Lactobacillus plantarum IOB602 for regulating bone metabolism and application thereof

Abstract

The present invention provides a plant lactobacillus IOB602 for regulating bone metabolism and its application, and relates to the field of microbial technology. Lactiplantibacillus plantarum , was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on June 29, 2018, referred to as CGMCC, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 16021. The bacteria reduces the content of lipid peroxidation product malondialdehyde and increases the content of antioxidant enzymes, which is of great significance for reducing ROS damage to bone cells, regulating the balance of osteoblasts and osteoclasts, improving bone metabolism, and preventing and treating diabetic osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Lactobacillus plantarum IOB602 for regulating bone metabolism and an application thereof. Background Art

[0002] Type 2 diabetes is a chronic metabolic disease with a high incidence in the elderly. Diabetic osteoporosis (DOP) is a common complication of diabetes and the main cause of fragility fractures and trabecular bone degeneration in diabetic patients. Increased cortical porosity, imbalanced bone metabolism, and distorted bone microarchitecture are the three main characteristics of DOP.

[0003] There are several bone cells in the bone microenvironment that work together to maintain bone homeostasis, including osteoblasts, osteoclasts, etc. Osteoblasts and osteoclasts are the main cells for bone formation and bone resorption, and the two coordinate with each other to ensure bone renewal and regeneration.

[0004] Reactive oxygen species (ROS) are natural byproducts of normal oxygen metabolism. When their concentration exceeds a certain range, they will disrupt the oxidation-antioxidation balance and induce a variety of diseases. High glucose levels can lead to a significant increase in intracellular ROS levels. ROS can promote osteoblast apoptosis and osteoclast differentiation through pathways such as mitogen-activated protein kinase (MAPK) and NF-KB, thereby destroying the dynamic balance between bone formation and bone resorption to a certain extent. Malondialdehyde (MDA) is the final product of lipid peroxidation. Glutathione (GSH) is involved in the removal of ROS and can be used to reflect the antioxidant level of cells. Therefore, it is an important oxidative stress indicator. When the oxidative factors in the cells increase, the expression of the main antioxidant enzyme regulator (Nrf-2) increases. Nrf-2 can reduce the differentiation ability of osteoblasts by inhibiting the Runt-related transcription factor 2 (RUNX2)-dependent pathway, thereby affecting the level of bone metabolism.

[0005] At present, most people take hypoglycemic drugs, adjust their diet, and supplement calcium to lower their blood sugar, increase their bone mass, and relieve osteoporosis symptoms. However, these methods have limited effects on improving patients' blood sugar and bone metabolism. Therefore, it is important to continue to find relevant indicators and mechanisms that affect the development of the disease. Probiotics refer to living microorganisms that bring health benefits to the host when given in sufficient amounts. Studies have shown that some probiotics and their fermentation products can increase the activity of antioxidant enzymes and protect cells from oxidative stress. In addition, probiotics can also be used to ferment solid fermentation substrates to enhance the biological activity of the fermentation substrates.

[0006] Therefore, it is urgent to find and develop a preparation that can regulate bone metabolism and its microecological properties. Summary of the invention

[0007] In view of the technical problems existing in the prior art, the present invention aims to provide a Lactobacillus plantarum IOB602 for regulating bone metabolism and application thereof.

[0008] One of the purposes of the present invention is to provide a plant lactobacillus IOB602 for regulating bone metabolism, wherein the plant lactobacillus IOB602 is classified as plant lactobacillus Lactiplantibacillus plantarum It was deposited on June 29, 2018 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.16021.

[0009] Preferably, the Lactobacillus plantarum IOB602 has at least one of the following functions:

[0010] (1) Reduce the content of malondialdehyde, a lipid peroxidation product, increase the content of antioxidant enzymes, and improve oxidative stress response;

[0011] (2) Improve the differentiation and proliferation ability of osteoblasts;

[0012] (3) Inhibit osteoclast differentiation and proliferation;

[0013] (4) Up-regulate the relative expression levels of Runt-related transcription factor 2, osteopontin and bone morphogenetic protein 2 genes.

