A strain of Bifidobacterium longum capable of alleviating osteoporosis and its application

Abstract

The present invention relates to a strain of Bifidobacterium longum capable of alleviating osteoporosis and its application, belonging to the technical field of biomedicine. The present invention provides a strain of Bifidobacterium longum XA-1102 (GOLDGUT-BL23), which can increase the bone volume fraction, trabecular thickness, trabecular number and cortical bone thickness of ovariectomized mice and reduce the trabecular separation, reduce the content of CTX and TRACP in the serum of ovariectomized mice and increase the content of OCN and PINP, promote polarized THP-1 cells to secrete IIL-10, TNFα, IL-1β, IL-1Ra, IL-12p40, IL-12p70, IL-23 and reduce the ratio of TNFa / IL-10, IL-6 / IL-10, IL-1Ra / IL-1β, reduce the permeability of a monolayer epithelial inflammatory injury model, and restore the content of ZO-1 in a monolayer epithelial inflammatory injury model.

Description

Technical Field

[0001] The invention relates to a strain of Bifidobacterium longum capable of alleviating osteoporosis and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Osteoporosis is a systemic bone disease caused by a variety of reasons, which leads to a decrease in bone density and quality, destruction of bone microstructure, increased bone brittleness, and easy fracture. At present, there are three clinical types of osteoporosis: primary, secondary and idiopathic. Among them, primary osteoporosis is a physiological degenerative disease that occurs with age, mainly manifested as postmenopausal osteoporosis and senile osteoporosis; secondary osteoporosis is a pathological degenerative disease induced by metabolic diseases or long-term use of certain drugs and other factors, and the inducing factors include hyperthyroidism or hypothyroidism, acromegaly, diabetes, etc.; idiopathic osteoporosis is more common in adolescents, often accompanied by a genetic family history, mainly manifested as adolescent osteoporosis and osteoporosis in women during pregnancy and lactation.

[0003] At present, bisphosphonates, selective estrogen receptor modulators, estrogen and calcitonin are mainly used in the clinic to treat osteoporosis. These drugs can effectively promote the formation of osteoblasts and inhibit the formation of osteoclasts, but there are certain potential complications and side effects. For example, bisphosphonates can cause severe bone, joint or muscle discomfort in some patients, and a small number of patients have an increased risk of jaw osteonecrosis when suffering from dental diseases or receiving invasive dental treatment; long-term use of alendronic acid is associated with the occurrence of subtrochanteric and femoral shaft fractures; selective estrogen receptor modulator raloxifene can increase the risk of venous thromboembolism and stroke; estrogen can increase the risk of endometrial hyperplasia and cancer in patients with intact uterus, and the incidence of gallstone disease and venous thromboembolism is also increased by 2 to 3 times; calcitonin may cause allergic reactions, severe patients may experience anaphylactic shock, and large-scale use of calcitonin can cause patients to have hypocalcemia, and some patients may also experience joint pain and skeletal muscle pain.

[0004] Therefore, there is an urgent need to find a drug that can effectively relieve osteoporosis and will not cause complications and side effects to patients with long-term use. Summary of the invention

[0005] To solve the above problems, the present invention provides a strain of Bifidobacterium longum (Bifidobacterium longum) XA-1102 (GOLDGUT-BL23), wherein the Bifidobacterium longum XA-1102 (GOLDGUT-BL23) is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.23468 and a deposit date of September 22, 2021.

[0006] The Bifidobacterium longum XA-1102 (GOLDGUT-BL23) was derived from fresh fecal samples of healthy people in Shenzhen. The strain was sequenced and analyzed, and its 16S rDNA sequence was shown in SEQ ID NO.3. The sequence obtained by sequencing was compared with the nucleic acid sequence in GeneBank, and the result showed that the strain was Bifidobacterium longum, and was named Bifidobacterium longum XA-1102 (GOLDGUT-BL23).

[0007] The present invention also provides the use of the above Bifidobacterium longum XA-1102 (GOLDGUT-BL23) in the preparation of medicines for preventing and / or treating osteoporosis.

[0008] In one embodiment of the present invention, in the drug, the viable count of the above-mentioned Bifidobacterium longum XA-1102 (GOLDGUT-BL23) is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0009] In one embodiment of the present invention, the medicine contains the above-mentioned Bifidobacterium longum XA-1102 (GOLDGUT-BL23), a drug carrier and / or a pharmaceutical excipient.

[0010] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0011] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0012] In one embodiment of the present invention, the excipient comprises a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an absorbent, a diluent, a flocculant, a deflocculant, a filter aid and / or a release retardant.

[0013] In one embodiment of the present invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and / or refined lecithin.

[0014] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0015] The present invention also provides a product, which contains the above-mentioned Bifidobacterium longum XA-1102 (GOLDGUT-BL23).

