, adjust the pH to 5.0-6.8 after dissolving, sterilize at 117.5-118.5℃ for 20min, and adjust the pH to 6.0-7.3 after sterilization to obtain the freeze-drying protectant.

[0041] Example 4 This embodiment provides a YM1921-TWK10 probiotic powder, which is prepared by: preparing Streptococcus salivarius thermophilic subsp. YM1921, Bifidobacterium animalis lactis subsp. BAL-28, and Lactobacillus plantarum TWK10 strains respectively according to the method in Example 3, with a viable count of 1.0 × 10⁻⁶. 10 Probiotic powder with CFU / g, by mixing three types of probiotic powder in a mass ratio of 1:3:1, yields YM1921-TWK10 probiotic powder (live count 1.0 × 10⁻⁶). 10 CFU / g).

[0042] Example 5 This embodiment verifies the ability of the metabolites of Streptococcus salivarius thermophilic subspecies YM1921 and YM1921-TWK10 in an intestinal in vitro fermentation simulation system to promote the growth of mouse mesenchymal stem cells C3H10T1 / 2.

[0043] 1. Preparation of fermentation supernatant of Streptococcus salivarius subsp. thermophilus YM1921, Streptococcus salivarius subsp. S131, Streptococcus salivarius subsp. SS003, and Streptococcus salivarius-TWK10 under in vitro fermentation conditions of intestinal flora. This experiment consisted of a control group (CK), a blank control group, an experimental group, an S131 control group, an SS003 control group, and a YM1921-TWK10 bacterial group. Fermentation supernatants were prepared for all groups except the CK control group. A three-stage continuous culture intestinal model system was used. The human colon three-stage continuous culture model consisted of three fermenters simulating the proximal (V1, 280 mL), transverse (V2, 300 mL), and distal colon (V3, 320 mL). The three fermenters in series were maintained at 37°C, pH values ​​of 5.5 (V1), 6.2 (V2), and 6.8 (V3), and anaerobic conditions were introduced via continuous N2. V1 was connected to intestinal model culture medium (CMGM medium) supplemented with D-gluconic acid (100 mg / L) via a peristaltic pump. Fresh fecal samples were collected from infants and stored in an anaerobic chamber (10% H2, 10% CO2, 80% N2). After collection, a 1:5 (w / w) fecal dilution was prepared in anaerobic PBS (0.1 mol / L PBS, pH 7.4) within a maximum of 15 minutes. 10 mL of fecal slurry was inoculated at each stage of the colon model. The total system throughput time was set to 48 h based on the average retention time of healthy individuals. After inoculation, the colon model was run in batch culture for 24 hours to stabilize the bacterial population before the inoculation of CMGM medium. After 24 hours, the system was run for 8 full-volume cycles to achieve a steady state. The blank control group received no intervention and underwent eight volume cycles to reach a steady state for 3 hours. After three consecutive days, the fermentation culture collected daily was centrifuged at 12,000 rpm / min at 4°C for 10 minutes, and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain the fermentation supernatant required for the blank control group. In the experimental group, YM1921 probiotic powder (prepared in Example 3) was added to V1 at 1% (w / v) daily, and eight volume cycles were performed to reach a steady state for 3 hours. Then, sampling was carried out for three consecutive days, and the fermentation culture collected daily was centrifuged at 12,000 rpm / min at 4°C for 10 minutes. The supernatant was filtered through a 0.22 μm sterile filter membrane to obtain the fermentation supernatant required for the experimental group. The YM131 control group, YM003 control group, and YM1921-TWK10 bacterial group were prepared using probiotic powder of Streptococcus thermophilus subsp. YM131 (preparation method as in Example 3), probiotic powder of Streptococcus thermophilus subsp. YM003 (preparation method as in Example 3), and probiotic powder of YM1921-TWK10 bacterial group (obtained from Example 4), respectively, with the same preparation method as the experimental group.