[0014] Preferably, the Lactobacillus plantarum IOB602 can be used to prepare Lactobacillus plantarum IOB602 postbiotics.

[0015] Preferably, the preparation method of the Lactobacillus plantarum IOB602 powder and the Lactobacillus plantarum IOB602 postbiotics comprises:

[0016] Inoculate the plant lactobacillus IOB602 in the cryopreserved tube into the slant culture medium and place it at 35-39°C for 18-22 hours of activation time, take the fresh slant bacteria and inoculate it into the seed liquid basic culture medium, and culture it at 35-39°C in a closed state for 16-20 hours; inoculate the cultured seed liquid into the fermentation medium at an inoculation rate of 3±0.5%, and culture it at a temperature of 34-38°C for 16-20 hours;

[0017] The bacterial liquid after fermentation is inactivated and dried to obtain Lactobacillus plantarum IOB602 fermentation postbiotics.

[0018] Preferably, the formula of the slant culture medium is: peptone 10.0 g, beef extract 10.0 g, yeast extract 4.5 g, glucose 20.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate (7H2O) 2.0 g, sodium acetate (3H2O) 5.0 g, triammonium citrate 2.0 g, magnesium sulfate (7H2O) 0.2 g, manganese sulfate (4H2O) 0.05 g, agar 15.0 g, pH = 6.0 ± 0.2, and the amount of water added is 1 L.

[0019] Preferably, the formula of the seed liquid basic culture medium and the fermentation culture medium is: peptone 10.0g, beef extract 10.0g, yeast extract 4.5g, glucose 20.0g, Tween 80 1.0mL, dipotassium hydrogen phosphate·7H2O2.0g, sodium acetate·3H2O5.0g, triammonium citrate 2.0g, magnesium sulfate·7H2O0.2g, manganese sulfate·4H2O0.05g, pH=6.0±0.2, and the amount of water added is 1L.

[0020] Preferably, the Lactobacillus plantarum IOB602 can be used to prepare medicines for regulating bone metabolism.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a plant lactobacillus IOB602 for regulating bone metabolism, which reduces the content of malondialdehyde, a lipid peroxidation product, and increases the content of antioxidant enzymes. In the human body, the generation of free radicals (ROS) is part of normal metabolism, but excessive ROS can trigger lipid peroxidation, resulting in an increase in the content of malondialdehyde, a lipid peroxidation product. At the same time, the role of antioxidant enzymes is to remove ROS. When the content of antioxidant enzymes is increased, the level of ROS can be reduced, lipid peroxidation can be reduced, and the content of malondialdehyde can be reduced. In the process of bone metabolism, osteoblasts are responsible for bone formation, and osteoclasts are responsible for bone absorption, and the two jointly maintain the stability of bone mass. However, excessive ROS can cause oxidative damage to osteoblasts and osteoclasts, inhibit osteoblast activity and proliferation, reduce bone matrix synthesis, and promote osteoclast activity and proliferation, increase bone absorption, and lead to bone loss. In the diabetic state, high blood sugar can lead to increased oxidative stress in the body and produce a large amount of ROS. This not only aggravates lipid peroxidation and increases the content of malondialdehyde, but also destroys the antioxidant enzyme system, reduces the content of antioxidant enzymes, and cannot effectively remove excessive ROS. At the same time, oxidative stress caused by high blood sugar will disrupt the balance of bone metabolism, inhibit osteoblast function, stimulate osteoclasts, and aggravate diabetic osteoporosis. Therefore, reducing the content of lipid peroxidation product malondialdehyde and increasing the content of antioxidant enzymes are of great significance for reducing ROS damage to bone cells, regulating the balance of osteoblasts and osteoclasts, improving bone metabolism, and preventing and treating diabetic osteoporosis.

