Calcium-containing antarctic krill oligopeptide nutritional composition for promoting bone cell growth and application thereof

Abstract

The invention discloses a calcium-containing antarctic krill oligopeptide nutritional composition for promoting bone cell growth and application thereof, and belongs to the technical field of functional foods.The calcium-containing antarctic krill oligopeptide nutritional composition is composed of antarctic krill oligopeptide and a calcium compound, and the mass ratio of the antarctic krill oligopeptide to the calcium compound is (0.5-5): 1; or the composition is composed of the euphausia superba oligopeptide, the calcium compound and the vitamin D, and the mass ratio of the euphausia superba oligopeptide to the calcium compound to the vitamin D is (0.5-5): 1: (2.5 * 10 <-5 >)-(15 * 10 <-5 >); the mass of the calcium compound is calculated according to the content of the calcium element in the antarctic krill oligopeptide; the antarctic krill oligopeptide is obtained by treating antarctic krill, antarctic krill powder or defatted antarctic krill powder by utilizing an enzymolysis method; the content of a peptide fragment with the molecular weight of less than 1000Da in the antarctic krill The raw materials of the nutritional composition are easy to obtain, the safety is high, and the nutritional composition can be used for preparing products with the effect of promoting bone cell growth.

Description

Technical Field

[0001] The invention relates to the technical field of functional foods, and in particular to a calcium-containing Antarctic krill oligopeptide nutritional composition for promoting bone cell growth and an application thereof. Background Art

[0002] Osteoblasts are the precursors of osteocytes. They are undifferentiated cells with the potential for proliferation and differentiation. The main functions of osteoblasts are proliferation and differentiation. During bone growth and repair, osteoblasts can rapidly proliferate and increase the number of cells to meet the needs of bone formation. At the same time, under the regulation of various cytokines (such as bone morphogenetic protein, BMP) and hormones (such as parathyroid hormone, PTH), osteoblasts can differentiate into osteoblasts. Osteocytes play a variety of important functions in maintaining bone homeostasis. For example, osteocytes can regulate the mineralization of bone matrix and sense the mechanical stress of bone tissue. Osteocytes are also involved in the regulation of calcium-phosphorus balance in the body. When the blood calcium concentration changes, osteocytes can promote the release or deposition of bone calcium. At present, a variety of functional food raw materials have been proven to have the effect of promoting the growth and development of bones and related tissues, such as milk mineral salts, seaweed powder, mushroom powder, casein phosphopeptide, colostrum basic protein, hydrolyzed collagen, lysine, vitamin D3, vitamin K2, etc.

[0003] As is well known, calcium and vitamin D play a vital role in promoting the growth and development of bone cells, but relying solely on dietary supplements is often difficult to meet the needs of the human body at different growth stages and specific health conditions. In response to this, researchers have developed some nutritional compositions with bone health care effects. For example, the Chinese patent literature with publication number CN102133395A discloses a bone health care product or composition that can increase bone density, relieve bone pain, and restore bone function, and its main components include calcium carbonate, vitamin D3, casein phosphopeptide, chondroitin sulfate, glucosamine hydrochloride, collagen, and manganese chloride. Each component in its formula cooperates with each other and has high bioavailability. The Chinese patent literature with publication number CN114832020A discloses a composition composed of calcium carbonate, vitamin D3, vitamin K2, zinc gluconate, bacillus coagulans and amino acids, and this composition can be used in coordination with bacillus coagulans and amino acids to promote the body's absorption and utilization of calcium, zinc, vitamin D3, and vitamin K2, enhance bone formation, and promote growth and development. The Chinese patent document with publication number CN103892282A discloses a protein powder having the function of promoting bone growth and a preparation method thereof, wherein the protein powder is prepared from lactoferrin powder, whey protein isolate powder, vitamin E and calcium carbonate through cation exchange chromatography purification, size exclusion chromatography purification, ingredient mixing, product drying and other steps, and the protein powder has the activity of promoting osteoblast proliferation. The nutritional compositions in the above inventions are all prepared from a variety of raw materials, and the composite of a variety of raw materials is used to synergistically exert the bone health care effect.

