Application of animal Bifidobacterium lactis subspecies CP-9 in preparing products for promoting bone growth and development or improving bone health

Abstract

The present invention provides an application of animal Bifidobacterium lactis subspecies CP-9 in the preparation of a product that promotes bone growth and development or improves bone health. After long-term research and a large number of experiments, the present invention found that animal Bifidobacterium lactis subspecies CP-9 can improve the body's bone volume fraction, trabecular thickness, bone density and bone mass, and can also effectively increase the level of growth hormone and insulin-like growth factor 1, thereby promoting bone growth and development. At the same time, animal Bifidobacterium lactis subspecies CP-9 can effectively increase the level of type I procollagen N-terminal propeptide, osteocalcin level, bone morphogenetic protein level, and osteoprotegerin level in the body, and significantly reduce the level of type I collagen amino-terminal peptide and nuclear factor κB receptor activator ligand level, thereby promoting osteoblast differentiation, inhibiting osteoclast differentiation, and then promoting bone formation, inhibiting bone resorption, improving bone metabolism, and ultimately achieving the effect of improving bone health.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of animal Bifidobacterium lactis subspecies CP-9 in preparing a product that promotes bone growth and development or improves bone health. Background Art

[0002] The skeleton is a vital support and protection system for the human body. It forms the human frame, maintaining upright posture and supporting muscle movement. It protects vital organs, such as the skull, which protects the brain, and the thorax, which protects the heart, lungs, and other vital organs. It participates in mineral storage and regulation, storing the majority of the body's calcium and phosphorus and regulating calcium and phosphorus metabolism through a balance between bone formation and resorption. The bone marrow within the skeleton also plays a role in hematopoiesis, producing red blood cells, white blood cells, and platelets to maintain immune and circulatory function.

[0003] However, poor bone growth and development or impaired bone health can affect the normal function of bones and can easily lead to diseases such as growth retardation and limited mobility, seriously affecting quality of life. For example, it can cause short stature, fractures, bone cracks, and osteoporosis. Therefore, how to provide a product that can promote bone growth and development and improve bone health is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] The present invention provides an application of animal Bifidobacterium lactis subspecies CP-9 in preparing a product for promoting bone growth and development or improving bone health;

[0005] Bifidobacterium animalis subsp. lactis CP-9 was deposited in China Center for Type Culture Collection on November 24, 2014, with the deposit number CCTCC NO: M2014588;

[0006] The product is a health product or medicine;

[0007] The application form of the animal Bifidobacterium lactis subspecies CP-9 is at least one of live bacteria and freeze-dried powder.

[0008] The above application, wherein the viable count of animal Bifidobacterium lactis subspecies CP-9 in the product is 1×10 6 -10 10 CFU / mL or 1×10 6 -10 10 CFU / g.

[0009] The application as described above, wherein promoting bone growth and development comprises:

[0010] Improve bone volume fraction; and / or,

[0011] Improved trabecular bone thickness; and / or,

[0012] Improved growth hormone levels; and / or,

[0013] Improved insulin-like growth factor 1 levels; and / or,

[0014] Improve bone mass.

[0015] The use as described above, wherein improving bone health comprises:

[0016] Improve the level of type I procollagen N-terminal propeptide; and / or,

[0017] Improvement of type I collagen amino-terminal peptide levels; and / or,

[0018] Improved osteocalcin levels; and / or,

[0019] Improve bone morphogenetic protein levels; and / or,

[0020] Improved osteoprotegerin levels; and / or,

[0021] Improve the level of receptor activator of nuclear factor-κB ligand.

[0022] The application as described above, wherein the product form includes at least one of oral liquid, tablet, granule, granule, powder, capsule, pill, aqueous solution, powder, soft capsule, and film.

[0023] The application as described above, wherein the health care product comprises at least one of a functional beverage, a functional granule, and a functional capsule; and / or,

[0024] The drugs include drugs for promoting osteoblast differentiation, inhibiting osteoclast differentiation, treating or assisting in the promotion of bone formation, treating or assisting in the inhibition of bone resorption, improving bone metabolism, preventing or treating osteoporosis, preventing or treating abnormal bone growth, and preventing or treating bone dysplasia.