[0023] Runt-related transcription factor 2 is a key transcription factor for osteoblast differentiation. It can activate the expression of osteoblast-specific genes and promote the differentiation of mesenchymal stem cells into osteoblasts. It plays an indispensable role in bone development and bone repair. Osteopontin is a secreted phosphorylated glycoprotein that participates in the mineralization and absorption of bone tissue. It can adsorb on the surface of hydroxyapatite and regulate the deposition and dissolution of calcium salts. At the same time, osteopontin can also act as a cell adhesion molecule to promote the interaction between cells and cells and cells and matrix. In bone metabolism, osteopontin is mainly secreted by osteoblasts, and the upregulation of its gene expression contributes to the maturation and mineralization of bone matrix. Bone morphogenetic protein 2 is a member of the transforming growth factor-β (TGF-β) superfamily and an important inducing factor for bone formation. It can induce undifferentiated mesenchymal stem cells to differentiate into osteoblasts and promote the proliferation and differentiation of osteoblasts. In the study of bone defect repair, exogenous addition of bone morphogenetic protein 2 can significantly promote the formation of new bone. When the relative expression of bone morphogenetic protein 2 gene is upregulated, the process of bone formation will be accelerated.

[0024] Diabetic osteoporosis is caused by oxidative stress caused by hyperglycemia, which leads to an increase in ROS and affects the balance of bone metabolism. Upregulating the relative expression of Runt-related transcription factor 2, osteopontin and bone morphogenetic protein 2 genes can promote bone formation. Under oxidative stress, this effect of promoting bone formation can, to a certain extent, counteract the inhibition of osteoblast function and bone loss caused by ROS. For example, even in the presence of a large amount of ROS caused by hyperglycemia, upregulating the relative expression of bone morphogenetic protein 2 gene can stimulate the differentiation of osteoblasts and try to restore the damaged bone metabolism balance. At the same time, the upregulation of these factors indirectly affects the antioxidant enzyme system. The improvement of osteocyte function may feedback regulate the intracellular redox state, promote the production of antioxidant enzymes, and thus reduce the content of lipid peroxidation product malondialdehyde. After the function of osteoblasts is enhanced, the metabolic environment in the cells changes, which may activate certain signaling pathways, resulting in the upregulation of the expression of antioxidant enzyme genes, thereby increasing the content of antioxidant enzymes and reducing the damage of oxidative stress to osteocytes. This has positive significance for improving the condition of diabetic osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The morphology of osteoclast RAW264.7 cells;

[0026] Figure 2 This is the morphology of osteoblast MC3T3-E1 cells;

[0027] Figure 3 is the malondialdehyde content;

[0028] Figure 4 is the glutathione content;

[0029] Figure 5 Alizarin red staining for osteoblastic MC3T3-E1 cells;

[0030] Figure 6 Tartrate-resistant acid phosphatase staining for osteoclast RAW264.7;

[0031] Figure 7 Cell proliferation of osteoblasts MC3T3-E1 after 48 h of induction;

[0032] Figure 8 This is the cell proliferation of osteoclast RAW264.7 after 48 hours of induction;

[0033] Fig. 9 is the relative expression level of RUNX2 gene;

[0034] Fig.10 is the relative expression of BMP2 gene;

[0035] Fig.11 is the relative expression level of OPN gene.

[0036] The Lactobacillus plantarum IOB602 used in the present invention is classified and named as Lactobacillus plantarum Lactiplantibacillus plantarum It was deposited on June 29, 2018 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.16021. DETAILED DESCRIPTION

[0037] The present invention provides a plant lactobacillus IOB602 for regulating bone metabolism, wherein the plant lactobacillus IOB602 is classified and named as plant lactobacillus Lactiplantibacillus plantarum It was deposited on June 29, 2018 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.16021.

[0038] In a preferred embodiment of the present invention, the Lactobacillus plantarum IOB602 has at least one of the following functions:

[0039] (1) Reduce the content of malondialdehyde, a lipid peroxidation product, increase the content of antioxidant enzymes, and improve oxidative stress response;

[0040] (2) Improve the differentiation and proliferation ability of osteoblasts;

[0041] (3) Inhibit osteoclast differentiation and proliferation;

[0042] (4) Up-regulate the relative expression levels of Runt-related transcription factor 2, osteopontin and bone morphogenetic protein 2 genes.

[0043] In a preferred embodiment of the present invention, the Lactobacillus plantarum IOB602 can be used to prepare Lactobacillus plantarum IOB602 postbiotics.