[0016] In one embodiment of the present invention, in the product, the viable count of the above-mentioned Bifidobacterium longum XA-1102 (GOLDGUT-BL23) is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0017] In one embodiment of the present invention, the product comprises food or medicine; the food is a special medical food, a health product or a functional beverage.

[0018] In one embodiment of the present invention, the medicine contains the above-mentioned Bifidobacterium longum XA-1102 (GOLDGUT-BL23), a drug carrier and / or a pharmaceutical excipient.

[0019] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0020] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0021] In one embodiment of the present invention, the excipient comprises a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an absorbent, a diluent, a flocculant, a deflocculant, a filter aid and / or a release retardant.

[0022] In one embodiment of the present invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and / or refined lecithin.

[0023] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0024] In one embodiment of the present invention, the food contains the above-mentioned Bifidobacterium longum XA-1102 (GOLDGUT-BL23) and / or food additives.

[0025] In one embodiment of the present invention, the food additive comprises an antioxidant, a bleaching agent, a colorant, a color protecting agent, an enzyme preparation, a flavor enhancer, a preservative and / or a sweetener.

[0026] The technical solution of the present invention has the following advantages:

[0027] 1. The present invention provides a strain of Bifidobacterium longum (Bifidobacterium longum) XA-1102 (GOLDGUT-BL23), the deposit number of which is CGMCC No. 23468. The Bifidobacterium longum XA-1102 (GOLDGUT-BL23) can relieve osteoporosis, relieve bone loss, inhibit inflammation and repair intestinal barrier, which is specifically embodied in:

[0028] (1) It can significantly increase the bone volume fraction, trabecular thickness, trabecular number and cortical bone thickness of ovariectomized mice, and at the same time, significantly reduce the trabecular separation of ovariectomized mice;

[0029] (2) It can significantly reduce the content of type I collagen carboxyl terminal peptide in the serum of ovariectomized mice;

[0030] (3) It can significantly increase the level of osteocalcin in the serum of ovariectomized mice;

[0031] (4) It can significantly increase the level of type I procollagen amino-terminal propeptide in the serum of ovariectomized mice;

[0032] (5) It can significantly reduce the level of tartrate-resistant acid phosphatase in the serum of ovariectomized mice;

[0033] (6) It can dose-dependently promote the secretion of IL-10, TNFα, IL-1β, IL-1Ra, IL-12p40, IL-12p70, and IL-23 by polarized THP-1 cells, and dose-dependently reduce the ratios of TNFa / IL-10, IL-6 / IL-10, and IL-1Ra / IL-1β;

[0034] (7) In the absence of LPS stimulation, it can promote the secretion of CCL-17 by polarized THP-1 cells in a dose-dependent manner, while in the presence of LPS stimulation, it can inhibit the secretion of CCL-17 by polarized THP-1 cells;

[0035] (8) It can significantly reduce the cell membrane permeability of the monolayer epithelial inflammatory injury model;

[0036] (9) It can significantly restore the content of tight junction protein ZO-1 in the monolayer epithelial inflammatory injury model.

[0037] It can be seen that this Bifidobacterium longum XA-1102 (GOLDGUT-BL23) has great application prospects in the preparation of products (such as food or medicine, etc.) for preventing and / or treating osteoporosis, bone loss, inflammation and intestinal barrier damage.

[0038] 2. This Bifidobacterium longum XA-1102 (GOLDGUT-BL23) belongs to Bifidobacterium longum subsp. longum in Bifidobacterium longum. In the list of strains that can be used for food, it can be seen that the Bifidobacterium longum XA-1102 (GOLDGUT-BL23) of the present invention and the product containing Bifidobacterium longum XA-1102 (GOLDGUT-BL23) as an active ingredient have the advantage of high safety, and long-term use will not cause complications and side effects in patients.

[0039] Biomaterial Deposit

[0040] A strain of Bifidobacterium longum (Bifidobacterium longum) XA-1102 (GOLDGUT-BL23), taxonomically named Bifidobacterium longum, was deposited in the General Microbiology Center of China Culture Collection Administration on September 22, 2021, with the deposit number CGMCC No.23468, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Results of phylogenetic tree analysis of the core genome of Bifidobacterium longum XA-1102 (GOLDGUT-BL23).

[0042] Figure 2 : MLST analysis results of Bifidobacterium longum XA-1102 (GOLDGUT-BL23).

[0043] Figure 3 : Bone tissue morphology of different groups of ovariectomized mice.

[0044] Figure 4 : Bone volume fraction levels in different groups of ovariectomized mice.

[0045] Figure 5 : Trabecular bone thickness levels in different groups of ovariectomized mice.

[0046] Figure 6 :The levels of trabecular bone number in different groups of ovariectomized mice.

[0047] Figure 7 :The level of trabecular bone separation in different groups of ovariectomized mice.

[0048] Figure 8 : Cortical bone thickness levels in different groups of ovariectomized mice.

[0049] Fig. 9: The content of type I collagen carboxyl terminal peptide in serum of different groups of ovariectomized mice.

