Probiotic agent capable of improving physiological activity function of vitamin D and maintaining bone health and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a probiotic agent capable of improving the physiological activity function of vitamin D and maintaining bone health and application of the probiotic agent. The invention provides a probiotic agent composed of bifidobacterium adolescentis and bifidobacterium longum, the probiotic agent can significantly improve the bone mineral density, the bone trabecula thickness, the bone trabecula number, the bone trabecula connectivity and the bone volume fraction of an osteoporosis mouse, and meanwhile, the bone trabecula separation degree of the osteoporosis-removed mouse is significantly reduced; the level of vitamin D metabolites such as 25-hydroxy vitamin D and 1, 25-dihydroxy vitamin D in mouse serum is improved; the content of serum calcium and alkaline phosphatase in serum of mice is reduced, and the level of osteocalcin and type I procollagen amino terminal peptide in the serum is improved. The probiotic agent can relieve bone calcium loss, maintain bone health, repair bone injury, increase osteoblasts and reduce the osteoporosis degree, and can be used for preparing drugs for preventing and / or treating osteoporosis or preparing health care products or food beneficial to improving bone mineral density.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a probiotic agent capable of improving the physiological activity of vitamin D and maintaining bone health, and an application thereof. Background Art

[0002] Osteoporosis is a systemic metabolic disease caused by various reasons, which leads to bone loss and reduction, changes in bone tissue microstructure, and increased bone brittleness, which can easily cause fractures in patients. Osteoporosis is divided into two major categories: primary osteoporosis and secondary osteoporosis. Primary osteoporosis is further divided into two major categories: senile osteoporosis and postmenopausal osteoporosis. Common symptoms of osteoporosis are pain, shortened stature, hunchback, fractures and decreased respiratory function. Calcium supplementation is a basic measure for the treatment of osteoporosis, but calcium supplementation alone is far from enough for the treatment of osteoporosis, and medications need to be added according to the patient's condition.

[0003] Currently, osteoporosis is primarily treated clinically with drugs such as bisphosphonates, selective estrogen receptor modulators (SERMs), estrogen, and calcitonin. These drugs effectively promote osteoblast formation and inhibit osteoclast formation. However, bisphosphonates can cause severe bone, joint, or muscle discomfort in some patients, and a small number of patients are at increased risk of jaw osteonecrosis during dental work or invasive dental treatment. Long-term use of bisphosphonates has been associated with subtrochanteric and femoral shaft fractures. The 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 an intact uterus and increase the incidence of gallstones and venous thromboembolism by 2-3 times. Calcitonin can cause allergic reactions, including anaphylactic shock in severe cases. Excessive use of calcitonin can lead to hypocalcemia, and some patients experience joint and skeletal muscle pain. Therefore, there is an urgent need for a drug or treatment that can effectively alleviate osteoporosis without complications or side effects with long-term use.

[0004] Vitamin D (VD) is an important element for maintaining the physiological function of bones and bone growth and development. It plays an important role in calcium absorption by bones. It can absorb calcium from the intestines into the blood and improve the absorption of calcium. At the same time, it can also help guide calcium from the blood into the bones, so that the calcium in osteoblasts will not be excessively lost, avoiding the occurrence of osteoporosis and bone growth and development disorders in children. Secondly, it can also promote the formation of osteoblasts. The active form of VD, 1,25-dihydroxyvitamin D (1,25(OH)2D), can also act as a direct agonist to bind to the vitamin D receptor (VDR) for regulation in the human body. Appropriate supplementation of VD in children and the elderly will have direct benefits on bones, such as improving muscle strength and regulating the body's balance ability. It plays an important role in promoting human bone health.

[0005] Patients with osteoporosis often have reduced vitamin D metabolism. Vitamin D is a fat-soluble vitamin that can be synthesized through skin exposure to ultraviolet light or through dietary intake. Vitamin D undergoes two key metabolic steps in the body to function: first, vitamin D synthesized in the skin is converted to 25-hydroxyvitamin D (25(OH)D), which is then further converted to its active form, 1,25(OH)2D, in the kidneys. Reduced vitamin D metabolism in osteoporosis can occur for a variety of reasons, the most common of which are aging and decreased renal function. With aging, the skin's ability to synthesize vitamin D decreases, leading to a decrease in levels of 25(OH)D, the storage form of vitamin D. Furthermore, decreased renal function may hinder the production of 1,25(OH)2D, further reducing vitamin D metabolism. Therefore, elderly individuals and those with osteoporosis require additional vitamin D intake to compensate for reduced vitamin D synthesis and improve vitamin D metabolism. Compared to medications, probiotics have no significant side effects and can be used long-term, overcoming the risks associated with long-term medication for osteoporosis. However, no probiotics or products with probiotics as active ingredients have been found so far that can improve the body's metabolism of vitamin D and effectively alleviate osteoporosis. Summary of the Invention