[0044] In a preferred embodiment of the present invention, the preparation method of the Lactobacillus plantarum IOB602 postbiotics comprises:

[0045] Inoculate the plant lactobacillus IOB602 in the cryopreserved tube into the slant culture medium and place it at 35-39°C for 18-22 hours of activation time, take the fresh slant bacteria and inoculate it into the seed liquid basic culture medium, and culture it at 35-39°C in a closed state for 16-20 hours; inoculate the cultured seed liquid into the fermentation medium at an inoculation rate of 3±0.5%, and culture it at a temperature of 34-38°C for 16-20 hours;

[0046] The bacterial liquid after fermentation is inactivated and dried to obtain Lactobacillus plantarum IOB602 fermentation postbiotics.

[0047] In a preferred embodiment of the present invention, the formula of the slant culture medium is: peptone 10.0g, beef extract 10.0g, yeast extract 4.5g, glucose 20.0g, Tween 80 1.0mL, dipotassium hydrogen phosphate (7H2O) 2.0g, sodium acetate (3H2O) 5.0g, triammonium citrate 2.0g, magnesium sulfate (7H2O) 0.2g, manganese sulfate (4H2O) 0.05g, agar 15.0g, pH=6.0±0.2, and the amount of water added is 1L.

[0048] In a preferred embodiment of the present invention, the formula of the seed liquid basic culture medium and the fermentation culture medium is: peptone 10.0g, beef extract 10.0g, yeast extract 4.5g, glucose 20.0g, Tween 80 1.0mL, dipotassium hydrogen phosphate·7H2O2.0g, sodium acetate·3H2O5.0g, triammonium citrate 2.0g, magnesium sulfate·7H2O0.2g, manganese sulfate·4H2O0.05g, pH=6.0±0.2, and the amount of water added is 1L;

[0049] In a preferred embodiment of the present invention, the Lactobacillus plantarum IOB602 can be used to prepare medicines for regulating bone metabolism.

[0050] Example 1

[0051] Preparation method of postbiotics of Lactobacillus plantarum IOB602

[0052] (1) Strain activation: Inoculate the plantarum Lactobacillus IOB602 in the cryopreserved tube into the slant culture medium and place it at 37±2℃ for 20±2h. After activation, the colonies are milky white, round, moist, opaque, and have neat edges; microscopic examination shows that the bacteria are rod-shaped, arranged singly or in pairs, and are Gram-positive.

[0053] The slant culture medium used was MRS modified culture medium, whose formula was: peptone 10.0 g, beef extract 10.0 g, yeast extract 4.5 g, glucose 20.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate (7H2O) 2.0 g, sodium acetate (3H2O) 5.0 g, triammonium citrate 2.0 g, magnesium sulfate (7H2O) 0.2 g, manganese sulfate (4H2O) 0.05 g, agar 15.0 g, pH = 6.0 ± 0.2, and the amount of water added was 1 L.

[0054] (2) Seed liquid culture: Take a fresh slant culture and inoculate it into the seed liquid basic culture medium. Culture it in a closed container at 37±2℃ for 18±2h.

[0055] (3) Fermentation: The seed liquid was inoculated into the fermentation medium at an inoculum rate of 3±0.5%, the culture temperature was 36±2°C, the culture time was 18±2h, and the activity of the final liquid fermentation product was 1×10 11 CFU / g.

[0056] The basic culture medium for seed liquid and fermentation culture medium used MRS culture medium, whose formula was: peptone 10.0g, beef extract 10.0g, yeast extract 4.5g, glucose 20.0g, Tween 80 1.0mL, dipotassium hydrogen phosphate (7H2O) 2.0g, sodium acetate (3H2O) 5.0g, triammonium citrate 2.0g, magnesium sulfate (7H2O) 0.2g, manganese sulfate (4H2O) 0.05g, pH=6.0±0.2, and the amount of water added was 1L.

[0057] (4) Preparation of postbiotic powder: After fermentation, the postbiotic powder is inactivated and dried to obtain the postbiotic powder fermented by Lactobacillus plantarum IOB602.