[0004] Antarctic krill ( Antarctic Krill) is a shrimp animal of the genus Euphausiaceae of the order Euphausiaceae. It is generally 2-6 cm long, and the largest is 15 cm. It is widely distributed in the waters of the Southern Ocean around the Antarctic and is a unique aquatic product in the Antarctic waters. It contains rich protein, mineral elements and a variety of unsaturated fatty acids. It also contains a variety of active ingredients, such as enzymes, chitin and astaxanthin, which have good medical and health functions for the human body. After Antarctic krill is made into Antarctic krill peptides, it has the advantages of small molecular weight, high human absorption rate, good water solubility and high stability. At present, researchers have conducted more explorations on the physiological functions of Antarctic krill oligopeptides. For example, the Chinese patent document with publication number CN114507702A discloses a marine Antarctic krill oligopeptide and its application. It is made from fresh or frozen Antarctic krill as raw materials. After crushing, it is enzymatically hydrolyzed with trypsin, pepsin and papain to obtain Antarctic krill protein hydrolyzed peptides, which have the effect of lowering cholesterol. The Chinese patent document with publication number CN117568430A provides an Antarctic krill oligopeptide with uric acid-lowering activity. Animal experiments show that the Antarctic krill oligopeptide can significantly reduce the blood uric acid level of mice, increase the uric acid excretion level in the urine and feces of mice, and has a certain degree of protective effect on the kidney function of rats. Although Antarctic krill oligopeptides have multiple potential physiological functions, there are few reports on their research on promoting bone cell growth. Summary of the invention

[0005] The invention provides a calcium-containing Antarctic krill oligopeptide nutritional composition for promoting bone cell growth. The raw materials of the composition are easy to obtain and have high safety, and the composition can be used to prepare a product having the effect of promoting bone cell growth.

[0006] The specific technical solutions adopted are as follows: The calcium-containing Antarctic krill oligopeptide nutritional composition for promoting bone cell growth is composed of Antarctic krill oligopeptide and calcium compound, the mass ratio of Antarctic krill oligopeptide to calcium compound is 0.5-5:1, and the mass of calcium compound is calculated by the calcium content therein; or it is composed of Antarctic krill oligopeptide, calcium compound and vitamin D, the mass ratio of Antarctic krill oligopeptide, calcium compound and vitamin D is 0.5-5:1:2.5×10 -5 -15×10 -5 , the mass of calcium compounds is measured by the calcium content therein; The Antarctic krill oligopeptide is obtained by treating Antarctic krill, Antarctic krill powder or defatted Antarctic krill powder by enzymatic hydrolysis, and the content of peptide segments with a molecular weight of less than 1000 Da in the Antarctic krill oligopeptide is greater than 85%.

[0007] The calcium compound includes but is not limited to calcium carbonate, milk calcium or seaweed calcium, and seaweed calcium is more preferred.

[0008] Preferably, the Antarctic krill oligopeptide is obtained by enzymatically treating defatted Antarctic krill powder, and the content of peptide segments with a molecular weight of less than 1000 Da in the Antarctic krill oligopeptide is greater than 90%.

[0009] Furthermore, when the calcium-containing Antarctic krill oligopeptide nutritional composition consists of Antarctic krill oligopeptide and calcium compound, the mass ratio of Antarctic krill oligopeptide to calcium compound is 1-3.5:1; more preferably 2:1; the mass of calcium compound is calculated based on the calcium content therein.

[0010] Furthermore, when the calcium-containing Antarctic krill oligopeptide nutritional composition is composed of Antarctic krill oligopeptide, calcium compound and vitamin D, the mass ratio of Antarctic krill oligopeptide, calcium compound and vitamin D is 1-3.5:1:3.5×10 -5 -8×10 -5 ; More preferably 2:1:5.1×10 -5 ; The mass of a calcium compound is measured by the calcium content therein.