[0050] Fig.10 : The content of osteocalcin in serum of different groups of ovariectomized mice.

[0051] Fig.11 : The content of amino-terminal propeptide of type I procollagen in serum of different groups of ovariectomized mice.

[0052] Fig.12 : The levels of tartrate-resistant acid phosphatase in serum of ovariectomized mice in different groups.

[0053] Fig.13 :The content of IL-6 (interleukin-6) in the supernatant of different cell culture.

[0054] Fig.14 :The content of IL-10 (interleukin-10) in the supernatant of different cell cultures.

[0055] Fig.15 : The content of TNFα (tumor necrosis factor-α) in the supernatant of different cell cultures.

[0056] Fig.16 :The content of IL-1β (interleukin-1β) in the supernatant of different cell cultures.

[0057] Fig.17 : The content of IL-1Ra (interleukin-1 receptor antagonist) in the supernatant of different cell culture.

[0058] Fig.18 : The ratio of IL-6 / IL-10 in the supernatants of different cell cultures.

[0059] Fig.19 : The ratio of TNFa / IL-10 in the supernatants of different cell cultures.

[0060] Fig. 20 : The ratio of IL-1Ra / IL-1β in the supernatants of different cell cultures.

[0061] Fig.21 :The content of IL-12p40 (interleukin-12p40) in the supernatant of different cell cultures.

[0062] Fig. 22 :The content of IL-12p70 (interleukin-12p70) in the supernatant of different cell cultures.

[0063] Fig.23 :The content of IL-23 (interleukin-23) in the supernatant of different cell cultures.

[0064] Fig.24 : The content of CCL-17 in the supernatant of different cell cultures.

[0065] Fig.25 : FITC-dextran cell membrane permeability in different groups of monolayer epithelial inflammatory injury models.

[0066] Fig.26 : The content of tight junction protein ZO-1 in different groups of monolayer epithelial inflammatory injury models.

[0067] Figures 4 to 26 In the table, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0068] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0069] The culture medium involved in the following examples is as follows:

[0070] MRS solid culture medium: peptone 10g / L, beef extract powder 5g / L, yeast extract powder 4g / L, glucose 20g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, Tween 80 1g / L, agar 15g / L, cysteine ​​salt 0.5g / L, pH 6.8.

[0071] MRS liquid culture medium: peptone 10g / L, beef extract powder 5g / L, yeast extract powder 4g / L, glucose 20g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, Tween 80 1g / L, cysteine ​​salt 0.5g / L, pH 6.8.

[0072] The detection methods involved in the following embodiments are as follows:

[0073] Detection method of viable bacteria count: adopt the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0074] The preparation method of the Bifidobacterium longum suspension involved in the following examples is as follows:

[0075] The long bifidobacterium was streaked on MRS solid culture medium, and statically cultured at 37°C in an anaerobic workstation (Electrotek AW500TG) for 48 hours to obtain a single colony; a single colony was picked and inoculated into MRS liquid culture medium, and cultured at 37°C in an anaerobic workstation (Electrotek AW500TG) for 18 hours to activate, and an activation solution was obtained; the activation solution was inoculated into MRS liquid culture medium at an inoculum amount of 4% (v / v), and cultured at 37°C in an anaerobic workstation (Electrotek AW500TG) for 18 hours to obtain a bacterial solution; the bacterial solution was centrifuged at 3000g for 10 minutes to obtain long bifidobacterium cells; the long bifidobacterium cells were washed with physiological saline and resuspended in PBS buffer (purchased from Beijing Solaibao Biotechnology Co., Ltd.) to a bacterial concentration of 1×10 8 CFU / mL, and obtain Bifidobacterium longum bacterial suspension, which is stored at -80°C for later use.

[0076] Experimental Example 1: Acquisition of Bifidobacterium longum

[0077] The specific steps are as follows:

[0078] Fresh feces from healthy people in Shenzhen were used as samples. 0.5 mL of the sample was added to 5 mL of MRS liquid culture medium and cultured at 37 °C for 24 h to obtain the enriched sample. 0.5 mL of the enriched sample was added to 4.5 mL of sterile saline to obtain 10 -1 diluent, then pipette 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 Dilution: 100 μL of gradient dilution was applied on MRS solid medium, 10 -4 , 10 -5 , 10 -6One plate for each gradient was cultured at 37°C for 48 hours to obtain colonies; colonies with typical characteristics of Bifidobacterium longum on MRS solid culture medium were selected according to colony shape, size, edge, transparency, etc., and colonies were picked with an inoculation loop for streaking on MRS solid culture medium, and cultured at 37°C for 48 hours to obtain purified single colonies; purified single colonies were picked and inoculated into 5 mL of MRS liquid culture medium respectively, and cultured at 37°C for 24 hours to obtain bacterial liquid; after numbering each strain corresponding to each bacterial liquid, Gram staining, strain identification, physiological and biochemical experiments and genome identification analysis were performed according to the steps recorded in the textbook "Microbiology" (edited by Shen Ping and Chen Xiangdong), and hemolytic activity test was performed according to the steps recorded in 3.7 of the "Technical Guidelines for Safety Inspection and Evaluation of Bacterial Species for Health Food Raw Materials" (2020 edition), and strains with typical characteristics of Bifidobacterium longum were selected to obtain strain XA-1102, numbered GOLDGUT-BL23;

