Nutritional composition for improving osteoblast development condition and application thereof

Abstract

The invention discloses a nutritional composition for improving osteoblast development and application thereof, and belongs to the technical field of functional foods.The nutritional composition is prepared from, by weight, 1-98 parts of colostrum basic protein, 1-98 parts of hydrolyzed collagen, 0.01-10 parts of phosphatidylserine and 0.01-10 parts of hydrolyzed yolk powder; the hydrolyzed collagen is derived from bovine bones. A product obtained by compounding the nutritional composition and a matrix can remarkably promote proliferation of osteogenic precursor cells, enhance the calcium absorption capacity of the osteogenic precursor cells, remarkably improve the collagen secretion effect and the cell matrix mineralization capacity of the osteogenic precursor cells, and is beneficial to body skeletal development and body height improvement.

Description

Technical Field

[0001] The invention relates to the technical field of functional foods, and in particular to a nutritional composition for improving osteoblast development and application thereof. Background Art

[0002] With the improvement of social living conditions and the increasing awareness of health among people, the market demand for functional dairy products continues to increase. Milk powder and formula milk powder are one of the functional food categories with more comprehensive research, higher public recognition and lower education costs. Bones are the basic structure of the body, which can protect internal organs and participate in metabolic processes. Formula milk powder that strengthens bone development can meet the nutritional needs of specific groups of people, such as the growth and development needs of adolescents and children, the needs of the elderly to prevent osteoporosis, etc., improve bone health and prevent bone-related diseases. Although many milk powders on the market claim to have the function of promoting bone growth, their nutritional functions are not comprehensive enough.

[0003] The patent document with publication number CN111543496A discloses a milk powder that helps children's height development and bone growth, and its active ingredients mainly include hydrolyzed egg yolk powder, lactoferrin, docosahexaenoic acid, animal bifidobacterium lactis subspecies Bb-12, bovine colostrum powder and milk mineral salts; the patent document with publication number CN116584544A discloses a formula of children's growth goat milk powder and its preparation process, and the main raw materials of the formula are raw goat milk and desalted goat whey powder, and the auxiliary materials are phosphatidylserine, lactoferrin, hawthorn powder, yam powder, hydrolyzed egg yolk powder, probiotic powder, etc. However, none of the above inventions conducts in vivo or in vitro efficacy verification experiments on the effect of milk powder on promoting bone growth and development, so the rigor, scientificity and effectiveness of the above formula need to be further verified.

[0004] The patent document with the publication number CN113575695A discloses a formula milk powder that can promote the increase of tibia bone density and the growth of femur length. Its raw material composition is complex, including whole milk powder, desalted whey powder, lactose, oligomalto-oligosaccharide, oligofructose, 3 parts of vitamin package, 2 parts of minerals, phosphatidylserine, milk mineral salt, etc. A 28-day rat feeding experiment shows that the formula milk powder can increase the content of 25-hydroxyvitamin D, osteocalcin, bone alkaline phosphatase and type I procollagen amino terminal peptide in rat serum, and can promote the increase of tibia bone density and the growth of femur length in rats. However, the formula milk powder has complex effective ingredients, and there is no obvious increase trend in tibia length and femur bone density. There is no in-depth discussion on the effects of different concentrations of bone growth promoting ingredients in the formula milk powder and different combinations of the ingredients on rat bones.

[0005] Therefore, it is necessary to develop a nutritional composition that can improve the development of osteoblasts in order to develop products that promote bone development in the body, and to conduct in-depth research on the actual efficacy of the composition to confirm the scientificity and effectiveness of the composition ingredients and formula. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a nutritional composition for improving the development of osteoblasts. The ingredients of the nutritional composition are easy to obtain, and the product obtained after compounding with a matrix can significantly promote the proliferation of osteoblast precursor cells and enhance their ability to absorb calcium, significantly increase the collagen secretion effect of osteoblast precursor cells and the cell matrix mineralization ability, and can promote bone growth and development.

[0007] The specific technical solutions adopted are as follows:

[0008] A nutritional composition for improving the development of osteoblasts, comprising, by weight, 1 to 98 parts of colostrum alkaline protein, 1 to 98 parts of hydrolyzed collagen, 0.01 to 10 parts of phosphatidylserine and 0.01 to 10 parts of hydrolyzed egg yolk powder; the hydrolyzed collagen is derived from bovine bones, and the specific preparation process includes raw material pretreatment, steaming, filter press separation, decolorization, deodorization, desalination, concentration, spray drying and other processes.