[0011] Specifically, the preparation method of Antarctic krill oligopeptide includes: (1) dissolving defatted Antarctic krill powder in water to obtain a raw material solution; (2) Adjusting the pH value of the raw material solution, adding protease, and performing enzymolysis at the optimal temperature of the protease; (3) After inactivating the enzyme, filter and cool the supernatant; (4) The solution obtained in step (3) is decolorized, and further solid-liquid separation is performed to obtain a supernatant, and the supernatant is sterilized and then dried to obtain the Antarctic krill oligopeptide.

[0012] Preferably, the weight ratio of the defatted Antarctic krill powder to water is 1:8-12.

[0013] The protease includes at least one of a composite protease (commercially available), alkaline protease, neutral protease, flavor protease, papain, and bromelain, preferably a mixed protease of a composite protease (commercially available) and alkaline protease.

[0014] Based on the amount of defatted Antarctic krill powder, the amount of protease added is 0.5-3wt%, preferably 2-3wt%, and the enzymolysis time is preferably 3-6 h. Under the above conditions, the degree of enzymolysis is relatively high.

[0015] Preferably, the drying method is spray drying. Spray drying has a fast speed and helps to maintain the activity of Antarctic krill oligopeptide in the product.

[0016] The content of peptide segments with a molecular weight of less than 1000 Da in the Antarctic krill oligopeptide prepared by the method of the present invention is greater than 85%, and further greater than 90%. After the Antarctic krill oligopeptide is compounded with a calcium compound or vitamin D, it is more easily absorbed by the body on the one hand, and has a better effect of promoting bone cell growth on the other hand.

[0017] Compared with the minimum purity limit (≥75%) for "marine fish collagen oligopeptide powder" in the national standard "GB / T 22729 Marine fish oligopeptide powder", the purity of the Antarctic krill oligopeptide provided by the present invention is more than 15% higher than the minimum limit of this national standard.

[0018] The present invention also provides the use of the calcium-containing Antarctic krill oligopeptide nutritional composition in the preparation of a product promoting bone cell growth.

[0019] Furthermore, promoting bone cell growth refers to improving the proliferation and differentiation of MC3T3-E1 osteoblast precursor cells, specifically including at least one of the following methods: (1) Promote the proliferation of MC3T3-E1 cells; (2) Promote calcium absorption in MC3T3-E1 cells; (3) Increase the collagen secretion level of MC3T3-E1 cells; (4) Increase the matrix mineralization capacity of MC3T3-E1 cells.

[0020] The invention also provides a product for promoting bone cell growth, comprising the calcium-containing Antarctic krill oligopeptide nutritional composition.

[0021] The product for promoting bone cell growth is a food or a medicine, and its forms include but are not limited to powders, tablets, capsules, granules or drinks, etc., and its ingredients also include pharmaceutically acceptable excipients or food-acceptable additives.

[0022] Furthermore, the product for promoting bone cell growth is a soft capsule or a gel candy, and the content of the calcium-containing Antarctic krill oligopeptide nutritional composition in the soft capsule or the gel candy is 20-50wt% (excluding the rubber).

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention compounds Antarctic krill oligopeptides and calcium compounds or Antarctic krill oligopeptides, calcium compounds and vitamin D to obtain a calcium-containing Antarctic krill oligopeptide nutritional composition for promoting bone cell growth. The raw materials of the composition are easy to obtain and have high safety. The composition can improve the proliferation and differentiation of MC3T3-E1 osteoblast precursor cells, promote the proliferation of MC3T3-E1 cells and their calcium absorption, and improve their collagen secretion level and matrix mineralization ability. The composition can be further prepared into common food, health products or medicines for long-term consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The proliferation rates of MC3T3-E1 cells after being treated with different concentrations of the composition for different time periods, A is 24 h, B is 48 h, C is 72 h, and D is 96 h; Note: *P<0.05, **P<0.01, compared with the control group.