[0006] To address the above-mentioned problems, the present invention provides a probiotic containing Bifidobacterium adolescentis (Bifidobacterium adolescentiscens) and Bifidobacterium longum (Bifidobacterium longum). This probiotic has the effects of enhancing the physiological activity of vitamin D and alleviating osteoporosis, as embodied in: (1) significantly increasing the levels of vitamin D metabolites in the blood of osteoporotic mice; (2) significantly improving bone-related indicators in osteoporotic mice; and (3) significantly improving the bone health of osteoporotic mice. Therefore, this probiotic has great application prospects in the preparation of products for enhancing the physiological activity of vitamin D, preventing and / or treating osteoporosis, and / or preventing and / or treating bone loss.

[0007] The technical solutions of the present invention are as follows:

[0008] The present invention provides a probiotic agent capable of improving the physiological activity function of vitamin D and maintaining bone health. The probiotic agent comprises Bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM1447 strain with a preservation number of GDMCC No: 65382 and Bifidobacterium longum subsp. longum (Bifidobacterium longum subsp. longum) CCFM1448 strain with a preservation number of GDMCC No: 65383, wherein the ratio of the viable cell count of the CCFM1447 strain to that of the CCFM1448 strain is 2:1 to 1:2.

[0009] The Bifidobacterium adolescens CCFM1447 strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 31, 2024, with the deposit number GDMCC No: 65382, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0010] The Bifidobacterium adolescens CCFM1447 strain was isolated from human feces. Sequencing analysis showed that the 16S rRNA sequence of the strain was shown as SEQ ID NO. 1. Comparison of the sequence in GenBank showed that the strain was Bifidobacterium adolescens, and was named Bifidobacterium adolescens CCFM1447.

[0011] The Bifidobacterium adolescens CCFM1447 strain is a Gram-positive bacillus that does not produce spores and is arranged singly, in pairs, or in a V-shape. It is anaerobically cultured on an MRS (lactic acid bacteria culture medium) plate at 36° C. for 5 days to produce small colonies.

[0012] The Bifidobacterium longum subsp. longum CCFM1448 strain was deposited in Guangdong Provincial Microbiological Culture Collection on October 31, 2024, with the deposit number GDMCC No: 65383, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0013] The Bifidobacterium longum subsp. longum CCFM1448 strain was isolated from human feces. Sequencing analysis showed that the 16S rRNA sequence of the strain was shown as SEQ ID NO. 2. Comparison of the sequence in GenBank showed that the strain was Bifidobacterium longum, and the strain was named Bifidobacterium longum subsp. longum CCFM1448.

[0014] The Bifidobacterium longum subsp. longum CCFM1448 strain is a Gram-positive bacillus that does not produce spores and grows singly, in pairs, or in a V-shaped arrangement. After anaerobically cultured on an MRS (lactic acid bacteria culture medium) plate at 36° C. for 3 days, the colonies are milky white, round, convex, with smooth surfaces and neat edges.

[0015] Preferably, the viable count of the Bifidobacterium adolescentis CCFM1447 strain is not less than 1.0×10 6 CFU / ml, the number of viable bacteria of Bifidobacterium longum subspecies longum CCFM1448 strain is not less than 1.0×10 6 CFU / ml.

[0016] Preferably, the probiotics further include food raw materials, and / or food or medically acceptable excipients or additives.

[0017] Preferably, the dosage form of the probiotic includes lyophilized powder, capsule, tablet or granule.

[0018] A product containing any of the above-mentioned probiotics includes food, medicine or health care products.

[0019] Preferably, any of the above-mentioned probiotics and products are used in the preparation of medicines for preventing and / or treating osteoporosis.

[0020] Preferably, the prevention and / or treatment of osteoporosis includes any of the following functions:

[0021] (1) Increase the levels of vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in serum;

[0022] (2) Increase total bone density, cortical bone density, distal femoral trabecular bone density, trabecular thickness, trabecular number, trabecular connectivity, bone surface area, bone volume, and bone volume fraction; and reduce trabecular separation;

[0023] (3) Reduce serum calcium and alkaline phosphatase levels;

[0024] (4) Increase the levels of osteocalcin and type I procollagen amino-terminal propeptide in serum.