[0009] Furthermore, the nutritional composition for improving the development of osteoblasts comprises, by weight, 1 to 50 parts of colostrum basic protein, 1 to 40 parts of hydrolyzed collagen, 0.01 to 5 parts of phosphatidylserine and 0.01 to 4 parts of hydrolyzed egg yolk powder.

[0010] Furthermore, the nutritional composition for improving the development of osteoblasts comprises, by weight, 10 parts of colostrum basic protein, 20 parts of hydrolyzed collagen, 1.5 parts of phosphatidylserine and 1 part of hydrolyzed egg yolk powder.

[0011] Colostrum alkaline protein is a small molecule protein composition made from bovine colostrum, which can directly act on bone cells and promote bone metabolism; hydrolyzed egg yolk powder is made from egg yolk polypeptides after concentration and drying, which can maintain the balance of bone components and inhibit the division of osteoclasts; phosphatidylserine is processed from soy lecithin and L-serine, which can regulate cell metabolism and maintain cell integrity and stability; hydrolyzed collagen can provide high-quality amino acid raw materials for the synthesis of human collagen, and has a significant effect on improving the health of bones and joints. After colostrum alkaline protein, hydrolyzed egg yolk powder, hydrolyzed collagen and phosphatidylserine are compounded, they can provide the body with high-quality amino acid raw materials, which is beneficial to improving the development of osteoblasts, promoting osteoblast proliferation and calcium absorption.

[0012] The present invention also provides the use of the nutritional composition for improving the development of osteoblasts in the preparation of products for promoting the development of bones in the body.

[0013] The present invention also provides a product for promoting the development of bones in the body, the ingredients of which include the nutritional composition for improving the development of osteoblasts.

[0014] Specifically, the product for promoting bone development includes but is not limited to powders, tablets, capsules, granules or drinks, etc., and the ingredients also include pharmaceutically acceptable excipients or food-acceptable matrices or additives.

[0015] Preferably, the product for promoting bone development of the body is milk powder, comprising 30-40wt% of the nutritional composition for improving the development of osteoblasts and 60-70wt% of milk-based powder, wherein the milk-based powder comprises 30-40wt% whole milk powder, 20-30wt% skim milk powder, 3-5wt% vegetable fat powder, 10-20wt% desalted whey powder, 10-20wt% lactose, 1-5wt% vitamins and 1-5wt% minerals.

[0016] More preferably, the product for promoting bone development of the body comprises, by weight, 10 parts of colostrum alkaline protein, 1.5 parts of phosphatidylserine, 20 parts of hydrolyzed collagen, 1 part of hydrolyzed egg yolk powder, and 67.5 parts of milk-based powder.

[0017] Furthermore, the product for promoting bone development of the body promotes bone development of the body by improving the development of MC3T3-E1 cells, and improving the development of MC3T3-E1 cells specifically includes at least one of the following:

[0018] (1) Promote the proliferation of MC3T3-E1 cells;

[0019] (2) Improve the calcium absorption capacity of MC3T3-E1 cells;

[0020] (3) Increase the expression levels of calcium-binding proteins and plasma membrane calcium ion ATPase in MC3T3-E1 cells;

[0021] (4) Increase collagen secretion in MC3T3-E1 cells;

[0022] (5) Promote MC3T3-E1 cell matrix mineralization.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention obtains a nutritional composition capable of improving the development of osteoblasts by compounding colostrum alkaline protein, hydrolyzed collagen, phosphatidylserine, and hydrolyzed egg yolk powder in specific proportions. The raw materials of the composition are easy to obtain and have high safety. The composition can be used to prepare products that promote the body's bone development and can be consumed for a long time as a common food, health product, or medicine.

[0025] (2) The compound composition of colostrum alkaline protein, hydrolyzed collagen, phosphatidylserine and hydrolyzed egg yolk powder has a significant synergistic effect on promoting bone development. Removing any active ingredient will lead to the weakening or loss of osteoblast proliferation ability and calcium absorption.