[0025] Figure 2 The figure is a statistical diagram of the calcium absorption capacity of cells after being treated with different concentrations of the combination for 96 h, where A is the calcium content, B is the AKP activity, and C is the mRNA expression level of plasma membrane calcium ion ATPase and calcium binding protein d9k; Note: *P<0.05, **P<0.01, compared with the control group; ##P<0.01, compared with the combination group.

[0026] Figure 3 The diagrams are of collagen secretion of MC3T3-E1 cells after being treated with different concentrations of the composition, wherein A is the result of viscose red staining, and B is a statistical diagram, Note: *P<0.05, **P<0.01, compared with the control group.

[0027] Figure 4 The diagrams are of matrix mineralization of MC3T3-E1 cells after being treated with different concentrations of the composition, wherein A is the result of Alizarin Red staining, and B is a statistical diagram, Note: *P<0.05, **P<0.01, compared with the control group. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the examples and accompanying 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 performed under normal conditions or according to the conditions recommended by the manufacturer. The content that is not described in detail in this specification sheet belongs to the prior art known to professionals in the field. The experimental materials used in the examples, if not otherwise specified, can all be purchased by conventional biochemical reagent companies.

[0029] In the embodiment, the preparation method of Antarctic krill oligopeptide comprises the following steps: (1) dissolving defatted Antarctic krill powder in water, wherein the weight ratio of defatted Antarctic krill powder to water is 1:10, to obtain a raw material solution; (2) adjusting the pH value of the raw material solution, adding 2.5wt% protease (a mixed protease of a composite protease (commercially available) and an alkaline protease) of defatted Antarctic krill powder, and performing enzymolysis at the optimal temperature of the protease for 4 hours; (3) heating to 100°C to inactivate the enzyme for 20 minutes, filtering and cooling the supernatant; (4) further adding 15wt% activated carbon of defatted Antarctic krill powder, and decolorizing at 70°C for 1 hour; (5) after decolorization, centrifuging to separate the solid and liquid; (6) passing the obtained clear liquid through a ceramic membrane, a nanofiltration membrane, an evaporator, and an ultra-high temperature instantaneous sterilization process; (7) finally spray drying to obtain Antarctic krill oligopeptide. The test results show that the content of peptide segments with a molecular weight of <1000Da in the Antarctic krill oligopeptide is >90%.

[0030] Example 1 (soft capsule) According to the weight of the effective ingredients, 300 mg of Antarctic krill oligopeptide, seaweed calcium (including 150 mg of calcium), 7.65 μg of vitamin D, and flaxseed oil (an appropriate amount, flaxseed oil plays a filling and dispersing role, and can also be replaced with soybean oil, sunflower seed oil, MCT oil, etc., and the specific amount added can be customized according to market demand) are mixed uniformly in proportion to obtain the content, and gelatin, glycerin, and water are mixed in a ratio of 1:0.4:1 to prepare a gelatin solution, which is pelletized using an automatic soft capsule pelletizing machine, and dried to obtain a soft capsule product (the content (excluding the rubber skin) contains 20-50 wt% of the calcium-containing Antarctic krill oligopeptide nutritional composition).

[0031] Example 2 (jelly candy) According to the weight of the effective ingredients, 300 mg of Antarctic krill oligopeptide, seaweed calcium (including 150 mg of calcium), 7.65 μg of vitamin D, fruit powder (appropriate amount, the specific amount of addition can be customized according to market demand), linseed oil (appropriate amount, the specific amount of addition can be customized according to market demand), and glyceryl monostearate (appropriate amount, the specific amount of addition can be customized according to market demand) are uniformly mixed in proportion to obtain the content, and gelatin, glycerin, and sorbitol liquid are mixed in a ratio of 1:0.4:1 to prepare a glue liquid, which is filled with a molding machine to obtain a gel candy (the content (excluding the rubber) contains 20-50wt% of the calcium-containing Antarctic krill oligopeptide nutritional composition).