[0058] Example 2

[0059] 1. Establishment of cell model

[0060] Mouse bone marrow-derived macrophages RAW264.7 and mouse embryonic osteoblast precursor cells MC3T3-E1 were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0061] Morphological observation of RAW264.7 cells:

[0062] After being inoculated into the culture dish, RAW264.7 cells adhered to the wall and grew very quickly. Half an hour after inoculation, the adhered area reached about 60%, and after 2 hours, almost all of them could grow on the wall. The RAW264.7 cells after adhesion were spherical or irregular spherical, single-nucleated, and slightly smaller than normal cells. Figure 1 Shown are the cell morphologies of RAW 264.7 cells 24 h after seeding.

[0063] Morphological observation of MC3T3-E1 cells:

[0064] MC3T3-E1 cells were inoculated into the culture dish for 4 hours, and then the cells were observed to grow attached to the wall, and the cells gradually stretched. As time went on, MC3T3-E1 cells began to have more protrusions, and the shape gradually changed to spindle shape, and a few cross-links began to appear between cells. The cell nucleus was located in the center of the cell and was slightly bulging. The morphology of MC3T3-E1 cells after 24 hours of inoculation is as follows Figure 2 shown.

[0065] Induction of Osteoblasts / Osteoclasts:

[0066] Take out the cryovials containing MC3T3-E1 and RAW264.7 cells, set the electric constant temperature water bath at 37°C, wipe the taken out cryovials with alcohol cotton, quickly put them into the water bath and shake them. When the cells are completely thawed after about 1 min, use a pipette to extract the cell suspension and transfer it to a sterile centrifuge tube. Then slowly add DMEM complete culture medium to the centrifuge tube, mix well by blowing, and centrifuge at 1000 rpm for 3 min at room temperature. After centrifugation, the supernatant was removed and the precipitate obtained by centrifugation was resuspended in osteoblast / osteoclast induction culture medium complete culture medium and transferred to a 100 mm cell culture dish with a pipette. The transferred culture dish was placed in a CO2 constant temperature incubator for culture at 37°C and 5% CO2, and the osteoblast / osteoclast induction culture medium was replaced every 12 h (osteoblast induction medium: containing 50 μg / mL ascorbic acid and 10 mmol / L sodium β-glycerophosphate; osteoclast induction medium: 50 μg / mL RANK Ligand solution).

[0067] Cell grouping and treatment:

[0068] The successfully induced osteoblasts (MC3T3-E1) and osteoclasts (RAW264.7) were divided into 4 groups: control group; high glucose group; control-postbiotic group; high glucose-postbiotic group.

[0069] Modeling: The control group was cultured with a cell culture medium containing 5.5 mmol / L D-glucose for 3 days, and the high-glucose group was cultured with a cell culture medium containing 30 mmol / L D-glucose for 3 days.

[0070] The postbiotic powder fermented by Lactobacillus plantarum IOB602 was mixed with the osteoclast / osteoclast induction fluid of the control group and the high-sugar group at a ratio of 1:20, and after 5 days of induction, the control-postbiotic group and the high-sugar-postbiotic group were obtained.

[0071] 2. Effects of Lactobacillus plantarum IOB602 postbiotics on malondialdehyde content and antioxidant enzyme content in osteoblasts (MC3T3-E1)

[0072] Experimental materials: MDA detection kit S0131S, Shanghai Biyuntian Biotechnology Co., Ltd.;

[0073] GSH detection kit S0053, Shanghai Bio-Technology Co., Ltd.

[0074] Malondialdehyde (MDA) is the final product of lipid peroxidation, while glutathione (GSH) is involved in the scavenging of free radicals (ROS) and can be used to reflect the antioxidant level of cells, so it is an important indicator of oxidative stress response. Collect osteoblast samples from the control group, high glucose group, control-postbiotic group, and high glucose-postbiotic group: digest the cells with 0.25% trypsin for 2 min, add serum-containing culture medium to terminate the reaction, carefully blow the cells until all fall off, centrifuge at 3000 r / min for 1 min, and remove the supernatant. Add serum-containing culture medium to resuspend the cells. Take cell samples and detect them according to the steps of the MDA and GSH detection kits.