[0026] (3) The product for promoting bone development provided by the present invention can significantly promote the proliferation of osteoblast precursor cells and enhance their ability to absorb calcium. At the same time, it can significantly increase the collagen secretion effect and cell matrix mineralization ability of osteoblast precursor cells, which is beneficial to the body's bone development and improves height. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The proliferation of MC3T3-E1 cells after being treated with different concentrations of the milk powder of Example 1 and commercially available children's growth milk powder, the treatment time of A was 24h, the treatment time of B was 48h, the treatment time of C was 72h, and the treatment time of D was 96h; *P<0.05, **P<0.01, compared with the 0μg / mL control group; #P<0.05, compared with the commercially available children's growth milk powder at the same concentration.

[0028] Figure 2 The proliferation of MC3T3-E1 cells after being treated with different nutritional compositions, hydrolyzed collagen alone and the milk powder of Example 1, the treatment time of A was 24h, the treatment time of B was 48h, the treatment time of C was 72h, and the treatment time of D was 96h; *P<0.05, **P<0.01, compared with the negative control group.

[0029] Figure 3 The calcium absorption capacity of MC3T3-E1 cells after being treated with different concentrations of the milk powder of Example 1 and commercially available children's growth milk powder, A is the calcium content test result, B is the mRNA expression level of calcium binding protein and plasma membrane calcium ion ATPase; *P<0.05, **P<0.01, compared with the 0 μg / mL control group; ##P<0.01, compared with the commercially available children's growth milk powder at the same concentration.

[0030] Figure 4: Calcium content of MC3T3-E1 cells in different treatment groups, A is treatment with different concentrations of nutrient compositions, B is treatment with different nutrient compositions, hydrolyzed collagen alone and milk powder of Example 1, *P<0.05, **P<0.01, different concentration treatment groups compared with negative control group.

[0031] Figure 5 The collagen secretion of MC3T3-E1 cells after being treated with different concentrations of the milk powder of Example 1 and commercially available children's growth milk powder, A is a viscose red staining image, B is a statistical graph, *P<0.05, **P<0.01, compared with the 0 μg / mL control group; #P<0.05, compared with the same concentration of commercially available children's growth milk powder.

[0032] Figure 6 The matrix mineralization of MC3T3-E1 cells after being treated with different concentrations of milk powder of Example 1 and commercially available children's growth milk powder, A is an Alizarin red staining image, B is a statistical graph, *P<0.05, **P<0.01, compared with the 0 μg / mL control group; #P<0.05, compared with the same concentration of commercially available children's growth milk powder. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the examples and drawings. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Contents not described in detail in this specification belong to prior art known to professionals and technicians in the field.

[0034] In the following examples, colostrum alkaline protein, hydrolyzed collagen (beef bone), phosphatidylserine, and hydrolyzed egg yolk powder were purchased from the market; the components of the milk-based powder included 35wt% whole milk powder, 25wt% skim milk powder, 4wt% vegetable fat powder, 15wt% desalted whey powder, 15wt% lactose, 2.25wt% vitamins and 3.75wt% minerals.

[0035] Embodiment 1 (powder)

[0036] According to weight proportions, 10 parts of colostrum alkaline protein, 1.5 parts of phosphatidylserine, 20 parts of hydrolyzed collagen (derived from bovine bones), 1 part of hydrolyzed egg yolk powder and 67.5 parts of milk-based powder are mixed evenly according to proportions and packaged to obtain milk powder.

[0037] Embodiment 2 (powder)

[0038] According to weight proportions, 20 parts of colostrum alkaline protein, 3.5 parts of phosphatidylserine, 10 parts of hydrolyzed collagen (derived from bovine bones), 3 parts of hydrolyzed egg yolk powder, 2 parts of fruit powder and 61.5 parts of oligofructose are mixed evenly in proportion and packaged to obtain a powder product.

[0039] Example 3 (solid beverage)

[0040] According to weight proportions, 30 parts of colostrum alkaline protein, 5.5 parts of phosphatidylserine, 8 parts of hydrolyzed collagen (derived from bovine bones), 5 parts of hydrolyzed egg yolk powder, 13 parts of xylo-oligosaccharides, 15 parts of isomaltooligosaccharides and 23.5 parts of skimmed milk powder are uniformly mixed in proportion and packaged to obtain a solid beverage product.