[0025] Use of any of the above-mentioned probiotics and products in the preparation of health products that help improve bone density.

[0026] Beneficial effects:

[0027] The present invention provides a probiotic containing Bifidobacterium adolescens CCFM1447 strain and Bifidobacterium longum subsp. longum CCFM1448 strain. This probiotic has the effects of increasing the physiological activity of vitamin D and alleviating osteoporosis, which is specifically embodied in the following aspects:

[0028] (1) Significantly increased total bone density, cortical bone density, distal femoral trabecular bone density, trabecular thickness, trabecular number, trabecular connectivity, bone surface area, bone volume, and bone volume fraction in osteoporotic mice;

[0029] (2) Significantly reduced trabecular separation in osteoporotic mice;

[0030] (3) Increase the levels of vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in the serum of osteoporotic mice;

[0031] (4) Reduce the levels of serum calcium and alkaline phosphatase in the serum of osteoporotic mice, thereby increasing the levels of serum osteocalcin and type I procollagen amino-terminal propeptide.

[0032] Therefore, probiotics have great application prospects in the preparation of products for preventing and / or treating osteoporosis.

[0033] Preservation Instructions

[0034] Bifidobacterium adolescens CCFM1447 strain

[0035] Storage address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province

[0036] Deposit date: October 31, 2024

[0037] Species name: Bifidobacterium adolescentis

[0038] Latin name: Bifidobacterium adolescens

[0039] Strain ID: CCFM1447

[0040] Depository: Guangdong Provincial Microbial Culture Collection Center

[0041] Depository abbreviation: GDMCC

[0042] GDMCC registration number: GDMCC No:65382

[0043] Bifidobacterium longum subsp. longum CCFM1448 strain

[0044] Storage address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province

[0045] Deposit date: October 31, 2024

[0046] Species name: Bifidobacterium longum subsp. longum

[0047] Latin name: Bifidobacterium longum subsp.longum

[0048] Strain ID: CCFM1448

[0049] Depository: Guangdong Provincial Microbial Culture Collection Center

[0050] Depository abbreviation: GDMCC

[0051] GDMCC registration number: GDMCC No:65383 BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0053] Figure 1The figure shows the content of 25-hydroxyvitamin D in the serum of osteoporosis mice in different groups.

[0054] Figure 2 The figure shows the content of 1,25-dihydroxyvitamin D in the serum of osteoporosis mice in different groups.

[0055] Figure 3 The figure shows the serum calcium content in the serum of osteoporosis mice in different groups.

[0056] Figure 4 The graph shows the levels of alkaline phosphatase in the serum of osteoporosis mice in different groups.

[0057] Figure 5 The graph shows the content of osteocalcin in the serum of osteoporosis mice in different groups.

[0058] Figure 6 The figure shows the content of type I procollagen amino-terminal propeptide in the serum of osteoporosis mice in different groups.

[0059] Figure 7 Figure 2 shows the total bone density levels of osteoporotic mice in different groups.

[0060] Figure 8 Figure 2 shows the cortical bone density levels of osteoporotic mice in different groups.

[0061] Figure 9 Figure 3 shows the trabecular bone density levels of the distal femur in different groups of osteoporotic mice.

[0062] Figure 10 Figure 2 shows the trabecular separation levels of osteoporotic mice in different groups.

[0063] Figure 11 Figure 2 shows the trabecular thickness levels of osteoporotic mice in different groups.

[0064] Figure 12 Figure 2 shows the levels of trabecular bone in different groups of osteoporotic mice.

[0065] Figure 13 Figure 2 shows the trabecular connectivity levels of osteoporotic mice in different groups.

[0066] Figure 14 Horizontal graph of bone surface area in different groups of osteoporotic mice.

[0067] Figure 15 Figure 2 shows the bone volume levels of osteoporotic mice in different groups.

[0068] Figure 16 is the bone volume fraction level of osteoporotic mice in different groups. DETAILED DESCRIPTION

[0069] In order to better understand the present invention, the present invention is further described in detail below with reference to the embodiments and drawings. However, those skilled in the art will understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0071] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0072] The culture medium involved in the following examples is as follows:

[0073] MRS solid medium: peptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 g / L, agar 15 g / L, cysteine ​​amino acid salt 0.5 g / L, pH 6.8.