[0075] The results are as follows Figure 3 and Figure 4 As shown: Compared with the control group, the high sugar group had a higher MDA content and a lower GSH content. It can be concluded that the high sugar environment leads to an imbalance in the cellular oxidation-antioxidation level, which in turn causes cellular oxidative stress damage. Compared with the control group and the high sugar group, the MDA content of osteoblasts in the control-postbiotic group and the high sugar-postbiotic group after postbiotic treatment decreased by 32.14% and 18.76%, respectively, and the GSH content increased by 26.40% and 68.75%, respectively, indicating that postbiotic powder fermented with Lactobacillus plantarum IOB602 can improve cellular oxidative stress damage.

[0076] 3. Effects of Lactobacillus plantarum IOB602 postbiotics on osteoblast and osteoclast differentiation

[0077] Alizarin red staining of osteoblasts (MC3T3-E1) to identify the induction results:

[0078] Experimental materials: Alizarin red staining kit, Beijing Solebow Technology Co., Ltd.

[0079] Alizarin red staining:

[0080] Alizarin Red S is an anthraquinone derivative and is a sodium salt of alizarin sulfonic acid. It can chelate with calcium salts in calcium carbonate or calcium phosphate to form an orange-red complex. The culture medium of osteoblasts in the control group, high glucose group, control-postbiotic group, and high glucose-postbiotic group was discarded, washed twice with PBS, and 4% paraformaldehyde was used as a fixative, added to the cells for 15 min, the fixative was discarded, and the cells were rinsed with ultrapure water three times; the prepared alizarin red solution was added to stain the cells at room temperature for 30 min; after rinsing with distilled water to remove the alizarin red solution, the slides were dried and sealed, and observed and photographed under a microscope.

[0081] The results are as follows Figure 5 As shown: compared with the control group and the high sugar group, the osteoblast calcification nodules in the control-postbiotic group and the high sugar-postbiotic group after treatment with Lactobacillus plantarum IOB602 postbiotics were obvious and darker in color. It can be concluded that the postbiotic powder fermented by Lactobacillus plantarum IOB602 promotes the differentiation of osteoblasts.

[0082] Results of identification of osteoclast tartrate-resistant acid phosphatase induction:

[0083] Experimental materials: Alizarin red staining kit, Beijing Solebow Technology Co., Ltd.

[0084] Identification of osteoclasts: RAW264.7 cells differentiate into osteoclasts during induction and produce tartrate-resistant acid phosphatase. If the acid phosphatase (ACP) is active, it will be stained purple-red. The staining method is carried out according to the instructions of the tartrate-resistant acid phosphatase staining solution. The steps are as follows: (1) The osteoclasts in the control group, high glucose group, control-postbiotic group and high glucose-postbiotic group are fixed with TRAP fixative solution precooled at 2-8℃ for 60 seconds. (2) Wash with distilled water and drain off excess water (do not over-dry). (3) Add TRAP incubation solution to cover the cells, incubate in a 37℃ incubator for 45-60 minutes, and wash with distilled water. (4) Counterstaining: Stain with methyl green counterstain solution for 2-3 minutes. If hematoxylin staining solution is used for counterstaining, tap water must be used to return to blue for 10 minutes after staining. (5) Covering and observation: (Adherent cells) Wash with distilled water for a while, and observe under a microscope with water or PBS.

[0085] The results are as follows Figure 6 As shown: compared with the control group and the high sugar group, the number of dark-stained osteoclasts in the control-postbiotic group and the high sugar-postbiotic group decreased, which shows that the postbiotic powder fermented by Lactobacillus plantarum IOB602 has an inhibitory effect on the differentiation of osteoclasts.

[0086] 4. Effects of Lactobacillus plantarum IOB602 postbiotics on osteoblast and osteoclast proliferation

[0087] The postbiotics fermented by Lactobacillus plantarum IOB602 were mixed with the osteoclast induction solution of the control group and the high-glucose group at a ratio of 1:20. After 48 hours of induction, the cell proliferation of osteoclasts in each group was detected by MTT method.