[0025] The use as described above, wherein the drug further comprises a drug carrier and / or a pharmaceutically acceptable excipient;

[0026] The drug carrier includes at least one of microcapsules, microspheres, nanoparticles, and liposomes;

[0027] The excipients include at least one of fillers, flavoring agents, diluents, wetting agents, adhesives, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH regulators, and isotonic or isotonic regulators.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention has been found through long-term research and a large number of experiments that animal bifidobacterium lactis subspecies CP-9 can improve the bone volume fraction, trabecular thickness, bone density and bone mass of the body, and can also effectively improve growth hormone levels and insulin-like growth factor 1 levels, and then promote bone growth and development. Meanwhile, animal bifidobacterium lactis subspecies CP-9 can effectively improve type i procollagen N-terminal propeptide levels, osteocalcin levels, bone morphogenetic protein levels, osteoprotegerin levels in the body, and significantly reduce type i collagen amino terminal peptide levels, nuclear factor kappa B receptor activator ligand levels, thereby promoting osteoblast differentiation, inhibiting osteoclast differentiation, and then promoting bone formation, inhibiting bone resorption, improving bone metabolism, and finally achieving the effect of improving bone health. By the new use of animal bifidobacterium lactis subspecies CP-9 provided by the present invention, it is not only possible to fill the gaps in existing markets, break through the limitations of existing products, but also possible to promote bone growth and development, improve bone health, prevent or treat osteoporosis, prevent or treat bone growth abnormalities, and prevent or treat bone dysplasia and provide new ideas. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a scan of femoral bone mass in Example 3;

[0031] Figure 2 This is the bone volume fraction-body weight ratio result diagram in Example 3;

[0032] Figure 3 This is the result diagram of trabecular bone thickness in Example 3;

[0033] Figure 4 This is the result diagram of serum PINP levels in Example 4;

[0034] Figure 5 This is a graph showing the serum NTX level results in Example 4;

[0035] Figure 6 This is a graph showing the serum OCN level results in Example 5;

[0036] Figure 7 This is a graph showing the serum BMPs level results in Example 6;

[0037] Figure 8 This is a graph showing the serum OPG / RANKL level results in Example 7;

[0038] Figure 9 This is a graph showing the serum GH level results in Example 8;

[0039] Figure 10 This is a graph showing the serum IGF-1 level results in Example 9;

[0040] Figure 11 This is a microscopic fluorescence image of the skeleton of the zebrafish larva in Example 11;

[0041] Figure 12 This is the fluorescence intensity result of the vertebrae of zebrafish larvae in Example 11. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific embodiments listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present invention.

[0043] For providing a kind of product that can promote bone growth and development, improve bone health, the present invention has found through long-term research and a large number of experiments that animal bifidobacterium lactis subspecies CP-9 can improve the body's bone volume fraction, trabecular thickness, bone density and bone mass, and can also effectively improve growth hormone level and insulin-like growth factor 1 level, and then promote bone growth and development. Simultaneously, animal bifidobacterium lactis subspecies CP-9 can effectively improve type I procollagen N-terminal propeptide level, osteocalcin level, bone morphogenetic protein level, osteoprotegerin level in the body, and significantly reduce type I collagen amino terminal peptide level, nuclear factor kappa B receptor activator ligand level, thereby promoting osteoblast differentiation, inhibiting osteoclast differentiation, and then promoting bone formation, inhibiting bone resorption, improving bone metabolism, and finally achieving the effect of improving bone health. Animal bifidobacterium lactis subspecies CP-9 also has good biosafety, has no toxic side effects to the human body, and has broad market prospects.

[0044] Therefore, the present invention provides a use of Bifidobacterium animalis subsp. lactis CP-9 in preparing a product that promotes bone growth and development or improves bone health. The Bifidobacterium animalis subsp. lactis CP-9 was deposited with the China Center for Type Culture Collection (Wuhan) on November 24, 2014, with a deposit number of CCTCC NO: M2014588. The new use of Bifidobacterium animalis subsp. lactis CP-9 provided by the present invention not only fills a gap in the existing market and overcomes the limitations of existing products, but also provides new ideas for promoting bone growth and development, improving bone health, and preventing or treating osteoporosis, preventing or treating bone growth abnormalities, and preventing or treating bone dysplasia.