[0079] The Gram staining process is as follows:

[0080] A single colony of XA-1102 (GOLDGUT-BL23) was selected for bacterial smear, and then heated and fixed after drying; crystal violet was added for staining for 10 seconds, washed with water and then dried, iodine solution was added for staining for 10 seconds, washed with water and then dried, decolorizing solution was added for decolorization for 10 seconds, washed with water and then dried, and safranin solution was added for restaining for 10 seconds, and washed with water; after the specimen was dried naturally, 1 drop of cedar oil was added to the smeared area for oil microscope observation, and the observation results were as follows: XA-1102 (GOLDGUT-BL23) was purple in Gram staining, and it was a rod-shaped Gram-positive bacterium;

[0081] The strain identification process is as follows:

[0082] XA-1102 (GOLDGUT-BL23) cells were taken, and the genome of XA-1102 (GOLDGUT-BL23) was extracted using a bacterial genome extraction kit. The 27F / 1492R primer pair (27F: AGAGTTTGATCMTGGCTCA, 1492R: TGTACGGYTACCTTGTTACGACTT, in 27F and 1492R, M and Y are degenerate bases, M=A or C, Y=C orT) was used to amplify the extracted genome of XA-1102 (GOLDGUT-BL23) as a template to obtain the 16S rRNA of XA-1102 (GOLDGUT-BL23) (the 16S rDNA sequence of XA-1102 is shown in SEQ ID NO.3); the 16S The rDNA was sequenced using the Blastn program of NCBI, and the results showed that the strain was Bifidobacterium longum, which was named Bifidobacterium longum XA-1102 (GOLDGUT-BL23);

[0083] The physiological and biochemical experimental process is as follows:

[0084] A single colony of XA-1102 (GOLDGUT-BL23) was picked up with a sterile cotton swab and inoculated into sterile saline. 2DensiCHEKTM Plus was used to prepare homogenized bacterial suspension at a turbidity equivalent to McFarland 3.0, and the suspension was loaded onto the ANC card for physiological and biochemical identification within 30 minutes of preparation. The results showed that XA-1102 (GOLDGUT-BL23) could ferment D-galactose, D-glucose, D-mannose, D-maltose, sucrose, arbutin, maltotriose, L-arabinose, D-ribose, and D-xylose, but could not metabolize D-cellobiose;

[0085] The genome identification and analysis process is as follows:

[0086] Take XA-1102 (GOLDGUT-BL23) cells, extract DNA of XA-1102 (GOLDGUT-BL23) by SDS method, detect the quality of extracted DNA by agarose electrophoresis, and use 2.0 for DNA quantification; the genome was sequenced using the NanoporePromethION platform and the Illumina NovaSeq platform. The sequencing strategy was a 10Kb library, a sequencing depth of ≥100×, and an assembly standard of 1 contig and 0 gaps. The sequencing results were: XA-1102 (GOLDGUT-BL23) contains a circular genome with a size of 2.33M bp, and 3 circular plasmids, with sizes of 10.2kbp, 18.65kbp, and 3.92kbp respectively; Diamond software was used to compare the amino acid sequence encoded by the XA-1102 (GOLDGUT-BL23) genome with the VFDB database, and the sequence homology of the potential virulence factors obtained was less than 75%, and the main genes were genes encoding cell wall components, without virulence genes; the amino acid sequence encoded by the XA-1102 (GOLDGUT-BL23) genome was compared with the ARDB database to analyze the potential drug-resistant genes, and it was found that the XA-1102 (GOLDGUT-BL23) genome contained homologous genes (>99%) of the rifampicin resistance gene rpoB and the trimethoprim resistance gene dfr, suggesting that XA-1102 (GOLDGUT-BL23) may be resistant to rifampicin and trimethoprim;

[0087] There are four subspecies of Bifidobacterium longum, namely Bifidobacterium longumsubsp.longum, Bifidobacterium longum subsp.infantis, Bifidobacterium longum subsp.suis, and Bifidobacterium longum subsp.suillum. Only Bifidobacterium longum subsp.longum and Bifidobacterium longum subsp.infantis are included in the "List of Bacteria that Can Be Used in Food" (2022 Edition). Therefore, XA-1102 (GOLDGUT-BL23) was identified as a subspecies; core genome evolutionary tree analysis and literature Yanokura E.et al. (2015) MLST analysis (selecting 7 housekeeping genes including clpC, dnaG, dnaJ1, hsp60, purF, rpoC, xfp), both analysis methods showed that XA-1102 (GOLDGUT-BL23) belongs to Bifidobacterium longum subspecies longum, which is a species in the list of bacteria that can be used for food (analysis results are shown in Figures 1-2 ).