[0088] The results are as follows Figure 7 As shown in the figure, compared with the control group and the high sugar group, the differentiation of osteoblasts was promoted to different degrees after 48 hours of intervention with Lactobacillus plantarum IOB602 fermentation. Figure 8 As shown in the figure, the proliferation ability of osteoclasts was inhibited. In summary, the fermented raw material powder of Lactobacillus plantarum IOB602 can promote the proliferation of osteoblasts and inhibit the proliferation of osteoclasts.

[0089] 5. Effects of Lactobacillus plantarum IOB602 postbiotics on osteoblast gene expression

[0090] The osteoblast RNA of the control group, high glucose group, control-postbiotic group and high glucose-postbiotic group was extracted and reverse transcribed into cDNA, and the gene expressions of Runt-related transcription factor 2 (RUNX2), osteopontin (OPN) and bone morphogenetic protein 2 (BMP2) were detected by RT-qPCR.

[0091] The results are as follows Fig. 9 As shown in the figure, compared with the control group and the high glucose group, after postbiotic intervention, the relative expression of RUNX2 gene in osteoblasts in the control-postbiotic group and the high glucose-postbiotic group increased by 239.66% and 126.53%, respectively. Fig.10 As shown in Figure 2, the relative expression of BMP2 gene increased by 92.82% and 53.31% respectively. Fig.11 As shown, the relative expression levels of OPN gene increased by 58.70% and 41.03%, respectively, indicating that the fermentation of Lactobacillus plantarum IOB602 with the raw material powder has an up-regulating effect on the expression of osteoblast proliferation and differentiation-related genes, and can promote osteoblast differentiation.

[0092] Compared with the control group, the increase ratio of the control-metabiotic group (%) = (control-metabiotic group-control group) / control group × 100;

[0093] Compared with the high sugar group, the increase ratio (%) of the high sugar-postbiotic group = (high sugar-postbiotic group-control group) / high sugar group × 100.

Claims

1. Application of a postbiotic of Lactobacillus plantarum IOB602 in the preparation of a medicine for preventing and treating diabetic osteoporosis, wherein the classification name of the Lactobacillus plantarum IOB602 is Lactobacillus plantarum ( Lactiplantibacillus plantarum ), the deposit number is CGMCC No.16021; The preparation method of the Lactobacillus plantarum IOB602 postbiotics comprises: Inoculate the plant lactobacillus IOB602 in the cryopreserved tube into the slant culture medium and place it at 35-39°C for 18-22 hours of activation time, take the fresh slant bacteria and inoculate it into the seed liquid basic culture medium, and culture it at 35-39°C in a closed state for 16-20 hours; inoculate the cultured seed liquid into the fermentation medium at an inoculation rate of 3±0.5%, and culture it at a temperature of 34-38°C for 16-20 hours; After the fermentation is completed, the bacterial liquid is inactivated and dried to obtain the Lactobacillus plantarum IOB602 fermented raw material powder.

2. The use according to claim 1, characterized in that The formula of the slant culture medium is: peptone 10.0g, beef extract 10.0g, yeast extract 4.5g, glucose 20.0g, Tween 80 1.0mL, dipotassium hydrogen phosphate·7H2O2.0g, sodium acetate·3H2O5.0g, triammonium citrate 2.0g, magnesium sulfate·7H2O0.2g, manganese sulfate·4H2O0.05g, agar 15.0g, pH=6.0±0.2, and the amount of water added is 1L.

3. The use according to claim 1, characterized in that The formula of the seed liquid basic culture medium and the fermentation culture medium is: 10.0g of peptone, 10.0g of beef extract, 4.5g of yeast extract, 20.0g of glucose, 1.0mL of Tween 80, 2.0g of dipotassium hydrogen phosphate·7H2O, 5.0g of sodium acetate·3H2O, 2.0g of triammonium citrate, 0.2g of magnesium sulfate·7H2O, 0.05g of manganese sulfate·4H2O, pH=6.0±0.2, and the amount of water added is 1L.

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