[0045] In addition, during childhood and adolescence, bones continue to grow and develop, and bone mass, bone quality, bone density and bone strength also increase with age; after adulthood, bone growth and development gradually stop, and the bones enter a stable state. Therefore, good bone growth and development and bone health play a very important role in the growth process of the human body, and it is very important to take relevant measures in time to promote bone development and maintain bone health during childhood and adolescence. The present invention has found that animal Bifidobacterium lactis subspecies CP-9 is particularly suitable for mammals in the early stages of life, such as infants, children and adolescents. Therefore, in a preferred embodiment, the present invention provides an application of animal Bifidobacterium lactis subspecies CP-9 in the preparation of a product that promotes bone growth and development in mammals in the early stages of life or improves the bone health of mammals in the early stages of life.

[0046] In the above technical solution, the application form of animal Bifidobacterium lactis subsp. lactis CP-9 includes at least one of live bacteria, inactivated bacteria, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, exosomes, metabolites, cell lysate, and lyophilized powder. For example, animal Bifidobacterium lactis subsp. lactis CP-9 can be used in the form of a fermentation broth in products that promote bone growth and development or improve bone health.

[0047] It is understandable that Bifidobacterium animalis subsp. lactis CP-9 can be used alone as a microbial preparation in products that promote bone growth and development or improve bone health, or it can be combined with other microorganisms to form a microbial preparation for products that promote bone growth and development or improve bone health.

[0048] When Bifidobacterium animalis subsp. lactis CP-9 is used alone as a microbial preparation, or is combined with other microorganisms to form a microbial preparation, the microbial preparation can be in a solid form or a liquid form.

[0049] In the above technical solution, the number of viable bacteria of animal Bifidobacterium lactis subspecies CP-9 in the product is 1×10 6 -10 10 CFU / mL or 1×10 6 -10 10 CFU / g. For example, the viable count can be 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL and any value between any two of the above ranges; the number of viable bacteria can also be 1×10 6 CFU / g, 1×10 7 CFU / g, 1×108 CFU / g, 1×10 9 CFU / g, 1×10 10 CFU / g and any value between any two numerical ranges mentioned above.

[0050] In the above technical solution, animal Bifidobacterium lactis subsp. lactis CP-9 can be used to prepare a product for promoting bone growth and development, wherein promoting bone growth and development includes:

[0051] Improved bone volume fraction, improved trabecular thickness, improved growth hormone levels, improved insulin-like growth factor 1 levels and bone mass.

[0052] Specifically, experiments have shown that animal Bifidobacterium lactis subspecies CP-9 can increase the bone volume fraction of early-life mammals (such as young mice), enhance their bone density, increase the thickness of their femoral trabeculae, and increase the levels of growth hormone and insulin-like growth factor 1 in their serum; it can also promote the bone development of zebrafish larvae and increase their bone mass, thereby promoting bone growth and development.

[0053] It is understandable that animal Bifidobacterium lactis subsp. lactis CP-9 can be used to prepare products that promote bone growth and development, and can also be used to prepare bone volume fraction enhancers, trabecular thickness enhancers, bone density enhancers, bone mass enhancers, growth hormone agonists or insulin-like growth factor 1 agonists.

[0054] In the above technical solution, animal Bifidobacterium lactis subsp. lactis CP-9 can be used to prepare a product for improving bone health, wherein improving bone health includes:

[0055] Improve the level of type I procollagen N-terminal propeptide, improve the level of type I collagen amino-terminal peptide, improve the level of osteocalcin, improve the level of bone morphogenetic protein, improve the level of osteoprotegerin, and improve the level of nuclear factor κB receptor activator ligand.

[0056] Specifically, experiments have verified that animal Bifidobacterium lactis subspecies CP-9 can increase the levels of type I procollagen N-terminal propeptide, osteocalcin, bone morphogenetic protein, and osteoprotegerin in the serum of early-life mammals (such as young mice), and reduce the levels of type I collagen amino-terminal peptide and nuclear factor κB receptor activator ligand in the serum, and increase the ratio of osteoprotegerin to nuclear factor κB receptor activator ligand in the serum, thereby promoting osteoblast differentiation and inhibiting osteoclast differentiation, thereby promoting bone formation, inhibiting bone resorption, improving bone metabolism, and ultimately improving bone health.

[0057] It is understandable that animal Bifidobacterium lactis subspecies CP-9 can be used to prepare products for improving bone health, and can also be used to prepare type I procollagen N-terminal propeptide agonists, osteocalcin agonists, bone morphogenetic protein agonists, osteoprotegerin agonists, type I collagen amino-terminal peptide inhibitors, nuclear factor κB receptor activator ligand inhibitors, osteoblast promoters or osteoclast inhibitors.