[0041] Example 4 (granules)

[0042] According to weight proportions, 25 parts of colostrum alkaline protein, 4.5 parts of phosphatidylserine, 9 parts of hydrolyzed collagen (derived from bovine bones), 4 parts of hydrolyzed egg yolk powder and 57.5 parts of lactose are uniformly mixed in proportion, granulated, dried and then packaged to obtain a granule product.

[0043] Example 5 (Tablet)

[0044] According to weight proportions, 15 parts of colostrum alkaline protein, 2.5 parts of phosphatidylserine, 15 parts of hydrolyzed collagen (derived from bovine bones), 4 parts of hydrolyzed egg yolk powder, 23.5 parts of galacto-oligosaccharide and 40 parts of maltodextrin are uniformly mixed in proportion, granulated and dried, and then tabletted to obtain a tablet product.

[0045] Embodiment 6 (hard capsule)

[0046] According to weight proportions, 30 parts of colostrum alkaline protein, 6 parts of phosphatidylserine, 60 parts of hydrolyzed collagen (derived from bovine bones), and 4 parts of hydrolyzed egg yolk powder are mixed evenly in proportion, and then filled with a capsule filling machine to obtain a hard capsule product.

[0047] Example 7 (microcapsules)

[0048] According to weight proportions, 15 parts of colostrum basic protein, 3 parts of phosphatidylserine, 30 parts of hydrolyzed collagen (derived from bovine bones), 2 parts of hydrolyzed egg yolk powder, 24.5 parts of maltodextrin, and 25.5 parts of whey protein powder are taken; the colostrum basic protein, phosphatidylserine, hydrolyzed collagen and hydrolyzed egg yolk powder are uniformly mixed, and then the mixed solution of maltodextrin and whey protein powder is used to embed the mixed solution, and the mixed solution is low-temperature dried and then packaged to obtain a microcapsule product.

[0049] Embodiment 8 (soft capsule)

[0050] According to weight parts, 11 parts of colostrum basic protein, 1.6 parts of phosphatidylserine, 22 parts of hydrolyzed collagen (derived from bovine bones), 1.1 parts of hydrolyzed egg yolk powder, 8 parts of gelatin, 6 parts of vegetable oil, and 50.3 parts of water are uniformly mixed in proportion to obtain the contents, and gelatin, glycerin, and water are mixed in a ratio of 1:0.4:1 to prepare a gelatin solution, which is pressed into pills using an automatic soft capsule pill press, and a soft capsule product is obtained after drying.

[0051] Example 9 (liquid beverage)

[0052] According to weight proportions, 10 parts of colostrum alkaline protein, 1.5 parts of phosphatidylserine, 20 parts of hydrolyzed collagen (derived from bovine bones), 1 part of hydrolyzed egg yolk powder, 7.5 parts of whole milk powder and 60 parts of purified water are mixed evenly in proportion and packaged to obtain a formula dairy product.

[0053] Sample analysis

[0054] The milk powder of Example 1, commercially available children's growing milk powder (mainly comprising whole milk powder, skim milk powder, lactose, desalted whey powder and colostrum alkaline protein) and the nutritional composition sample of Example 1 were tested for their effects. The specific methods and test results are as follows:

[0055] 1.1 Cell culture and osteogenic induction

[0056] Osteoblast precursor cells (MC3T3-E1 cell line, Haixing Biotechnology Co., Ltd.) were used for the experiment. α-MEM medium containing 10% fresh bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin was used, and cultured at 37°C, 5% CO2, and saturated humidity, and routine medium changes and passages were performed. During the culture process, osteogenic induction medium was used to induce cell osteogenic differentiation, wherein the osteogenic induction medium was the above medium with 5 mmol / L β-glycerophosphate and 50 μg / mL ascorbic acid added.

[0057] 1.2 Determination of calcium absorption

[0058] The cells were cultured at 1×10 4The cells were inoculated in a 12-well plate at a density of 100 cells / mL and cultured for 24 hours before being treated with different concentrations of milk powder of Example 1 and commercially available children's growth milk powder. For the screening experiment of the nutritional composition in the milk powder of Example 1, cells were treated with different concentrations of nutritional composition and hydrolyzed collagen alone; for the screening experiment of nutritional composition of different active ingredients, 4 combinations of ingredients and 3 combinations of ingredients (combination 1: colostrum alkaline protein powder, phosphatidylserine and hydrolyzed collagen; combination 2: colostrum alkaline protein powder, phosphatidylserine and hydrolyzed egg yolk powder; combination 3: colostrum alkaline protein powder, hydrolyzed collagen and hydrolyzed egg yolk powder; combination 4: phosphatidylserine, hydrolyzed collagen and hydrolyzed egg yolk powder) and hydrolyzed collagen alone and the positive control 600 μg / mL milk powder of Example 1 were used to treat cells. After 96 h, the cells were collected, resuspended in 0.3 mL of deionized water, disrupted by ultrasound, vortexed and centrifuged at 2500 rpm for 10 min. The supernatant was taken and the calcium content was determined according to the instructions of the calcium test kit (Nanjing Jiancheng).