[0032] Example 3 (Powder) In terms of weight, every 20g of Antarctic krill oligopeptide formulated milk powder includes 13.6g of whole milk powder, 2.0g of desalted whey powder, 1.5g of lactose, 1.0g of galacto-oligosaccharide, 1.0g of inulin, 300mg of Antarctic krill oligopeptide, and milk calcium (including 150mg of calcium). By dry mixing the above raw materials and auxiliary materials, Antarctic krill oligopeptide formulated milk powder can be obtained.

[0033] Example 4 (Tablets) By weight, every 3.0 g of Antarctic krill oligopeptide chewable tablets includes 1.28 g of whole milk powder, 0.533 g of xylitol, 0.14 g of strawberry fruit powder, 0.075 g of ferric pyrophosphate, 0.072 g of magnesium stearate, 300 mg of Antarctic krill oligopeptide, milk calcium (including 150 mg of calcium), and 7.65 μg of vitamin D. The above raw and auxiliary materials are dry mixed and directly tableted to obtain Antarctic krill oligopeptide chewable tablets.

[0034] Sample analysis 1 Materials and methods 1.1 Cell culture and osteogenic induction The MC3T3-E1 cell line (Haixing Biotechnology Co., Ltd.) was used for the experiment. The cells were cultured in α-MEM medium containing 10% newborn calf serum, 100 IU / mL penicillin, and 100 µg / mL streptomycin at 37°C, 5% CO2, and saturated humidity. The medium was changed and passaged routinely. During the culture process, osteogenic induction medium was used to induce cell osteogenesis, in which 5 mmol / L β-glycerophosphate and 50 µg / mL ascorbic acid were added to the above medium.

[0035] 1.2 Cell proliferation The cells were cultured at 1.5 × 10 3 The cells were inoculated in a 96-well plate at a density of 100 cells / mL. After culturing for 24 h, the cells were treated with three compositions of different concentrations for 24, 48, 72, and 96 h, respectively. The three compositions specifically included: a composite group of Antarctic krill oligopeptides and seaweed calcium, referred to as the "phosphorus·calcium group", in which the mass ratio of Antarctic krill oligopeptides to seaweed calcium (calculated by calcium content) was 2:1; a composite group of Antarctic krill oligopeptides, seaweed calcium and vitamin D, referred to as the "phosphorus·complex group", in which the mass ratio of Antarctic krill oligopeptides, seaweed calcium (calculated by calcium content) to vitamin D was 2:1:5.1×10 -5 ; The seaweed calcium and vitamin D composite group, referred to as the "composite group", the mass ratio of seaweed calcium (calculated by calcium content) to vitamin D was 1: 5.1×10 -5; The blank control group was an equal amount of culture medium without functional components (the functional component solution of the cell experiment was basically prepared with the culture medium); PBS was rinsed once, and 20 μL MTT (5 g / L) and 200 μL serum-free α-MEM culture medium were added to each well. After incubation at 37 °C for 4 h, the supernatant was discarded, and 150 μL dimethyl sulfoxide was added to each well to dissolve the generated crystals, and the cells were shaken at room temperature for 20 min, and then the optical density (OD) value was measured at 490 nm.

[0036] 1.3 Calcium absorption assay The cells were cultured at 1×10 4 The cells were inoculated in 12-well plates at a density of 100 μg / mL and treated with three compositions at different concentrations after culturing for 24 h. After 96 h, the cells were collected, resuspended in 0.3 mL of deionized water, ultrasonically disrupted, vortexed, centrifuged at 2500 rpm for 10 min, and the supernatant was taken to measure the calcium content according to the instructions of the calcium test kit (Nanjing Jiancheng, Nanjing).

[0037] 1.4 Alkaline phosphatase (AKP) activity assay The cells were cultured at 1×10 4 The cells were inoculated in 12-well plates at a density of 1000 cells / well and treated with three compositions at different concentrations after 24 h of culture. The culture medium was collected after 96 h and centrifuged at 1000 rpm for 10 min. The supernatant was collected and the AKP activity of the cells was detected using an AKP kit (Nanjing Jiancheng, Nanjing).