[0058] In the above technical solution, the form of the product for promoting bone growth and development or improving bone health includes at least one of oral liquid, tablets, granules, granules, powders, capsules, pills, aqueous solutions, powders, soft capsules, and film preparations.

[0059] In the above technical solution, the product that promotes bone growth and development or improves bone health is at least one of a health product and a medicine.

[0060] Furthermore, the health care product may include a health care product for assisting in promoting bone mass increase. Specifically, the health care product may include at least one of a functional beverage, a functional granule, and a functional capsule.

[0061] Furthermore, the drug may include at least one of a drug for promoting osteoblast differentiation, inhibiting osteoclast differentiation, treating or assisting in the promotion of bone formation, treating or assisting in the inhibition of bone resorption, improving bone metabolism, preventing or treating osteoporosis, preventing or treating abnormal bone growth, and preventing or treating bone dysplasia.

[0062] The above-mentioned drugs also include drug carriers and / or pharmaceutically acceptable excipients (i.e., pharmaceutical excipients), wherein the drug carriers may include at least one of microcapsules, microspheres, nanoparticles, and liposomes; the excipients may include at least one of fillers, flavoring agents, diluents, wetting agents, adhesives, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal chelating agents, inert gases, preservatives, local analgesics, pH regulators, and isotonic or isotonic regulators.

[0063] When the above-mentioned drugs are actually used, they can be achieved by at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, and transdermal administration.

[0064] The present invention also provides a use of animal Bifidobacterium lactis subspecies CP-9 in the preparation of children's milk powder, children's dietary supplement, children's snack, adolescent milk powder, adolescent dietary supplement or adolescent snack that promotes bone growth and development or improves bone health;

[0065] Bifidobacterium animalis subsp. lactis CP-9 was deposited in China Center for Type Culture Collection on November 24, 2014, with the deposit number CCTCC NO: M2014588;

[0066] The application form of animal Bifidobacterium lactis subsp. lactis CP-9 is at least one of live bacteria and freeze-dried powder.

[0067] The technical solutions of this application are further explained below with reference to specific examples. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. All reagents used, unless otherwise specified, were commercially available or publicly available.

[0068] In the following examples, “ " indicates significant difference compared with the blank control group, among which, indicates p<0.05, Indicates p<0.01, indicates p<0.001, indicates p<0.0001;" " indicates significant difference compared with the model group, among which, indicates p<0.05, Indicates p<0.01, indicates p<0.001, Indicates p < 0.0001, and the error is presented as Mean ± SEM.

[0069] Example 1: Preparation of Bifidobacterium animalis subsp. lactis CP-9 bacterial suspension

[0070] A culture of Bifidobacterium animalis subsp. lactis CP-9 was streaked onto Lactobacillus delbrueckii (MRS) solid medium from a cryovial and incubated anaerobically at 37°C for 48 h to obtain a single colony. A single colony was selected and inoculated into MRS liquid medium and incubated anaerobically at 37°C for 24 h to activate the culture. This activated culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum volume and incubated anaerobically at 37°C for 24 h. This activation process was repeated three times to obtain the activated culture. The activated culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum volume and incubated at 37°C for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged, washed twice with phosphate-buffered saline (PBS) buffer (pH 7.4), and resuspended in saline to adjust the viable count to 1×10. 9 CFU / mL, and obtain the bacterial suspension of Bifidobacterium animalis subsp. lactis CP-9.

[0071] Among them, Bifidobacterium animalis subsp. lactis CP-9 (CCTCC NO: M2014588) was deposited in the China Center for Type Culture Collection in Wuhan, China on November 24, 2014. The formula of the above-mentioned MRS liquid medium includes: tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 20 g / L, anhydrous sodium acetate 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, Tween 80 1 mL / L, and cysteine ​​hydrochloride 0.5 g / L. The formula of the above-mentioned MRS solid medium includes: tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 20 g / L, anhydrous sodium acetate 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, Tween 80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L, and agar 20 g / L.