[0074] MRS liquid medium: peptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 g / L, cysteine ​​amino acid salt 0.5 g / L, pH 6.8.

[0075] The detection methods involved in the following embodiments are as follows:

[0076] Method for detecting viable bacteria count: adopt the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0077] The present invention will be further described below with reference to specific embodiments.

[0078] Example 1 Screening, Identification and Cultivation of Bifidobacterium adolescentis

[0079] 1. Screening

[0080] Human feces were used as samples. After pretreatment, the samples were stored in a -80°C refrigerator in about 20% glycerol. After being taken out and thawed, 0.5 mL of the sample was mixed and added to 4.5 mL. Gradient dilutions were performed with 0.9% saline containing 0.05% cysteine. Appropriate gradient dilutions were selected and spread on MRS plates added with 0.05% cysteine. The plates were cultured at 37°C for 48 hours. Typical colonies were picked and streaked onto MRS plates for purification. Single colonies were picked and transferred to MRS liquid culture medium (containing 0.05% cysteine) for bacterial enrichment and preserved in 30% glycerol to obtain strain CCFM1447.

[0081] 2. Identification

[0082] The genome of CCFM1447 was extracted, and the 16S rDNA of CCFM1447 (shown as SEQ ID NO. 1) was amplified and sequenced (Shanghai Sangon Biotechnology Co., Ltd.). The sequence was compared in GenBank, and the results showed that the bacterium was Bifidobacterium adolescens, and was named Bifidobacterium adolescens CCFM1447.

[0083] 3. Cultivation of Bifidobacterium adolescentis

[0084] Bifidobacterium adolescens CCFM1447 strain was inoculated into MRS solid medium (containing 0.05% cysteine) and cultured at 37°C for 48 hours. The colonies were observed and the bacteria were observed under a microscope. It was found that it was a Gram-positive bacillus that did not produce spores and was arranged singly, in pairs, or in a V-shape. The colonies were small and the bacteria were short, forked rods.

[0085] Bifidobacterium adolescens CCFM1447 was inoculated into MRS liquid medium (containing 0.05% cysteine) and cultured at 37°C for 48 hours. A growth curve was prepared, and it was found that it reached a stable phase after being cultured at 37°C for 12 hours.

[0086] Bifidobacterium adolescens CCFM1447 was inoculated into MRS liquid culture medium (containing 0.05% cysteine) and cultured at 10, 15, 20, 25, 30, 35, 40, 45, and 50°C for 48 hours, and its growth was observed. It grew well at 20-35°C and was still able to grow at 45°C, but hardly grew at 15°C or below or 50°C. It was able to survive when stored at -80°C in a 30% glycerol tube.

[0087] Bifidobacterium adolescens CCFM1447 was inoculated into MRS liquid medium (containing 0.05% cysteine) and cultured at 37°C for 48 h. The culture was then transferred to fresh MRS medium (containing 0.05% cysteine) and cultured under the same conditions for 30 h. The cells were centrifuged at 6000 g for 15 min, washed with 0.9% saline, and centrifuged again at 6000 g for 10 min to obtain the cells. The cells were resuspended in 30% sucrose solution and frozen at -80°C until use.

[0088] Example 2 Screening, Identification and Cultivation of Bifidobacterium longum subspecies longum

[0089] 1. Screening

[0090] Human feces were used as samples. After pretreatment, the samples were stored in a -80°C refrigerator in about 20% glycerol. After being taken out and thawed, 0.5 mL of the sample was mixed and added to 4.5 mL. Gradient dilutions were performed with 0.9% saline containing 0.05% cysteine. Appropriate gradient dilutions were selected and spread on MRS plates added with 0.05% cysteine. The plates were cultured at 37°C for 48 hours. Typical colonies were picked and streaked onto MRS plates for purification. Single colonies were picked and transferred to MRS liquid culture medium (containing 0.05% cysteine) for bacterial enrichment and preserved in 30% glycerol to obtain strain CCFM1448.

[0091] 2. Identification

[0092] The genome of CCFM1448 was extracted, and the 16S rDNA of CCFM1448 (shown as SEQ ID NO. 2) was amplified and sequenced (Shanghai Sangon Biotechnology Co., Ltd.). The sequence was compared in GenBank, and the results showed that the bacterium was Bifidobacterium longum subsp. longum (B. longum subsp. longum), and was named Bifidobacterium longum subsp. longum (B. longum subsp. longum) CCFM1448.