[0059] 1.3 Cell proliferation ability

[0060] The cells were cultured at 1.5 × 10 3 The cells were inoculated in a 96-well plate at a density of 100 μg / mL. After culturing for 24 h, the cells were treated with different concentrations of the milk powder of Example 1 and commercially available children's growth milk powder for 24, 48, 72, and 96 h. For the screening experiment of different active ingredient combinations of the milk powder of Example 1, the cells were treated with a combination of 4 ingredients (colostrum basic protein, phosphatidylserine, collagen solution (cow bone), and hydrolyzed egg yolk powder) and a combination of 3 ingredients (colostrum basic protein, phosphatidylserine, collagen solution (cow bone), and any three of hydrolyzed egg yolk powder) and a positive control of 600 μg / mL of the milk powder of Example 1 for 24, 48, 72, and 96 h. The cells were then rinsed once with a buffer solution PBS, and 20 μL of MTT (5 g / L) and 200 μL of serum-free α-MEM culture medium were added to each well. After incubation at 37°C for 4 h, the supernatant was discarded, 150 μL of dimethyl sulfoxide was added to each well to dissolve the generated crystals, and the cells were shaken at room temperature for 20 min. The optical density (OD) was then measured at 490 nm.

[0061] 1.4 Real-time fluorescence quantitative polymerase chain reaction

[0062] Total RNA was extracted from cells using Trizol reagent (Novizan, Nanjing). RNA concentration was determined using a Nano-300 spectrophotometer (Aosheng, Hangzhou), and cDNA was synthesized using a cDNA reverse transcriptase kit (Qingke, Beijing). Quantitative real-time PCR was performed using SYBR Green real-time PCR MasterMix (Qingke, Beijing) on ​​a CFX Connectreal-time PCR system (Bio-rad, USA). β-actin transcription was used as a housekeeping gene for data normalization, and the gene primer sequences are shown in the following table:

[0063] Table 1 RT-qPCR primer sequences (SEQ ID NO.1-SEQ ID NO.6)

[0064]

[0065] 1.5 Cell collagen secretion assay

[0066] Collagen secretion was measured by viscoelastic red staining. 5 × 10 cells were plated per well. 4 The cells were inoculated in a 6-well plate at a density of 100 cells / mL. After culturing for 24 hours, different concentrations of milk powder of Example 1 and commercially available children's growth milk powder were used for treatment, and milk-based powder was used as a control group. After the cells were close to fusion, they were replaced with osteogenic induction medium and cultured for 21 days until they differentiated into mature osteoblasts. The cells were fixed with 4% paraformaldehyde for 30 minutes, rinsed with PBS 3 times, and stained with a modified Sirius red staining kit (Beijing Solebow Technology Co., Ltd.). For quantitative analysis, sodium hydroxide was added to dissolve the stain, and the OD value was measured at 570 nm.

[0067] 1.6 Cell matrix mineralization assay

[0068] Alizarin red staining was used to determine the mineralization of the cell matrix. The cells were seeded in a 6-well plate at a density of 5×104 cells per well. After culturing for 24 hours, they were treated with different concentrations of milk powder in Example 1 and commercially available children's growth milk powder, and the milk-based powder was used as the control group. After the cells were close to fusion, they were replaced with osteogenic induction medium and cultured for 28 days until the cell matrix was mineralized. The cells were rinsed with PBS three times and fixed with 75% ethanol for 1 hour. 40mmol / L Alizarin red dye solution (pH=4.2) was used for staining at room temperature for 20 minutes. After rinsing with PBS five times, the cells were dissolved and stained with 10% cetylpyridinium chloride, and the OD value was measured at 570nm.