[0038] 1.5 Real-time fluorescence quantitative polymerase chain reaction Total RNA was extracted from cells using Trizol reagent (Novizan, Nanjing). RNA concentration was measured 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 Master Mix (Qingke, Beijing) on ​​a CFX Connectreal-time PCR system (Bio-rad, USA). β-actin transcripts were used as housekeeping genes for data normalization, and the gene primer sequences are shown in the following table: Table 1 RT-qPCR primer sequences

[0039] 1.6 Cell collagen secretion assay Collagen secretion was determined by viscose red staining. 4Cells were seeded in 6-well plates at a density of 100 cells / mL and cultured for 24 h before being treated with three compositions at different concentrations. After the cells were close to confluence, the medium was 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 min, rinsed with PBS three 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.

[0040] 1.7 Cell matrix mineralization assay Alizarin red staining was used to determine cell matrix mineralization. 4 The cells were seeded in 6-well plates at a density of 100 cells / well and treated with three compositions at different concentrations after culturing for 24 h. After the cells were close to confluence, 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 h. The cells were stained with 40 mmol / L Alizarin Red solution (pH=4.2) at room temperature for 20 min, rinsed with PBS five times, and then dissolved and stained with 10% cetylpyridinium chloride. The OD value was measured at 570 nm.

[0041] 1.8 Statistical analysis All experimental data were expressed as "mean ± standard error", and statistical analysis was performed using one-way analysis of variance followed by Student-Newman-Keuls test. P <0.05 indicates a significant difference (indicated by * / #); P When the value is <0.01, the difference is extremely significant (indicated by ** / ##).

[0042] 2 Experimental Results 2.1 Cell proliferation assay results Compared with the control group, at 24 h after treatment, the three compositions at 200, 400 and 600 µg / mL could significantly increase the proliferation of MC3T3-E1 cells ( Figure 1 800 and 1000 µg / mL of the phosphorus·calcium group and phosphorus·complex group could significantly increase cell proliferation after 48 h ( Figure 1 B in the figure); while the 100 and 800 µg / mL compound groups and the 100 µg / mL phosphorus·calcium group and phosphorus·complex group significantly increased cell proliferation after 72 h ( Figure 1CD in). In addition, this proliferation showed dose-dependency and time-dependency within a certain range. On the other hand, the phosphorus·calcium group and the phosphorus·complex group had similar effects on cell proliferation. Among them, the cell proliferation rates of the 200, 400 and 600 μg / mL complex groups reached 119.5%, 123.7% and 120.9% after 96 h of treatment, respectively. In contrast, the cell proliferation rates of the corresponding concentrations of phosphorus·calcium group after 96 h of treatment were 120.4%, 123.5% and 126.2%, respectively, while the cell proliferation rates of the phosphorus·complex group were 122.7%, 126.9% and 126.9%, respectively. The phosphorus·calcium group and the phosphorus·complex group had better effects on promoting the proliferation of MC3T3-E1 cells.

[0043] 2.2 Results of calcium absorption capacity test Three compositions with concentrations of 200 µg / mL and 400 µg / mL were selected for subsequent experiments. Figure 2 As shown in A, after 96 h of treatment with different concentrations of the combination, the calcium content in MC3T3-E1 cells increased. Among them, after treatment with the 400 µg / mL compound group, the cell calcium content was 0.81 mmol / gprot, which was 171.2% higher than that of the control group, slightly lower than that of the phosphorus·calcium group and the phosphorus·compound group at the same concentration, the cell calcium content of which increased by 220.8% and 233.3% respectively compared with the control group. On the other hand, treatment with the three combinations at 400 µg / mL promoted the activity of alkaline phosphatase (AKP) of the cells after 96 h ( Figure 2 B in the figure). qPCR results showed that the compound group, phosphorus·calcium group and phosphorus·complex group could significantly increase the plasma membrane calcium ion ATPase ( PMCA1b ) mRNA expression level, and the improvement effect of 400 µg / mL phosphorus·calcium group and phosphorus·complex group was significantly better than that of the same concentration of complex group ( Figure 2 In addition, calcium binding protein d9k ( CaBP-d9k ) was also significantly upregulated after treatment with 400 µg / mL of the phosphate·calcium group and the phosphate·complex group ( Figure 2 C in.