[0093] 3. Cultivation of Bifidobacterium longum subsp. longum

[0094] Bifidobacterium longum subsp. longum CCFM1448 was inoculated into MRS solid medium (containing 0.05% cysteine) and cultured at 37°C for 48 hours. Microscopic observation of the colonies revealed that the bacterium was Gram-positive, non-spore-forming, and arranged singly, in pairs, or in a V-shaped pattern. Anaerobically cultured on MRS (lactic acid bacteria culture medium) plates at 36°C for three days revealed milky white, round, raised colonies with smooth, neatly marginated surfaces.

[0095] The Bifidobacterium longum subsp. longum CCFM1448 strain was inoculated into MRS liquid medium (containing 0.05% cysteine) and cultured at 37°C for 48 hours. A growth curve was prepared, and it was found that it reached a stable phase after being cultured at 37°C for 12 hours.

[0096] The longum subsp. longum CCFM1448 strain was inoculated into MRS liquid culture medium (containing 0.05% cysteine) and cultured at 10, 15, 20, 25, 30, 35, 40, 45, and 50°C for 48 hours, and its growth was observed. It grew well at 20-35°C and was still able to grow at 45°C, but hardly grew at 15°C or below or 50°C.

[0097] The longum subsp. longum CCFM1448 strain was inoculated into MRS liquid medium (containing 0.05% cysteine) and cultured at 37°C for 48 h. The culture was then transferred to fresh MRS liquid medium (containing 0.05% cysteine) and cultured under the same conditions for 30 h. The cells were centrifuged at 6000 g for 15 min, washed with 0.9% saline, and centrifuged again at 6000 g for 10 min to obtain the cells. The cells were resuspended in 30% sucrose solution and frozen at -80°C until use.

[0098] Example 3 Preparation of probiotics

[0099] Preparation of probiotic dry powder: Bifidobacterium adolescentis CCFM1447 strain and Bifidobacterium longum subspecies longum CCFM1448 strain were respectively inoculated into MRS liquid culture medium (containing 0.05% mass percentage cysteine) and cultured at 37°C for 48 hours, then transferred to fresh MRS culture medium (containing 0.05% mass percentage cysteine), cultured under the same conditions for 30 hours, centrifuged at 6000g for 15 minutes, washed with 0.9% saline and centrifuged again at 6000g for 10 minutes to obtain bacteria, which were placed in a vacuum freeze dryer for freeze-drying into bacterial powder. The bacterial powders of Bifidobacterium adolescentis CCFM1447 strain and Bifidobacterium longum subspecies longum CCFM1448 strain were compounded according to the viable bacterial count of 1:1. The ratio of the viable bacterial count of CCFM1447 strain to CCFM1448 strain of the present invention can achieve the technical effects described in the embodiment in the range of 2:1 to 1:2. 25% curcumin and 50% galacto-oligosaccharide were added to a final concentration of 3.75% bacterial powder. The remainder was supplemented with 5% maltodextrin, 6.25% erythritol, and 10% red grape juice powder. After crushing, sieving, magnetic separation, and mixing, a probiotic dry powder was obtained. The viable count of Bifidobacterium adolescentis CCFM1447 was 200 billion CFU / g, and the viable count of Bifidobacterium longum subsp. longum CCFM1448 was 100 billion CFU / g.

[0100] Preparation of probiotic solution: The above probiotic dry powder was dissolved in physiological saline to prepare a solution with a mass fraction of 50%, which was used for the intragastric gavage solution in the subsequent examples.

[0101] In the above-mentioned probiotics, the viable bacterial count of Bifidobacterium adolescentis CCFM1447 is adjusted to 50-100 billion CFU / g, the viable bacterial count of Bifidobacterium longum subspecies longum CCFM1448 is adjusted to 100-200 billion CFU / g, the curcumin content is 10%-25%, and the galacto-oligosaccharide content is 25%-50%, all of which can achieve effects similar to those of the above-mentioned probiotics.

[0102] Example 4 Effect of probiotics on vitamin D metabolite levels in osteoporotic mice 3-4 week old SPF C57BL / 6J male mice were divided into three groups: a blank group (CON), a model group (MOD), a positive control group (VD), and an intervention group (P experimental group), with 8 mice in each group. The animals were housed at the Jiangnan University Laboratory Animal Center, fed a standard diet, maintained at a constant temperature of 21-26°C, humidity of 40-70%, noise level ≤60 dB, and illumination of 15-20 lux. (All animal experimental procedures were reviewed and approved by the Jiangnan University Animal Welfare and Ethics Committee.)