[0088] Experimental Example 1: Effect of Bifidobacterium longum on osteoporosis in ovariectomized mice

[0089] The specific steps are as follows:

[0090] Eighteen 8-week-old SPF-grade C57BL / 6 female mice (purchased from Beijing Weitonglihua Biotechnology Co., Ltd., weighing 22±5g) were randomly divided into three groups, with 6 mice in each group. The three groups were: sham group (sham), model control group (OVX), and probiotic intervention group (OVX+XA-1102) gavaged with Bifidobacterium longum XA-1102 (GOLDGUT-BL23) bacterial suspension. After 3 days of adaptive feeding, the mice were fasted for 12 hours on the third day and prepared for ovariectomy. The mice in the model control group and the probiotic intervention group were anesthetized by intraperitoneal injection of sodium pentobarbital, and the ovariectomy was performed from the back opening and sutured. The mice in the sham group underwent sham surgery (incision and suture at the corresponding position), and recovered for 5 weeks after surgery. After 5 weeks of recovery, gavage was started until the end of the experiment. The mice in the sham operation group and the model control group were gavaged with PBS buffer at a dose of 10 μL / g*weight (0.2 mL), and the mice in the probiotic intervention group were gavaged with Bifidobacterium longum XA-1102 (GOLDGUT-BL23) bacterial suspension (bacterial concentration was 1×10 8 CFU / mL), once every 2 days, for a total of 5 weeks. After 5 weeks of gavage, mice were anesthetized by intraperitoneal injection of sodium pentobarbital, and the mice were killed by cervical dislocation after blood collection. The femurs of the mice were taken, wrapped with gauze soaked in saline, and placed vertically in the sample cup, and then placed in a μ-CT scanner for bone tissue microstructure analysis. The observation parameters included bone volume fraction (bone volume / total volume, BV / TV), trabecular bone thickness (trabecular bone thickness, Tb.Th), trabecular bone number (trabecular bone number, Tb.N), trabecular bone separation (trabecular bone separation, Tb.Sp) and cortical thickness (Cortical thickness, Ct.Th). The observation results are shown in Figures 3 to 8. Among them, the number of trabeculae, trabecular thickness and trabecular separation are the main indicators for evaluating the spatial morphological structure of trabeculae. When bone catabolism is greater than bone anabolism, such as when osteoporosis occurs, the values ​​of Tb.N and Tb.Th decrease, and the value of Tb.Sp increases. The blood of mice was collected, separated and serum was prepared, and the ELISA kit (purchased from NOVUS Biologicals) was used to determine the levels of type I collagen carboxyl terminal peptide (Collagen type I C-Telopeptide, CTX), osteocalcin (Osteocalcin, OCN), type I procollagen amino terminal propeptide (Procollagen type I N-propeptide, PINP), and tartrate-resistant acid phosphatase (Tartrate-resistant acid phosphatase, TRACP) in the serum. The test results are shown in Figures 9 to 12 Among them, CTX is the most widely used collagen degradation marker, reflecting the effective marker of metabolic bone disease characterized by osteoclast bone resorption. CTX levels are elevated in diseases such as osteoporosis, osteophyte, multiple myeloma and tumor bone metastasis; OCN, also known as bone-hydroxyglutamate protein (BGP), is the main component of bone non-collagenous protein, a specific protein of bone tissue, and a hormone-like polypeptide produced and secreted by osteoblasts. It is currently believed that OCN in serum is a biochemical marker reflecting osteoblast function. About 20% of osteocalcin synthesized by osteoblasts is released into the blood. Serum OCN and bone tissue OCN are positively correlated. Therefore, the determination of OCN in blood can reflect Functional status of osteoblasts; the expression level of PINP reflects the formation of new bone. With the synthesis of new type I collagen in osteoblasts, the proteases outside the osteoblasts will cleave PINP from type I procollagen. Part of the cleaved PINP can be directly deposited in the bone matrix, and the rest enters the blood circulation. When osteoblast synthesis decreases, the PINP level reflecting the changes in newly synthesized type I collagen decreases; TRACP is a bone resorption marker, which is mainly released by osteoclasts. Increased TRACP is seen in primary hyperthyroidism, chronic renal insufficiency, osteitis deformans, tumor bone metastasis, high-turnover osteoporosis (postmenopausal osteoporosis), diabetes, etc.