[0044] 2.3 Results of cell collagen secretion assay Viscose red staining was used to evaluate the collagen secretion of cells. Figure 3 As shown, after being treated with different concentrations of the combination, the collagen secretion of MC3T3-E1 cells increased, and the secretion of collagen fibers (green arrows) and muscle fibers (blue arrows) increased ( Figure 3Among them, the collagen secretion effect of the cells treated with 400 µg / mL compound group increased by 37.1% compared with the control group, while the collagen secretion effect of the cells treated with the same concentration of phosphorus·calcium group and phosphorus·complex group increased by 47.8% and 46.6% respectively compared with the control group ( Figure 3 B in the figure) was better than the compound group. The effects of the phosphorus·calcium group and the phosphorus·complex group on cell collagen secretion were similar.

[0045] 2.4 Cell matrix mineralization assay 40 mmol / L Alizarin red staining was used to determine the mineralization of the cell matrix. Figure 4 As shown. Among them, the cells in the 400 µg / mL compound treatment group were significantly stained with alizarin red, and the OD value of the matrix mineralization quantitative analysis was 1.962±0.063, which was 23.6% higher than that of the control group ( Figure 4 In addition, the matrix mineralization of cells was significantly increased after treatment with the phosphorus·calcium group and the phosphorus·complex group. Specifically, the coloring effect of the 200 and 400 µg / mL phosphorus·calcium groups increased by 17.3% and 27.9%, respectively, compared with the control group, while the coloring effect of the phosphorus·complex group increased by 16.4% and 27.1%, respectively. Figure 4 AB in the group), is better than the composite group.

[0046] 3 Experimental conclusions The study found that at 24 h after treatment with the three compositions, the 200, 400 and 600 µg / mL treatment groups significantly increased the proliferation of MC3T3-E1 cells, and the effect increased with the increase of treatment concentration within a certain range. Over time, the 1000 µg / mL phosphorus·calcium group and phosphorus·complex group significantly increased cell proliferation after 48 h, while the 100 µg / mL three compositions significantly increased the proliferation of MC3T3-E1 cells after 72 h, and the addition of vitamin D had no effect on cell proliferation. Calcium content and enzyme activity detection as well as qPCR results all showed that the Antarctic krill oligopeptide composition could promote calcium absorption of osteoblast precursor cells and facilitate the osteogenesis of precursor cells. The organic component of bone is mainly type I collagen, while viscose red staining can specifically detect type I and type III collagen and can identify collagen-producing cells in situ. This study showed that the 400 µg / mL compound group, phosphorus·calcium group and phosphorus·complex group significantly increased the collagen secretion of MC3T3-E1 cells. In addition, this study used Alizarin red staining and quantitative analysis to detect the matrix mineralization ability of MC3T3-E1 cells. The results showed that the 200 and 400 µg / mL phosphorus·calcium group and phosphorus·complex group significantly increased the ability to induce cell mineralization, and at the same concentration, the composition containing Antarctic krill oligopeptides had a stronger ability to induce cell mineralization than the composite group of seaweed calcium and vitamin D. The results showed that the composition containing Antarctic krill oligopeptides could significantly increase the proliferation ability, calcium absorption ability, collagen secretion and matrix mineralization ability of MC3T3-E1 osteoblast precursor cells.

[0047] 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 similar substitutions 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 calcium-containing Antarctic krill oligopeptide nutritional composition for promoting bone cell growth, characterized in that: The invention is composed of Antarctic krill oligopeptide and calcium compound, wherein the mass ratio of Antarctic krill oligopeptide to calcium compound is 0.5-5:1, and the mass of calcium compound is calculated by the calcium content therein; or the invention is composed of Antarctic krill oligopeptide, calcium compound and vitamin D, wherein the mass ratio of Antarctic krill oligopeptide, calcium compound and vitamin D is 0.5-5:1:2.5×10 -5 -15×10 -5 , the mass of calcium compounds is measured by the calcium content therein; The Antarctic krill oligopeptide is obtained by treating Antarctic krill, Antarctic krill powder or defatted Antarctic krill powder by enzymatic hydrolysis, and the content of peptide segments with a molecular weight of less than 1000 Da in the Antarctic krill oligopeptide is greater than 85%.