[0044] 2. Culture of C3H10T1 / 2 cells Thaw cryovials containing 1 mL of C3H10T1 / 2 cell suspension rapidly by shaking in a 37°C water bath. Add 4 mL of DMEM medium and mix thoroughly. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant, add 1-2 mL of medium, and resuspend. Pour the resulting cell suspension into a 10 cm culture dish, add approximately 8 mL of DMEM medium, and incubate overnight. Change the medium the next day and check the cell density. Once the cell density reaches 80%-90%, passage the cells. Discard the culture supernatant and wash the cells 1-2 times with calcium- and magnesium-free PBS. Add 1 mL of digestion solution (0.25% Trypsin-0.53 mM EDTA) to the culture flask and incubate at 37°C for 1-2 minutes. Observe the cell digestion under a microscope. If most cells become rounded and detach, add a small amount of DMEM medium to stop the digestion. Add 6-8 mL of DMEM medium to each bottle, gently mix, aspirate, centrifuge at 1000 rpm for 4 minutes, discard the supernatant, add 1-2 mL of DMEM medium, and mix well. Transfer the cell suspension to new culture dishes containing 8 mL of DMEM medium at a 1:2 ratio.

[0045] 3. Validation of the ability of in vitro fermentation system metabolites to promote the proliferation of C3H10T1 / 2 cells. After culturing C3H10T1 / 2 cells to passage 3, the cell concentration was adjusted to 2×10⁻⁶. 5 Cells were cultured at a density of 100 µL / well in a 96-well plate. After cell adhesion, 100 µL of fresh DMEM medium was added. The wells were divided into several groups: CK control group, blank control group (inoculated with the fermentation supernatant required for the blank control group), experimental group (inoculated with the fermentation supernatant of *Streptococcus thermophilus* subsp. YM1921), YM131 control group (inoculated with the fermentation supernatant of *Streptococcus thermophilus* subsp. YM131), YM003 control group (inoculated with the fermentation supernatant of *Streptococcus thermophilus* subsp. YM003), and YM1921-TWK10 group (inoculated with the fermentation supernatant of the YM1921-TWK10 group). 10 μL of fermentation supernatant from each group was seeded into each well, and 10 μL of DMEM medium was seeded into the CK control group. All wells were incubated at 37°C for 24 h in a 5% CO2 incubator. Cell proliferation was then assessed according to the CCK-8 assay instructions: 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 1 h. The OD value was measured at 450 nm. The relative cell proliferation rate was calculated using the following formula.

[0046]

[0047] In the formula: Ac represents the OD value of the CK control group, and As represents the OD value of the blank control group, experimental group, YM131 control group, YM003 control group, or YM1921-TWK10 bacterial group.

[0048] All experiments were conducted in triplicate. Experimental data were statistically analyzed using Excel 2019, and one-way ANOVA was performed using SPSS 24.0. Multiple comparisons were conducted using the LSD method. Normally distributed measurement data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, with a significance level of α = 0.05. The results are shown in Table 1.

[0049] Table 1. Relative proliferation rate of C3H10T1 / 2 cells in each group

[0050] Note: 'a' indicates a highly significant difference compared to the blank control group. P <0.01), c indicates no significant difference compared to the blank control group ( P >0.05), the same below.

[0051] Example 6 This embodiment uses the azo coupling method to verify the ability of Streptococcus salivarius thermophilic subspecies YM1921 and YM1921-TWK10 bacterial groups to promote osteogenic differentiation of mouse mesenchymal stem cells C3H10T1 / 2.