[0072] Example 2: Construction of a young mouse model

[0073] The experimental animals used in this example were specific pathogen-free (SPF) BALB / C mice purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. These mice were early-life pups. They were gavage-treated starting at one week of age and divided into a blank control group and a Bifidobacterium animalis subsp. lactis CP-9 group. After weaning at three weeks of age, male and female pups were separated by cages and gavage-treated until five weeks of age, at which time they were sacrificed. The specific grouping and treatment were as follows:

[0074] (1) Blank control group: The pups were allowed to freely consume breast milk and were gavaged with 0.2 mL of normal saline every day from day 8 to day 21 after birth; and were allowed to freely consume food and were gavaged with 0.2 mL of normal saline every day from day 22 to day 35.

[0075] (2) Animal Bifidobacterium lactis subsp. CP-9 group: The pups were allowed to freely consume breast milk from day 8 to day 21 after birth and were gavaged daily with 0.2 mL of the bacterial suspension of Animal Bifidobacterium lactis subsp. CP-9 obtained in Example 1; and were allowed to freely consume food from day 22 to day 35 and were gavaged daily with 0.2 mL of the bacterial suspension of Animal Bifidobacterium lactis subsp. CP-9 obtained in Example 1.

[0076] Body weights of the two groups of mice were recorded on day 35, and the mice were subsequently sacrificed. Whole blood was collected from the mice and centrifuged to obtain serum samples. The left femur of the mice was removed, the muscle removed, and fixed in 4% paraformaldehyde solution for 48 hours to obtain femoral samples.

[0077] Example 3: Effect of CP-9 on bone volume fraction and trabecular thickness

[0078] Micro-CT scanning was used to scan the distal femoral trabecular bone region of the femoral specimen in Example 2 to obtain the bone volume fraction and trabecular thickness results. Figure 1 、 Figure 2 and Figure 3 .

[0079] Figure 1 The femoral bone mass scans of the blank control group and the CP-9 group. Figure 1 It can be found that the femoral bone mass of female and male mice in the CP-9 group was significantly higher than that in the blank control group, indicating that animal Bifidobacterium lactis subsp. CP-9 helps promote bone growth and development.

[0080] Figure 2 The figure shows the bone volume fraction-body weight ratio (BV / TV / body weight) of the blank control group and the CP-9 group. The formula for calculating bone volume fraction is: BV / TV×100%, where BV represents the volume of bone tissue (unit: mm 3 ), TV represents the tissue volume (unit: mm 3 Bone volume fraction reflects bone density and quality. A higher value indicates a greater proportion of bone tissue in the overall tissue structure, indicating relatively denser and healthier bones. The bone volume fraction-to-body weight ratio refers to the ratio of an individual's bone volume fraction to their body weight, eliminating any interference from their body weight in the experiment. The body weight refers to the body weight of the pups on day 35 measured in Example 2. Figure 2The bone volume fraction-body weight ratio of female mice in the blank control group was 0.6223; the bone volume fraction-body weight ratio of female mice in the CP-9 group was 0.6581; the bone volume fraction-body weight ratio of male mice in the blank control group was 0.6355; the bone volume fraction-body weight ratio of male mice in the CP-9 group was 0.6732. It can be seen that the bone volume fraction-body weight ratio of female and male mice in the CP-9 group was significantly higher than that in the blank control group, indicating that animal Bifidobacterium lactis subsp. CP-9 helps to improve bone density and quality, promote bone densification, and promote bone health.

[0081] Figure 3 The following graph shows the trabecular bone thickness (Tb.Th) results for the blank control group and the CP-9 group. Trabecular bone thickness refers to the average thickness of trabeculae. Thicker trabeculae indicate greater femoral bone strength and fracture resistance. Figure 3 The trabecular bone thickness of female mice in the blank control group was 0.1121 μm, the trabecular bone thickness of female mice in the CP-9 group was 0.1272 μm, the trabecular bone thickness of male mice in the blank control group was 0.1086 μm, and the trabecular bone thickness of male mice in the CP-9 group was 0.1267 μm. It can be seen that the trabecular bone thickness of female and male mice in the CP-9 group was significantly higher than that in the blank control group, indicating that animal Bifidobacterium lactis subsp. CP-9 helps to improve bone strength and anti-fracture ability.

[0082] Example 4: Effect of CP-9 on Bone Metabolism Markers in Serum

[0083] The serum samples in Example 2 were tested using the ELISA kit for type I procollagen amino terminal peptide (PINP) and the ELISA kit for type I collagen amino terminal peptide (NTX) produced by Shanghai ELISA Biotechnology Co., Ltd. to obtain the PINP level and NTX level in the serum. Figure 4 and Figure 5 .