[0103] The experimental period lasted 42 days. Modeling was performed on the 7th day. Except for the blank group, all mice were gavaged with 200 μL of 90 mg / kg BW / d retinoic acid once daily to induce osteoporosis for three weeks. The blank group was gavaged with an equal amount of normal saline. After modeling, the positive control group was gavaged with 200 μL of 0.06 μg / kg BW / d VD solution once daily, and the intervention group was gavaged with 0.2 mL of probiotic solution once daily. The normal group and the model group were gavaged with only an equal amount of sterile saline as a control. All groups had free access to water and food for two weeks until mice were sacrificed on the 42nd day.

[0104] The experimental animal groups and treatment methods are shown in Table 1:

[0105] Table 1 Experimental animal groups

[0106]

[0107] After killing the mice, blood samples were collected and centrifuged to separate the serum. The serum was detected using the Shanghai ELISA Mouse 25-hydroxyvitamin D ELISA Kit (ml038442) and Mouse 1,25-hydroxyvitamin D ELISA Kit (ml062981). Figure 1 、 Figure 2 It was found that osteoporosis led to a decrease in the levels of vitamin D metabolites in the blood of mice. The positive control group significantly increased the levels of vitamin D metabolites, with 25-hydroxyvitamin D and 1,25-hydroxyvitamin D levels increasing by 42.27% and 27.90% respectively compared to the model group. The P experimental group significantly increased the levels of vitamin D metabolites in osteoporotic mice compared to the model group, with the levels of the metabolites 25-hydroxyvitamin D and 1,25-hydroxyvitamin D increasing by 15.87% and 25.51% respectively compared to the model group.

[0108] The above experimental results show that probiotics have similar therapeutic effects as VD and can significantly increase the level of vitamin D metabolites in osteoporotic mice.

[0109] Example 5 Effect of probiotics on serum calcium levels in osteoporosis mice

[0110] The mice were grouped and modeled in the same manner as in Example 4.

[0111] On the 42nd day, mice were killed and blood samples were collected. Serum was separated by centrifugation and serum calcium levels were detected using Nanjing Jiancheng Serum Calcium Kit (C004-2-1). Figure 3 .

[0112] Depend on Figure 3It can be seen that the serum calcium level of osteoporosis model mice was significantly higher than that of blank group mice. The serum calcium level of the positive control group was reduced by 18.02% compared with the model group. The serum calcium level of osteoporosis mice in the P experimental group was significantly reduced by 11.71% compared with the model group, reaching a level equivalent to that of the blank group, indicating that the probiotics have the same effect as VD treatment in alleviating bone calcium loss.

[0113] Example 6 Effect of probiotics on serum alkaline phosphatase levels in osteoporotic mice

[0114] The mice were grouped and modeled in the same manner as in Example 4.

[0115] On the 42nd day, mice were killed, blood samples were collected, serum was separated by centrifugation, and serum alkaline phosphatase levels were detected using Nanjing Jiancheng Serum Alkaline Phosphatase Kit (A059-2-2). Figure 4 .

[0116] Depend on Figure 4 It can be seen that the serum alkaline phosphatase level of osteoporosis model mice was significantly higher than that of normal mice. The positive control group significantly reduced the serum alkaline phosphatase level of osteoporosis mice by 15.83%. Compared with the model group, the serum alkaline phosphatase level of osteoporosis mice in the P experimental group was reduced by 25.17%, indicating that probiotics play a role in maintaining bone health, repairing bone damage, and reducing the degree of osteoporosis, and the therapeutic effect is better than VD.

[0117] Example 7 Effect of probiotics on serum osteocalcin levels in osteoporotic mice

[0118] The mice were grouped and modeled in the same manner as in Example 4.

[0119] On day 42, mice were killed and blood samples were collected. Serum was separated by centrifugation and osteocalcin levels were detected using the Elabscience Mouse Osteocalcin (OC / BGP) ELISA Kit (E-EL-M0864). Figure 5 .