[0052] The C3H10T1 / 2 cells were cultured according to the method in step 2 of Example 5. After three generations, the cell concentration was adjusted to 2 × 10⁻⁶. 5 Cells were seeded at a density of 100 µL / well in a 24-well plate and incubated overnight. When the cells reached 70%–80% confluence, 100 µL of fresh DMEM medium was added. Cells were then divided into groups as described in Example 5. The control group (CK) was seeded with 10 µL of DMEM medium, while the other groups were seeded with the corresponding fermentation supernatant (prepared from step 1 of Example 5) as described in Example 5. Cells were incubated at 37°C for 3 days in a 5% CO2 incubator, followed by trypsin digestion. Cells from each group were collected and prepared into cell suspensions. 100 µL of each cell suspension was placed on a slide to prevent cell detachment. After drying at room temperature, the suspension was fixed with 4% paraformaldehyde. ALP incubation solution (containing the substrate AS-BI phosphate and a chromogenic agent coupled with diazonium salt) was added for ALP staining. Microscopic examination revealed that the active ALP sites were blue. The relative ALP activity in the culture supernatant of each group was calculated according to the ALP activity assay kit instructions using the following formula.

[0053] ALP relative enzyme activity = As / Ac × 100% In the formula: As represents the enzyme activity of the blank control group, experimental group, YM131 control group, YM003 control group or YM1921-TWK10 bacterial group, and Ac represents the enzyme activity of the CK control group.

[0054] Table 2 Relative enzyme activities of each group

[0055] Note: b indicates a significant difference compared to the blank control group. P <0.05), c indicates no significant difference compared to the blank control group ( P >0.05).

[0056] In summary, the results show that the thermophilic subspecies of Streptococcus salivarius YM1921 and YM1921-TWK10 can promote not only stem cell proliferation but also osteoblast differentiation in a simulated human intestinal environment, thereby promoting bone development, increasing bone density, and reducing cartilage wear.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Streptococcus salivarius thermophilic subspecies YM1921, characterized in that, Its classification name is *Streptococcus salivarius* subsp. *thermophilus* ( Streptococcus salivarius subsp. thermophilus It was deposited on September 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31927; the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The metabolites of Streptococcus salivarius subsp. YM1921 as described in claim 1.

3. A YM1921-TWK10 bacterial group, characterized in that, It contains the *Streptococcus salivarius* subsp. *thermophilus* YM1921 strain as described in claim 1 and *Lactobacillus plantarum* TWK10; the *Lactobacillus plantarum* TWK10 was deposited at the China General Microbiological Culture Collection Center on June 27, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.25190.

4. The YM1921-TWK10 bacterial group according to claim 3, characterized in that, It also contains Bifidobacterium animalis subsp. lactis BAL-28; the strain of Bifidobacterium animalis subsp. lactis BAL-28 was deposited on June 26, 2022, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 7.

462.

5. The YM1921-TWK10 bacterial group according to claim 4, characterized in that, The ratio of viable counts of Streptococcus salivarius subsp. thermophilus YM1921, Bifidobacterium animalis subsp. lactis BAL-28, and Lactobacillus plantarum TWK10 was 1:3:

1.

6. Metabolites of the YM1921-TWK10 bacterial community as described in any one of claims 3 to 5.

7. An epigenetic agent, characterized in that, It is prepared from the thermophilic subspecies of Streptococcus salivarius YM1921 strain as described in claim 1 or the YM1921-TWK10 bacterial group as described in any one of claims 3 to 5.

8. The use of the *Streptococcus salivarius* subspecies YM1921 strain of claim 1, the metabolite of claim 2, the YM1921-TWK10 bacterial group of any one of claims 3 to 5, or the metabolite of claim 6 in the preparation of products that promote the proliferation of mesenchymal stem cells and / or promote the differentiation of osteoblasts.

9. The application according to claim 8, characterized in that, The product is a food or medicine that promotes bone development; and / or The product is a health food that helps improve bone density; and / or The product is a medicine that reduces cartilage wear.

10. The use of the *Streptococcus salivarius* subspecies YM1921 strain of claim 1, the metabolite of claim 2, the YM1921-TWK10 bacterial group of any one of claims 3 to 5, or the metabolite of claim 6 in in vitro enhancement of alkaline phosphatase activity in mesenchymal stem cells.

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