[0091] Depend on Figures 3 to 8It can be seen that compared with the sham operation group, the BV / TV, Tb.Th, Tb.N and Ct.Th of the model control group mice decreased significantly, from 4.07%, 46.22μm, 0.00089 / mm and 186.51μm to 1.13%, 40.19μm, 0.00026 / mm and 171.17μm, respectively, while Tb.Sp increased significantly, from 313.96 / μm to 465.24 / μm; After the intervention with bacteria, BV / TV, Tb.Th, Tb.N and Ct.Th of ovariectomized mice increased significantly compared with the model control group mice, from 1.13%, 40.19μm, 0.00026 / mm and 171.17μm to 2.48%, 43.53μm, 0.00058 / mm and 176.02μm, respectively, while Tb.Sp decreased significantly from 465.24 / μm to 357.85 / μm. It can be seen that Bifidobacterium longum XA-1102 (GOLDGUT-BL23) can significantly increase the bone volume fraction, trabecular thickness, trabecular number level and cortical bone thickness of ovariectomized mice, and at the same time, significantly reduce the trabecular separation of ovariectomized mice. This result shows that Bifidobacterium longum XA-1102 (GOLDGUT-BL23) intervention has the effect of treating osteoporosis.

[0092] Depend on Figures 9 to 12 It can be seen that compared with the sham operation group, the levels of type I collagen carboxyl terminal peptide and tartrate-resistant acid phosphatase in the serum of mice in the model control group were significantly increased, from 12.81pg / mL and 99.61pg / mL to 23.79pg / mL and 194.44pg / mL, respectively; after probiotic intervention, the levels of type I collagen carboxyl terminal peptide and tartrate-resistant acid phosphatase in the serum of ovariectomized mice were significantly decreased compared with those of mice in the model control group, from 23.79pg / mL and 194.44pg / mL to 14.97pg / mL and 194.44pg / mL, respectively. 119.74pg / mL; Compared with the sham operation group, the levels of osteocalcin and type I procollagen amino-terminal propeptide in the serum of the model control group mice decreased significantly, from 63.7pg / mL and 190pg / mL to 60.9pg / mL and 147pg / mL, respectively; After probiotic intervention, the levels of osteocalcin and type I procollagen amino-terminal propeptide in the serum of ovariectomized mice were significantly increased compared with those of the model control group mice, from 60.9pg / mL and 147pg / mL to 63.17pg / mL and 179.36pg / mL, respectively. It can be seen that Bifidobacterium longum XA-1102 (GOLDGUT-BL23) can significantly reduce the levels of type I collagen carboxyl terminal peptide and tartrate-resistant acid phosphatase in serum, and increase the levels of osteocalcin and type I procollagen amino-terminal propeptide in the serum of ovariectomized mice. The results showed that Bifidobacterium longum XA-1102 (GOLDGUT-BL23) intervention has the efficacy in treating osteoporosis.

[0093] Experimental Example 2: Experiment on the immunomodulatory function of Bifidobacterium longum

[0094] The specific steps are as follows:

[0095] THP-1 cells (purchased from ATCC) were resuspended in complete cell culture medium (purchased from Thermo Fisher Scientific) to a cell density of 1×10 6 After the cells were inoculated with 200 μL per well, they were inoculated into a 96-well plate; PMA (phorbol ester) was added to the 96-well plate to a final concentration of 50 ng / mL, and the cells were polarized at 37°C for 48 h; after the supernatant was removed from the 96-well plate, a complete cell culture medium without PMA was added at a volume of 200 μL per well, and the cells were statically cultured at 37°C for 72 h; after the supernatant was removed from the 96-well plate, a complete cell culture medium containing 1 μg / mL LPS (lipopolysaccharide) was added at a volume of 200 μL per well, and Bifidobacterium longum XA-1102 (GOLDGUT-BL23) in the mid-logarithmic phase was added at a multiplicity of infection (MOI) of 0, 1, and 10, respectively, and the plates were statically interacted at 37°C for 2 h; after the supernatant was removed from the 96-well plate, it was first washed once with D-PBS buffer, and then 200 μL of 1 μg / mL The cells were cultured in a complete cell culture medium containing LPS (lipopolysaccharide) and 100 g / mL gentamicin, and then cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 22 h; the cell culture supernatant in the 96-well plate was collected, and the LegendPlexTM multifactor flow cytometry kit was used to detect the content of TNFα, IL-6, IL-10, IL-1β, IL-1Ra, IL-12p40, IL-12p70, IL-23, and CCL-17 in the cell culture supernatant. The test results are shown in Figures 13-24 .

[0096] like Figures 13-24 As shown, Bifidobacterium longum XA-1102 (GOLDGUT-BL23) can dose-dependently promote the secretion of IIL-10, TNFα, IL-1β, IL-1Ra, IL-12p40, IL-12p70, and IL-23 by polarized THP-1 cells, and dose-dependently reduce the ratios of TNFa / IL-10, IL-6 / IL-10, and IL-1Ra / IL-1β; for chemokine CCL-17, in the absence of LPS stimulation, Bifidobacterium longum XA-1102 (GOLDGUT-BL23) can dose-dependently promote the secretion of CCL-17 by polarized THP-1 cells, and in the presence of LPS stimulation, Bifidobacterium longum XA-1102 (GOLDGUT-BL23) can inhibit the secretion of CCL-17 by polarized THP-1 cells. This shows that Bifidobacterium longum XA-1102 (GOLDGUT-BL23) has the function of inhibiting inflammation.