[0120] Depend on Figure 5 It can be seen that the serum osteocalcin (OC) level of osteoporosis model mice was significantly lower than that of normal mice. The positive control group significantly increased the serum osteocalcin level of mice by about 134.09%. Compared with the model group, the serum osteocalcin level of osteoporosis mice in the P experimental group was significantly increased by 127.78%, indicating that the probiotics have a similar effect as VD treatment in enhancing osteoblast function, repairing bone damage and osteoporosis.

[0121] Example 8 Effect of probiotics on the level of type I procollagen amino-terminal propeptide in the serum of osteoporotic mice

[0122] The mice were grouped and modeled in the same manner as in Example 4.

[0123] On day 42, mice were killed, blood samples were collected, and serum was separated by centrifugation. The serum level of type I procollagen amino-terminal propeptide (PⅠPN) was detected using the Elabscience mouse type I procollagen amino-terminal propeptide (PⅠNP) enzyme-linked immunosorbent assay kit (E-EL-M0233). Figure 6 .

[0124] Depend on Figure 6 It can be seen that the level of serum type I procollagen amino-terminal propeptide in osteoporosis model mice was significantly lower than that in normal group mice, the positive control group significantly increased the serum PIPN level of mice by about 51.53%, and the P experimental group significantly increased the level of serum type I procollagen amino-terminal propeptide in osteoporosis mice by 50.76% compared with the model group, indicating that the probiotics have a similar effect to VD treatment in increasing osteoblast synthesis, promoting new bone formation, repairing bone damage and osteoporosis.

[0125] Example 9 Effects of Probiotics on Bone-Related Indicators in Osteoporosis Mice

[0126] The mice were grouped and modeled in the same manner as in Example 4.

[0127] The mice were killed on the 42nd day, and the femurs were taken for detection of bone-related indicators using Micro-CT. The observed parameters included total bone density (Bone Mean, BM), cortical bone density (Cortex Mean, CM), distal femoral trabecular bone density (Trabeculae Mean, TM), trabecular bone separation (Tb.Sp), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), trabecular connectivity (Conn.D), bone surface area (BS), bone volume (BV) and bone volume fraction (bone volume / total volume, BV / TV). The results are shown in Figures 7 to 16 .

[0128] Depend on Figure 7 It can be seen that compared with the blank group, the BM of mice in the osteoporosis model group was significantly reduced by 3.1%. After treatment with VD, the BM of the positive control group increased by 1.68% compared with the model group; after oral gavage of probiotics, the BM of mice increased by 3.19%; the total bone density of the P group was 1.01 times that of the VD group.

[0129] Depend on Figure 8Compared with the blank group, the CM of the osteoporosis model group decreased by 0.61%. After VD treatment, the CM of the positive control group increased by 2.44% compared with the model group. After oral administration of probiotics, the CM of the mice increased by 4.16%. The cortical bone density of the P group was 1.02 times that of the VD group.

[0130] Depend on Figure 9 It can be seen that compared with the blank group, the TM of mice in the osteoporosis model group was significantly reduced by 5.27%. After treatment with VD, the TM of the positive control group increased by 3.66% compared with the model group; after oral gavage of probiotics, the TM of mice increased significantly by 6.24%; the trabecular bone density of the P group was 1.02 times that of the VD group.

[0131] Depend on Figure 10 It can be seen that compared with the blank group, the Tp.Sp of mice in the osteoporosis model group was significantly increased by 18.58%. After treatment with VD, the Tp.Sp of the positive control group was reduced by 20.77% compared with the model group; after oral administration of probiotics, the Tp.Sp of mice was reduced by 23.43%; the trabecular separation degree of the P group was 0.96 times that of the VD group.

[0132] Depend on Figure 11 It can be seen that compared with the blank group, the Tb.Th of mice in the osteoporosis model group decreased by 10.61%. After treatment with VD, the Tb.Th of the positive control group increased by 5.82% compared with the model group; after oral gavage of probiotics, the Tb.Th of mice increased by 9.81%; the trabecular thickness of the P group was 1.04 times that of the VD group.

[0133] Depend on Figure 12 It can be seen that compared with the blank group, the Tb.N of mice in the osteoporosis model group decreased by 25.64%. After treatment with VD, the Tb.N of the positive control group increased by 40.66% compared with the model group; after oral gavage of probiotics, the Tb.N of mice increased by 42.92%; the number of trabeculae in the P group was 1.02 times that of the VD group.