[0084] Figure 4 This is the result graph of PINP levels in the serum of the blank control group and the CP-9 group. Figure 5 The following graph shows serum NTX levels in the blank control group and the CP-9 group. PINP is a marker that reflects a high sensitivity of bone formation; NTX is associated with osteoclast resorption of bone tissue and reflects the level of bone resorption. PINP and NTX are two commonly used bone metabolism markers in clinical practice.

[0085] Figure 4The serum PINP level of female pups in the blank control group was 148.24 ng / mL, and the serum PINP level of female pups in the CP-9 group was 171.35 ng / mL. The serum PINP level of male pups in the blank control group was 136.03 ng / mL, and the serum PINP level of male pups in the CP-9 group was 159.21 ng / mL. Figure 5 In the blank control group, the serum NTX level in female pups was 7.77 nmol / L, while that in the CP-9 group was 6.58 nmol / L. The serum NTX level in male pups was 6.83 nmol / L, while that in the CP-9 group was 5.83 nmol / L. Therefore, compared with the blank control group, CP-9 intervention significantly increased the serum level of PINP, a marker of bone formation, and significantly decreased the serum level of NTX, a marker of bone resorption. These results suggest that Bifidobacterium animalis subsp. lactis CP-9 can regulate bone metabolism, promoting bone formation metabolism to a certain extent over bone resorption metabolism, thereby promoting bone growth in early-life mammals.

[0086] Example 5: Effect of CP-9 on serum osteocalcin

[0087] The serum samples in Example 2 were tested using an osteocalcin (OCN) ELISA kit produced by Shanghai ELISA Biotechnology Co., Ltd. to obtain the OCN level in the serum. Figure 6 .

[0088] Figure 6 The figure shows the serum OCN levels in the blank control group and the CP-9 group. OCN is a sensitive marker reflecting osteoblast function. Figure 6 In the blank control group, the serum OCN level in female pups was 43.66 ng / mL, while that in the CP-9 group was 51.99 ng / mL. The serum OCN level in male pups was 41.87 ng / mL, while that in the CP-9 group was 47.37 ng / mL. Therefore, the serum OCN levels in both female and male pups in the CP-9 group were significantly higher than those in the blank control group. These results suggest that Bifidobacterium animalis subsp. lactis CP-9 can promote bone formation.

[0089] Example 6: Effect of CP-9 on Bone Morphogenetic Protein in Serum

[0090] The serum samples in Example 2 were tested using the bone morphogenetic protein (BMPs) ELISA kit produced by Shanghai ELISA Biotechnology Co., Ltd. to obtain the BMPs level in the serum. Figure 7 .

[0091] Figure 7 The results of the BMPs levels in the serum of the blank control group and the CP-9 group are shown in Figure 2. BMPs are key growth factors that regulate osteoblast differentiation and bone repair. Figure 7 In the blank control group, the serum BMPs level in female pups was 216.95 ng / mL, while that in the CP-9 group was 249.58 ng / mL. The serum BMPs level in male pups was 192.01 ng / mL, while that in the CP-9 group was 225.54 ng / mL. Thus, the serum BMPs levels in both female and male pups in the CP-9 group were significantly higher than those in the blank control group. These results suggest that Bifidobacterium animalis subsp. lactis CP-9 can promote bone regeneration and repair.

[0092] Example 7: Effect of CP-9 on serum osteoprotegerin / receptor activator of nuclear factor κB ligand

[0093] The serum samples in Example 2 were tested using an osteoprotegerin (OPG) ELISA kit and a receptor activator of nuclear factor κB ligand (RANKL) ELISA kit produced by Shanghai ELISA Biotechnology Co., Ltd. to obtain the serum OPG level, serum RANKL level and the ratio of the two (OPG / RANKL). Figure 8 .

[0094] Figure 8 The results of serum OPG / RANKL levels in the blank control group and the CP-9 group are shown. OPG maintains bone mass by inhibiting osteoclastogenesis and bone resorption; RANKL promotes osteoclast differentiation and activation, inducing osteoclast maturation by binding to RANK receptors on the surface of osteoclast precursors. The OPG / RANKL ratio reflects the degree of osteoclast differentiation; a high OPG / RANKL ratio indicates inhibited osteoclast differentiation and reduced bone resorption.