[0097] Experimental Example 3: Experiment on the intestinal barrier repair function of Bifidobacterium longum

[0098] The specific steps are as follows:

[0099] CaCO-2 cells (purchased from ATCC) and HT-29-MTX-E12 cells in the logarithmic growth phase were inoculated into the upper chamber of the Transwell chamber at a cell number ratio of 9:1, with a total inoculation volume of 2.5×10 5 cells; 100 μL and 600 μL of complete culture medium (purchased from Thermo Fisher) were added to the upper and lower chambers of the Transwell chamber, respectively, and then cultured in a cell culture incubator at 37°C and 5% (v / v) CO2. Fresh complete culture medium was replaced every other day, and the cell transmembrane resistance was measured using an EVOM3 cell resistance meter. The cells were cultured for 18 days until they were fully fused and the transmembrane resistance value no longer increased; the culture medium in the upper and lower chambers was removed, and DMEM high-glucose culture medium (purchased from Thermo Fisher) containing 10% (v / v) inactivated FBS was used as a negative control (untreated group); 100 μL and 600 μL of 100 ng / mL IL-1β, 40 ng / mL TNFα, 100 μg / mL The monolayer epithelial cells were treated with DMEM high-glucose medium containing LPS (lipopolysaccharide) and 10% (v / v) inactivated FBS, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 24 h to construct a monolayer epithelial inflammatory injury model (treatment group); the culture medium in the upper and lower chambers was removed, and the Transwell chamber was washed once with PBS buffer. DMEM high-glucose cell culture medium without double antibody and containing 10% (v / v) FBS was added to the lower chamber, and a sodium butyrate solution with a final concentration of 2 mM was used as a positive control, and DMEM high-glucose cell culture medium was used as a solvent control. 100 μL of Bifidobacterium longum XA-1102 (GOLDGUT-BL23) resuspended in DMEM high-glucose cell culture medium was added to the upper chamber at an infection multiplicity (MOI) of 10:1, and the cells were incubated anaerobicly. The cells were incubated at 37°C for 2 h. The culture medium in the upper and lower chambers was removed, and the Transwell chamber was washed once with PBS buffer. 100 μL and 600 μL of DMEM high-glucose cells without dual antibodies were added to the upper and lower chambers of the Transwell chamber, respectively, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 22 h. 20 μL of FITC-dextran (4 KDa, 100 μg / mL) was added to the upper chamber and incubated in a cell culture incubator at 37°C and 5% (v / v) CO2 for 1 h. 60 μL of the culture medium in the lower chamber was collected, and the fluorescence intensity of the substances in the culture medium in the lower chamber was detected using a multifunctional microplate reader under the conditions of excitation light of 490 nm and emission light of 520 nm, and the cell membrane permeability of FITC-dextran was calculated. The calculation results are shown in Fig.25; Remove the culture medium from the upper and lower chambers, wash the Transwell chambers three times with PBS buffer, add 100 μL of 4% (g / 100 mL) paraformaldehyde precooled to 4°C, and fix at 4°C for 25 min; after washing with PBS buffer, add 200 μL of PBS buffer containing 0.2% (g / 100 mL) Triton X-100 to each well and let stand on ice for 10 min; after washing with PBS buffer, add 300 μL of PBS buffer containing 5% (g / 100 mL) BSA to each well and block at room temperature (25°C) for 1 h; add anti-ZO-1 antibody according to the instructions and incubate at 4°C for 16 h; after washing with PBS buffer, add FITC-labeled secondary antibody and incubate at room temperature (25°C) in the dark for 2 h; after washing with PBS buffer, carefully peel off the membrane of the transwell chamber, add a sealing agent containing DAPI, place it in a glass-bottomed culture dish and observe it under a fluorescence microscope. The observation results are shown in Fig.26 ;

[0100] Wherein, FITC-dextran cell membrane permeability = FITC-dextran fluorescence value of experimental well / FITC-dextran fluorescence value of blank well × 100%.

[0101] like Fig.25 As shown in the figure, compared with the untreated group, the cell membrane permeability of FITC-dextran in the monolayer epithelial inflammatory injury model in the treated group was significantly increased after treatment with IL-1β, TNFα and LPS, while treatment with sodium butyrate or Bifidobacterium longum XA-1102 (GOLDGUT-BL23) could significantly reduce the cell membrane permeability of FITC-dextran to the level of the untreated group. Fig.26 As shown, the content of tight junction protein ZO-1 in the monolayer epithelial inflammatory injury model of the treated group was significantly lower than that of the untreated group, while the content of tight junction protein ZO-1 in the monolayer epithelial inflammatory injury model treated with sodium butyrate or Bifidobacterium longum XA-1102 (GOLDGUT-BL23) could be significantly restored. The above results show that Bifidobacterium longum XA-1102 (GOLDGUT-BL23) has the function of intestinal barrier repair.