[0134] Depend on Figure 13 It can be seen that compared with the blank group, the Conn.D of mice in the osteoporosis model group decreased by 42.60%. After treatment with VD, the Conn.D of the positive control group increased by 8.31% compared with the model group; after oral gavage of probiotics, the Conn.D of mice increased by 29.88%; the trabecular connectivity of the P group was 1.20 times that of the VD group.

[0135] Depend on Figure 14 It can be seen that compared with the blank group, the BS of mice in the osteoporosis model group decreased by 22.33%. After treatment with VD, the BS of the positive control group did not increase significantly compared with the model group. After oral administration of probiotics, the BS of mice increased by 1.66%. The bone surface area of ​​the P group was 1.07 times that of the VD group.

[0136] Depend on Figure 15 It can be seen that compared with the blank group, the BV of mice in the osteoporosis model group decreased by 23.93%. After treatment with VD, the BV of the positive control group did not increase significantly compared with the model group. After oral administration of probiotics, the BV of mice increased by 14.02%. The bone volume of the P group was 1.19 times that of the VD group.

[0137] Depend on Figure 16 It can be seen that compared with the blank group, the BV / TV of mice in the osteoporosis model group decreased by 18.18%. After treatment with VD, the BV / TV of the positive control group did not increase significantly compared with the model group. After oral administration of probiotics, the BV / TV of mice increased by 16.94%. The bone volume fraction of the P group was 1.21 times that of the VD group.

[0138] The results showed that probiotics significantly increased total bone density (BMD), cortical BMD, distal femoral trabecular BMD, trabecular thickness, trabecular number, trabecular connectivity, bone surface area, bone volume, and bone volume fraction in osteoporotic mice. Furthermore, they significantly reduced trabecular separation in osteoporotic mice. These results suggest that probiotic intervention has therapeutic effects on osteoporosis, and that the therapeutic effect is better than that of vitamin D.

[0139] In summary, the probiotic agent of the present invention has the effect of improving the physiological activity of vitamin D and alleviating osteoporosis, and can significantly improve the total bone density, cortical bone density, distal femoral trabecular bone density, trabecular thickness, trabecular number, trabecular connectivity, bone surface area, bone volume and bone volume fraction of osteoporotic mice; significantly reduce the trabecular separation of osteoporotic mice; increase the content of vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in the serum of osteoporotic mice; reduce the content of serum calcium and alkaline phosphatase in the serum of osteoporotic mice, thereby increasing the levels of osteocalcin and type I procollagen amino-terminal propeptide in the serum, and can be used to prepare drugs for preventing and / or treating osteoporosis or prepare health products or foods that help improve bone density.

[0140] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A probiotic agent that can enhance the physiological activity of vitamin D and maintain bone health, characterized in that: The probiotic comprises Bifidobacterium adolescens CCFM1447 strain with a preservation number of GDMCC No: 65382 and Bifidobacterium longum subsp. longum CCFM1448 strain with a preservation number of GDMCC No: 65383, and the ratio of the number of viable bacteria of the CCFM1447 strain to the CCFM1448 strain is 2:1 to 1:

2.

2. The probiotic according to claim 1, characterized in that The viable bacterial count of Bifidobacterium adolescentis CCFM1447 strain is not less than 1.0×10 6 CFU / ml, the number of viable bacteria of Bifidobacterium longum subspecies longum CCFM1448 strain is not less than 1.0×10 6 CFU / ml.

3. The probiotic according to claim 1, characterized in that The probiotics also include food raw materials, and / or food or medically acceptable auxiliary materials or additives.

4. The probiotic according to claim 1, characterized in that The dosage form of the probiotics includes freeze-dried powder, capsules, tablets or granules.

5. A product containing the probiotic according to any one of claims 1 to 4, wherein the product comprises food, medicine or health product.

6. Use of the probiotic according to any one of claims 1 to 4 and the product according to claim 5 in the preparation of a medicament for preventing and / or treating osteoporosis.

7. The use according to claim 6, characterized in that The prevention and / or treatment of osteoporosis includes any of the following functions: (1) Increase the levels of vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in serum; (2) Increase total bone density, cortical bone density, distal femoral trabecular bone density, trabecular thickness, trabecular number, trabecular connectivity, bone surface area, bone volume, and bone volume fraction; and reduce trabecular separation; (3) Reduce serum calcium and alkaline phosphatase levels; (4) Increase the levels of osteocalcin and type I procollagen amino-terminal propeptide in serum.

8. Use of the probiotic according to any one of claims 1 to 4 and the product according to claim 5 in the preparation of a health product that helps improve bone density.

Images