[0069] 1.7 Statistical analysis

[0070] All experimental data are expressed as "mean ± standard error", and statistical analysis was performed using one-way analysis of variance followed by Student-Newman-Keuls test. When P < 0.05, it was indicated that there was a significant difference (indicated by * / #), and when P < 0.01, it was indicated that the difference was extremely significant (indicated by ** / ##).

[0071] 2 Results and Analysis

[0072] 2.1 Cell proliferation ability

[0073] Compared with the 0 μg / mL group, at 24 h after treatment, 400 and 600 μg / mL of the milk powder of Example 1 significantly promoted the proliferation of osteoblast precursor cells ( Figure 1 A); 200 and 800 μg / mL Example 1 milk powder groups significantly promoted the proliferation of osteoblast precursor cells at 48h and 72h ( Figure 1 B and C in Figure ), while the 100 μg / mL milk powder group significantly promoted the proliferation of osteoblast precursor cells after 96 hours ( Figure 1 In addition, this proliferation was dose-dependent and time-dependent within a certain range. On the other hand, 400 and 600 μg / mL of commercial children's growth milk powder significantly promoted the proliferation of osteoblast precursor cells after 48 h, while 200 μg / mL of commercial children's growth milk powder significantly promoted the proliferation of osteoblast precursor cells after 72 h ( Figure 1 AD in). Relatively speaking, the same concentration of Example 1 milk powder is superior to the commercially available children's growth milk powder in promoting the proliferation of osteoblast precursor cells. For example, 400 μg / mL of Example 1 milk powder can significantly promote the proliferation of osteoblast precursor cells at the 24th hour, while 400 μg / mL of the commercially available growth milk powder needs 48 hours to show a significant promoting effect on the proliferation of osteoblast precursor cells. In addition, after 96 hours of treatment with 100, 200, 400, 600 and 800 μg / mL of Example 1 milk powder, the cell proliferation rates reached 123%, 130%, 139.7%, 133.4% and 129.9%, respectively, which were increased by 3.9%, 5.8%, 15.7%, 11.5% and 12.9% respectively compared with the same dose of commercially available children's growth milk powder.

[0074] In order to evaluate the effect of the combination of four different active ingredients in the milk powder of Example 1 on cell proliferation, the content of the four active ingredients in the milk powder of Example 1 at 600 μg / mL was set as "100× concentration", the milk powder of Example 1 at a concentration of 600 μg / mL was selected as the positive control, and the commercially available children's growth milk powder was selected as the negative control group. The combination of four active ingredients at a concentration of 100× was compared with different combinations of three ingredients. The results showed that compared with the negative control group, at 24 hours after treatment, the milk powder of Example 1 at 600 μg / mL could significantly promote the proliferation of osteoblast precursor cells ( Figure 2 A in the figure); 100× (195 μg / mL) of the four active ingredients combined can significantly promote the proliferation of osteoblast precursor cells after 48 h ( Figure 2 B); 100× (195 μg / mL) of the combination of the three active ingredients significantly promoted the proliferation of osteoblast precursor cells after 72 h, while 100× (120 μg / mL) of hydrolyzed collagen alone significantly promoted the proliferation of osteoblast precursor cells after 72 h ( Figure 2 C).

[0075] In addition, the combination of the three ingredients without hydrolyzed collagen had no effect on cell proliferation within 96 h of treatment ( Figure 2 AD in). Among them, after 96h of treatment with 600μg / mL of Example 1 milk powder and 100×(195μg / mL) of the combination of four active ingredients and 100×(120μg / mL) of hydrolyzed collagen alone, the cell proliferation rates reached 137.2%, 126.1% and 121.6%, respectively, which were increased by 25.4%, 15.3% and 11.2% respectively compared with the negative control group. It shows that the single hydrolyzed collagen has a better promoting effect on the proliferation of osteoblast precursor cells. At the same time, compared with the negative control group, the three active ingredient combinations without hydrolyzed collagen can not significantly promote the proliferation of osteoblast precursor cells within 24h to 96h, indicating that hydrolyzed collagen is indispensable in the combination of active ingredients.