[0095] Figure 8 The serum OPG / RANKL ratio of female mice in the blank control group was 0.0968, and that of female mice in the CP-9 group was 0.1328. The serum OPG / RANKL ratio of male mice in the blank control group was 0.1464, and that of male mice in the CP-9 group was 0.1815. Thus, the serum OPG / RANKL ratios of female and male mice in the CP-9 group were significantly higher than those in the blank control group. These results indicate that Bifidobacterium animalis subsp. lactis CP-9 can inhibit osteoclast differentiation, reduce bone resorption, and thus induce bone formation.

[0096] Example 8: Effect of CP-9 on Serum Growth Hormone

[0097] The serum samples in Example 2 were tested using the growth hormone (GH) ELISA kit produced by Shanghai ELISA Biotechnology Co., Ltd. to obtain the GH level in the serum. Figure 9 .

[0098] Figure 9 The following is a graph showing the serum GH levels in the blank control group and the CP-9 group. GH can promote growth and metabolism, stimulate the division and proliferation of bone and cartilage cells, and promote protein synthesis, thereby accelerating bone growth and development. Figure 9 In the blank control group, the serum GH level of female pups was 162.00 ng / mL, while that of female pups in the CP-9 group was 178.07 ng / mL. The serum GH level of male pups in the blank control group was 162.37 ng / mL, while that of male pups in the CP-9 group was 180.35 ng / mL. Therefore, the serum GH levels of female and male pups in the CP-9 group were significantly higher than those in the blank control group. These results suggest that Bifidobacterium animalis subsp. lactis CP-9 can promote growth and metabolism, and promote bone development.

[0099] Example 9: Effect of CP-9 on serum insulin-like growth factor-1

[0100] The serum samples in Example 2 were tested using the insulin-like growth factor 1 (IGF-1) ELISA kit produced by Shanghai ELISA Biotechnology Co., Ltd. to obtain the IGF-1 level in the serum. Figure 10 .

[0101] Figure 10 The following is a graph showing serum IGF-1 levels in the blank control group and the CP-9 group. IGF-1, also known as a "growth factor," promotes bone anabolism and maintains its normal structural function. Figure 10 In the blank control group, the serum IGF-1 level in female pups was 18.60 ng / mL, while that in the CP-9 group was 21.84 ng / mL. The serum IGF-1 level in male pups was 14.50 ng / mL, while that in the CP-9 group was 17.64 ng / mL. Therefore, the serum IGF-1 levels in both female and male pups in the CP-9 group were significantly higher than those in the blank control group. These results suggest that Bifidobacterium animalis subsp. lactis CP-9 can promote bone growth and maintain healthy bone balance.

[0102] Example 10: Construction of a zebrafish larvae model

[0103] Zebrafish is fertilized in vitro and develops rapidly. The embryo develops quickly, and the membrane is hatched about 3 days after fertilization, and the mouth opens to eat about 5 days later. The zebrafish used in this embodiment was purchased from Huante Biological. It is a Tg (OlaSp7: nlsGFP) cy25 transgenic teleost green fluorescent zebrafish. The skeletal structure with higher resolution can be seen under a fluorescence microscope, and it can avoid the situation where death during the staining process causes cell autolysis and the fluorescent dye cannot specifically bind to the bone. The above-mentioned zebrafish were divided into a blank control group, a model group, a CP-9 group, a positive drug group, and a BB12 group. The specific grouping process is as follows:

[0104] (1) Blank control group: zebrafish were cultured in conventional aquaculture water from the 5th to the 9th day after fertilization.

[0105] (2) Model group: Zebrafish were cultured in aquaculture water containing 10 μM dexamethasone (Dex) from day 5 to day 9 after fertilization.

[0106] (3) CP-9 group: zebrafish were fed with 10 μM Dex and 1×10 7 CFU / mL of aquaculture water of Bifidobacterium animalis subsp. lactis CP-9.

[0107] (4) Positive drug group: zebrafish were cultured in culture water containing 10 μM Dex and 10 μM alendronate sodium from day 5 to day 9 after fertilization.

[0108] (5) BB12 group: zebrafish were treated with 10 μM Dex and 1×10 7 CFU / mL of Bifidobacterium lactis BB-12 was cultured in aquaculture water. Bifidobacterium lactis BB-12 was identified as Bifidobacterium animalis subsp. lactis and purchased from Chr. Hansen. The preparation method of the bacterial suspension was as described in Example 1.