[0102] Experimental Example 4: Tolerance experiment of Bifidobacterium longum

[0103] The specific steps are as follows:

[0104] Experiment 1: Gastric juice resistance test

[0105] Gastric fluid simulation fluid was purchased from Xiaodong Yijian (Suzhou) Instrument Equipment Co., Ltd. and was prepared according to the instructions; 5 mL of Bifidobacterium longum XA-1102 (GOLDGUT-BL23) bacterial suspension was mixed with gastric fluid simulation fluid (SGF) in a volume ratio of 1:1, and the pH was adjusted to 3.0 with 6M hydrochloric acid solution to obtain a mixed solution; the mixed solution was allowed to stand at 37°C in an anaerobic workstation, and samples were taken after standing for 0, 0.5, 1, 2, 4, and 6 hours, respectively; the samples taken at different time points were diluted 10 times with sterile physiological saline to obtain a dilution; the dilution was spread on an MRS solid culture medium, placed in an anaerobic workstation at 37°C for culture, and the number of colonies on the MRS solid culture medium was counted after 48 hours of culture to calculate the survival rate of Bifidobacterium longum XA-1102 (GOLDGUT-BL23), and the calculation results are shown in Table 1; wherein, survival rate = (number of colonies at Tn / number of colonies at T0) × 100%.

[0106] Experiment 2: Intestinal fluid resistance test

[0107] Intestinal fluid simulation fluid was purchased from Xiaodong Yijian (Suzhou) Instrument Equipment Co., Ltd. and prepared according to the instructions; 5 mL of Bifidobacterium longum XA-1102 (GOLDGUT-BL23) bacterial suspension was mixed with gastric fluid simulation fluid (SGF) in a volume ratio of 1:1, and the pH was adjusted to 6.8 with 1M sodium hydroxide solution to obtain a mixed solution; the mixed solution was allowed to stand at 37°C in an anaerobic workstation, and samples were taken after standing for 0, 0.5, 1, 2, 4, and 6 hours, respectively; the samples taken at different time points were diluted 10 times with sterile physiological saline to obtain a dilution; the dilution was spread on an MRS solid culture medium, placed in an anaerobic workstation at 37°C for culture, and the number of colonies on the MRS solid culture medium was counted after 48 hours of culture to calculate the survival rate of Bifidobacterium longum XA-1102 (GOLDGUT-BL23), and the calculation results are shown in Table 1; wherein, survival rate = (number of colonies at Tn / number of colonies at T0) × 100%.

[0108] As shown in Table 1, Bifidobacterium longum XA-1102 (GOLDGUT-BL23) has certain resistance to gastric juice and intestinal juice, and the resistance to intestinal juice is stronger than the resistance to gastric juice.

[0109] Table 1 Survival rate of Bifidobacterium longum XA-1102 (GOLDGUT-BL23) after being placed in gastric juice and intestinal juice for different time periods

[0110] Group 0 hours 0.5 hours 1 hour 2 hours 4 hours 6 hours Survival rate in gastric fluid 100％ 16.26％ 12.30％ 1.88％ 2.17％ 1.71％ Survival rate in intestinal fluid 100％ 35.53％ 30.46％ 29.44％ 26.73％ 22.92％

[0111] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. Use of Bifidobacterium longum in the preparation of medicines, characterized in that: The Bifidobacterium longum is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No. 23468; the drug has the following functions: (a) prevention and / or treatment of osteoporosis; (b) preventing and / or treating bone loss; (c) preventing and / or treating inflammation; and, (d) preventing and / or treating intestinal barrier damage.

2. A strain of Bifidobacterium longum, characterized in that The Bifidobacterium longum is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.23468.

3. A medicine, characterized in that: The medicine contains the Bifidobacterium longum described in claim 2; the medicine has the following functions: (a) prevention and / or treatment of osteoporosis; (b) preventing and / or treating bone loss; (c) preventing and / or treating inflammation; and, (d) preventing and / or treating intestinal barrier damage.

4. A medicine according to claim 3, characterized in that: The medicine contains the Bifidobacterium longum as claimed in claim 2 and pharmaceutical excipients.

5. A medicine according to claim 4, characterized in that: The pharmaceutical excipients include pharmaceutical carriers, excipients and / or additives.

6. A medicine according to claim 5, characterized in that: The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

7. A medicine according to claim 5, characterized in that: The excipients include solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, absorbents, diluents, flocculants, deflocculating agents, filter aids and / or release retardants; the additives include microcrystalline cellulose, hydroxypropyl methylcellulose and / or refined lecithin.

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