[0076] 2.2 Calcium absorption capacity

[0077] 2.2.1 Effects of different concentrations of the milk powder of Example 1 on the calcium absorption capacity of cells

[0078] The milk powders of Example 1 with concentrations of 0, 100, 200, 400, and 600 μg / mL and the commercially available children's growth milk powder with a concentration of 600 μg / mL were selected for subsequent experiments, and the milk powder of Example 1 with a concentration of 0 μg / mL was used as the control group. Figure 3As shown in A, after being treated with different concentrations of the milk powder of Example 1 and commercially available children's growth milk powder for 96 hours, the calcium content in osteoblast precursor cells increased. Among them, the treatment with 600μg / mL of the milk powder of Example 1 had the most significant effect on improving calcium absorption capacity, with a calcium content of 1.06mmol / gprot, an increase of 221.8% compared with the control group and an increase of 65.6% compared with the commercial children's growth milk powder of the same concentration. In addition, qPCR results showed that the milk powder of Example 1 can significantly increase the mRNA expression levels of calcium binding protein d9k (CaBP-d9k) and plasma membrane calcium ion ATPase (PMCAlb) in osteoblast precursor cells, and the improvement effect of 600μg / mL of the milk powder of Example 1 is significantly better than that of the commercial children's growth milk powder of the same concentration ( Figure 3 B).

[0079] 2.2.2 Effects of different combinations of the four active ingredients on cellular calcium absorption capacity

[0080] In order to evaluate the regulatory effects of the four active ingredients in the milk powder of Example 1 on the calcium absorption capacity of cells, the content of the active ingredients in the milk powder of Example 1 (600 μg / mL) (195 μg / mL) was set as "100× concentration" and the multiple was magnified or reduced, and commercially available children's growth milk powder was selected as the negative control group. Figure 4 As shown in A, after being treated with different concentrations of the active ingredient combination of the milk powder of Example 1 for 96 hours, the calcium content in osteoblast precursor cells increased. Among them, the calcium contents of the 100×, 200× and 400× active ingredient combination treatment groups were 0.69, 0.92 and 0.94 mmol / gprot, respectively, which were increased by 122.6%, 196.8% and 203.2% respectively compared with the negative control group. The 100× (195 μg / mL) active ingredient combination was selected for screening of different active ingredient combinations, and the results are shown in FIG. Figure 4 As shown in Figure B, combination 1, combination 3 and combination 4 can significantly promote calcium absorption of cells, which are increased by 91.4%, 97.6% and 85.2% respectively compared with the negative control group. In addition, active ingredient combination 2 without hydrolyzed collagen cannot promote calcium absorption of cells, and there is no significant difference compared with the commercially available children's growth milk powder (negative control group), which proves that hydrolyzed collagen plays a key role in promoting the increase of calcium content in osteoblasts.

[0081] 2.3 Cellular collagen secretion

[0082] The collagen secretion of cells was evaluated by viscose red staining. Figure 5 As shown in the figure, after being treated with different concentrations of the milk powder of Example 1, the collagen secretion of osteoblast precursor cells increased, and the secretion of collagen fibers (green arrows) and muscle fibers (blue arrows) increased ( Figure 5Among them, the effect of 600 μg / mL of Example 1 milk powder on collagen secretion was the most significant, increasing by 39.5% compared with the control group, and the effect of 400 μg / mL of Example 1 milk powder on collagen secretion was increased by 24.1% compared with the control group, while the effect of 600 μg / mL of commercially available children's growth milk powder on collagen secretion was only increased by 17.2% compared with the control group ( Figure 5 B).

[0083] 2.4 Cell matrix mineralization assay

[0084] The cell matrix mineralization was determined by 40 mmol / L Alizarin red staining. Figure 6 As shown. Among them, the cells in the groups treated with 200, 400 and 600 μg / mL milk powder of Example 1 showed significant alizarin red staining ( Figure 6 The OD values ​​of matrix mineralization quantitative analysis were 1.852±0.078, 1.953±0.069 and 2.118±0.071, which were increased by 18.4%, 24.9% and 35.4% respectively compared with the control group ( Figure 6 B), and as the sample concentration increases, the cell matrix mineralization increases. In addition, the matrix mineralization of 600 μg / mL commercially available children's growth milk powder increased by 18.2% compared with the control group, but it was significantly lower than the promotion effect of 600 μg / mL Example 1 milk powder on cell matrix mineralization.