2. The calcium-containing Antarctic krill oligopeptide nutritional composition according to claim 1, characterized in that: When the calcium-containing Antarctic krill oligopeptide nutritional composition consists of Antarctic krill oligopeptide and calcium compound, the mass ratio of Antarctic krill oligopeptide to calcium compound is 1-3.5:1; When the calcium-containing Antarctic krill oligopeptide nutritional composition is composed of Antarctic krill oligopeptide, calcium compound and vitamin D, the mass ratio of Antarctic krill oligopeptide, calcium compound and vitamin D is 1-3.5:1:3.5×10 -5 -8×10 -5 ; The calcium compound includes calcium carbonate, milk calcium or seaweed calcium; Antarctic krill oligopeptides are obtained by enzymatically treating defatted Antarctic krill powder. The content of peptides with a molecular weight of less than 1000Da in Antarctic krill oligopeptides is greater than 90%.

3. The calcium-containing Antarctic krill oligopeptide nutritional composition according to claim 2, characterized in that: When the calcium-containing Antarctic krill oligopeptide nutritional composition consists of Antarctic krill oligopeptide and calcium compound, the mass ratio of Antarctic krill oligopeptide to calcium compound is 2:1; When the calcium-containing Antarctic krill oligopeptide nutritional composition is composed of Antarctic krill oligopeptide, calcium compound and vitamin D, the mass ratio of Antarctic krill oligopeptide, calcium compound and vitamin D is 2:1:5.1×10 -5 ; The calcium compound is seaweed calcium.

4. The calcium-containing Antarctic krill oligopeptide nutritional composition according to claim 1, characterized in that: The preparation method of Antarctic krill oligopeptide comprises: (1) dissolving defatted Antarctic krill powder in water to obtain a raw material solution; (2) Adjusting the pH value of the raw material solution, adding protease, and performing enzymolysis at the optimal temperature of the protease; (3) After inactivating the enzyme, filter and cool the supernatant; (4) The solution obtained in step (3) is decolorized, and further solid-liquid separation is performed to obtain a supernatant, and the supernatant is sterilized and then dried to obtain the Antarctic krill oligopeptide.

5. The calcium-containing Antarctic krill oligopeptide nutritional composition according to claim 4, characterized in that: The protease comprises at least one of composite protease, alkaline protease, neutral protease, flavor protease, papain and bromelain.

6. Use of the calcium-containing Antarctic krill oligopeptide nutritional composition according to any one of claims 1 to 5 in the preparation of a product that promotes bone cell growth.

7. The use of the calcium-containing Antarctic krill oligopeptide nutritional composition according to claim 6 in the preparation of a product that promotes bone cell growth, characterized in that: Promoting bone cell growth refers to improving the proliferation and differentiation of MC3T3-E1 osteoblast precursor cells, specifically including at least one of the following methods: (1) Promote the proliferation of MC3T3-E1 cells; (2) Promote calcium absorption in MC3T3-E1 cells; (3) Increase the collagen secretion level of MC3T3-E1 cells; (4) Increase the matrix mineralization capacity of MC3T3-E1 cells.

8. A product for promoting bone cell growth, characterized in that: The calcium-containing Antarctic krill oligopeptide nutritional composition comprises any one of claims 1-5.

9. The product for promoting bone cell growth according to claim 8, characterized in that: Product forms include powder, tablet, capsule, granule or drink.

10. The product for promoting bone cell growth according to claim 8, characterized in that: The product for promoting bone cell growth is a soft capsule or a gel candy, and the content of the calcium-containing Antarctic krill oligopeptide nutritional composition in the soft capsule or the gel candy is 20-50wt%.

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