[0109] The culture water of the above groups was replaced regularly every day. After the zebrafish were cultured for 5-9 days after fertilization, zebrafish juvenile samples were obtained.

[0110] Example 11: Effects of CP-9 on Skeletal Development

[0111] The skeletal development of zebrafish larvae was observed under a microscope, and the fluorescence intensity of the zebrafish vertebrae was calculated using ImageJ to characterize the bone mass. Figure 11 and Figure 12 .

[0112] Figure 11 This is a fluorescence micrograph of the skeleton of a zebrafish larva, where the red arrow indicates the staining of the skeleton; Figure 12This is the fluorescence intensity result of the vertebrae of zebrafish larvae. Figure 11 and Figure 12 Comparison of the blank control group with the model group revealed that dexamethasone treatment severely impaired the skeletal development of zebrafish larvae. Comparison of the model group with the positive drug group, CP-9 group, and BB12 group revealed that CP-9 treatment significantly inhibited the damage caused by dexamethasone to the skeletal development of zebrafish larvae and effectively improved the skeletal development of zebrafish larvae. This improvement was comparable to that of the positive drug group and significantly superior to that of the BB12 group. These results indicate that animal Bifidobacterium lactis subsp. CP-9 can promote skeletal development and is significantly more effective than BB12, also a member of the same animal Bifidobacterium lactis subsp. This suggests that animal Bifidobacterium lactis subsp. CP-9 has more significant activity and advantages in regulating skeletal growth and development.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of animal Bifidobacterium lactis subsp. CP-9 in the preparation of a product for promoting bone growth and development or improving bone health; The animal Bifidobacterium lactis subsp. lactis CP-9 was deposited in China Center for Type Culture Collection on November 24, 2014, with the deposit number of CCTCC NO: M2014588; The product is a health product or medicine; The application form of the animal Bifidobacterium lactis subspecies CP-9 is at least one of live bacteria and freeze-dried powder.

2. The use according to claim 1, characterized in that The viable count of animal Bifidobacterium lactis subspecies CP-9 in the product is 1×10 6 -10 10 CFU / mL or 1×10 6 -10 10 CFU / g.

3. The use according to claim 1, characterized in that The promotion of bone growth and development includes: Improve bone volume fraction; and / or, Improved trabecular bone thickness; and / or, Improved growth hormone levels; and / or, Improved insulin-like growth factor 1 levels; and / or, Improve bone mass.

4. The use according to claim 1, characterized in that Improved bone health includes: Improve the level of type I procollagen N-terminal propeptide; and / or, Improvement of type I collagen amino-terminal peptide levels; and / or, Improved osteocalcin levels; and / or, Improve bone morphogenetic protein levels; and / or, Improved osteoprotegerin levels; and / or, Improve the level of receptor activator of nuclear factor-κB ligand.

5. The use according to claim 1, characterized in that The product may be in the form of at least one of oral liquid, tablet, granule, powder, capsule, pill, aqueous solution, powder, and film.

6. The use according to claim 1, characterized in that The health care product includes at least one of a functional beverage, a functional granule, and a functional capsule; and / or, The drugs include at least one of drugs for promoting osteoblast differentiation, inhibiting osteoclast differentiation, treating or assisting in the promotion of bone formation, treating or assisting in the inhibition of bone resorption, improving bone metabolism, preventing or treating osteoporosis, preventing or treating abnormal bone growth, and preventing or treating bone dysplasia.

7. The use according to claim 1, characterized in that The drug further comprises a drug carrier and / or a pharmaceutically acceptable excipient; The drug carrier includes at least one of microcapsules, microspheres, nanoparticles, and liposomes; The auxiliary materials include at least one of fillers, flavoring agents, wetting agents, adhesives, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, inert gases, preservatives, pH regulators, and isotonic or isotonic regulators.

8. Use of animal Bifidobacterium lactis subsp. lactis CP-9 in the preparation of children's milk powder, children's dietary supplement, children's snack, adolescent milk powder, adolescent dietary supplement or adolescent snack for promoting bone growth and development or improving bone health; The animal Bifidobacterium lactis subsp. lactis CP-9 was deposited in China Center for Type Culture Collection on November 24, 2014, with the deposit number of CCTCC NO: M2014588; The application form of the animal Bifidobacterium lactis subspecies CP-9 is at least one of live bacteria and freeze-dried powder.

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