[0085] 3 Conclusion

[0086] In summary, at 24h after the treatment with the milk powder of Example 1, the treatment with 400 and 600 μg / mL of the milk powder of Example 1 significantly increased the proliferation of osteoblast precursor cells, and the effect increased with the increase of the treatment concentration within a certain range. Over time, the 200 μg / mL milk powder of Example 1 group significantly increased the proliferation of osteoblast precursor cells at 48h, while the 100 and 800 μg / mL milk powder of Example 1 groups could significantly increase the proliferation of osteoblast precursor cells at 96h, and were superior to the existing children's growth milk powder under each concentration treatment.

[0087] 600μg / mL Example 1 milk powder treatment significantly increased the proliferation of osteoblast precursor cells after 24h; over time, 100× (195μg / mL) of the four active ingredients in combination significantly increased the proliferation of osteoblast precursor cells at 48h, while 100× (120μg / mL) of the hydrolyzed collagen group alone significantly increased the proliferation of osteoblast precursor cells at 72h, and the four active ingredients had a synergistic effect on the proliferation and calcium absorption capacity of osteoblast precursor cells. In particular, hydrolyzed collagen plays an indispensable role in promoting osteoblast proliferation and increasing calcium content. Calcium content detection and qPCR results show that Example 1 milk powder can promote calcium absorption of osteoblast precursor cells, which is beneficial to the osteogenesis of precursor cells. At the same time, 400 and 600μg / mL Example 1 milk powders significantly increased the collagen secretion of osteoblast precursor cells. In addition, the experiment also used Alizarin red staining and quantitative analysis to detect the matrix mineralization ability of osteoblast precursor cells. The results showed that 400 and 600 μg / mL milk powder could induce a significant increase in cell mineralization ability, and it was concentration-dependent.

[0088] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or substitutions in a similar manner made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A nutritional composition for improving osteoblast development, characterized in that: According to weight, it comprises 1 to 98 parts of colostrum alkaline protein, 1 to 98 parts of hydrolyzed collagen, 0.01 to 10 parts of phosphatidylserine and 0.01 to 10 parts of hydrolyzed egg yolk powder; the hydrolyzed collagen is derived from bovine bones.

2. The nutritional composition for improving osteoblast development according to claim 1, characterized in that: Calculated by weight, it comprises 1 to 50 parts of colostrum alkaline protein, 1 to 40 parts of hydrolyzed collagen, 0.01 to 5 parts of phosphatidylserine and 0.01 to 4 parts of hydrolyzed egg yolk powder.

3. The nutritional composition for improving osteoblast development according to claim 1, characterized in that: Calculated by weight, the invention comprises 10 parts of colostrum alkaline protein, 20 parts of hydrolyzed collagen, 1.5 parts of phosphatidylserine and 1 part of hydrolyzed egg yolk powder.

4. Use of the nutritional composition for improving osteoblast development according to any one of claims 1 to 3 in the preparation of a product for promoting bone development in the body.

5. A product for promoting bone development, characterized in that: The ingredients include the nutritional composition for improving the development of osteoblasts as described in any one of claims 1-3.

6. The product for promoting bone development according to claim 5, characterized in that: The product for promoting the body's bone development includes powders, tablets, capsules, granules or drinks, and the ingredients also include pharmaceutically acceptable excipients or food-acceptable matrices or additives.

7. The product for promoting bone development according to claim 5, characterized in that: The product for promoting the development of bones in the body is milk powder, which comprises 30-40wt% of the nutritional composition for improving the development of osteoblasts and 60-70wt% of milk-based powder.

8. The product for promoting bone development according to claim 5, characterized in that: Calculated by weight, the invention comprises 10 parts of colostrum alkaline protein, 1.5 parts of phosphatidylserine, 20 parts of hydrolyzed collagen, 1 part of hydrolyzed egg yolk powder and 67.5 parts of milk-based powder.

9. The product for promoting bone development according to claim 5, characterized in that: The product for promoting bone development of the body promotes bone development of the body by improving the development of MC3T3-E1 cells. Improving the development of MC3T3-E1 cells specifically includes at least one of the following: (1) Promote the proliferation of MC3T3-E1 cells; (2) Improve the calcium absorption capacity of MC3T3-E1 cells; (3) Increase the expression levels of calcium-binding proteins and plasma membrane calcium ion ATPase in MC3T3-E1 cells; (4) Increase collagen secretion in MC3T3-E1 cells; (5) Promote MC3T3-E1 cell matrix mineralization.

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