Preparation method of yak bone collagen peptide, composition for promoting bone development and application thereof

Abstract

The application provides a preparation method of yak bone collagen peptide, a composition for promoting bone development and application thereof, and relates to the technical field of health foods.The preparation method comprises the following steps: after ultrasonic pretreatment of yak bone, supercritical CO2 is used for defatting to obtain a defatted product; the defatted product is mixed with a deodorizing agent to obtain a deodorized product; the deodorizing agent is an aqueous solution of a plant extract; the plant extract is at least one selected from the group consisting of Amomum tsao-ko extract, turmeric extract, green tea extract and momordica grosvenori extract; the deodorized product is subjected to enzymolysis reaction through a fixed immobilized enzyme reactor, the complex enzyme is prolyl endopeptidase, papain and ficin; a crude product is obtained; the crude product is subjected to ultrafiltration through a filter membrane with a molecular weight cut-off of 1kDa and is subjected to macroporous resin adsorption.The yak bone collagen peptide and the composition prepared by the method can significantly improve body length, promote bone development and promote the secretion of growth hormone, and meet the growth needs of children.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of health food, and particularly relates to a preparation method of yak bone collagen peptide, a composition for promoting bone development and application of the composition. BACKGROUND

[0002] Bone is an important structural component of the human body, bearing multiple functions such as supporting the body, protecting organs, coordinating movement, and storing minerals. The period from infancy to childhood is a critical period for bone development, and bone health during this period has a decisive influence on long-term health. First, the rapid increase in bone density, the continuous increase in bone length and hardness, and the formation of bone mass and the deposition of minerals are mainly completed during this period. Adequate nutrition intake and moderate exercise during this period are important factors for promoting bone health development. Second, the healthy development of bone health in childhood can prevent bone diseases such as osteoporosis in adulthood. A large number of studies have shown that the peak bone mass (PBM) reached by the body before and after puberty is an important indicator for predicting the risk of osteoporosis. The formation of PBM is mainly concentrated in the period from childhood to adolescence. Therefore, optimizing bone development during this period helps to increase peak bone mass and thus reduce the risk of bone disease in adulthood.

[0003] Collagen polypeptide is a small molecule protein obtained by hydrolysis of collagen or gelatin with protease, and the molecular weight is generally about 10 Da-10 kDa. Structurally, collagen polypeptide is a polypeptide mixture of short-chain peptides obtained by completely opening the triple helix structure of collagen and degrading the peptide chain. Collagen peptide is generally composed of a small number of amino acids, and the oligopeptide or small peptide product on the market has the effect of easy absorption due to its small molecular weight. In addition, collagen peptide has some special active functions, such as immune regulation, antioxidant, antibacterial, antihypertensive, anti-aging, prevention and treatment of osteoarthritis and osteoporosis, etc. Yak is a unique breed of cattle in alpine areas, and is a rare and precious local semi-wild unique population in China and the world. Compared with general cattle bones, yak bones have high bone density, excellent bone quality, and large amounts of collagen small peptides and high amino acid content, which are very suitable for the extraction of small molecule bone collagen peptides. Yak bone collagen peptide is a high-quality active bone collagen peptide extracted from yak bones naturally raised at an altitude of more than 3000 meters, and is a small molecule collagen peptide that is easily absorbed by the human body.

[0004] Gamma-aminobutyric acid (GABA) is a compound with the chemical formula C4H9NO2, also known as 4-aminobutyric acid, which is an amino acid widely found in vertebrates, plants, and microorganisms. Gamma-aminobutyric acid is an important central nervous system inhibitory neurotransmitter with good water solubility and thermal stability. It has been proven that GABA, as a small molecular weight non-protein amino acid, is safe for consumption and can be used in the production of food such as beverages. Studies have shown that consuming a certain amount of GABA can promote children's growth, so gamma-aminobutyric acid is an important nutrient element for children's growth.

[0005] Chinese patent application CN119753072A discloses a preparation method of yak bone collagen peptide for increasing bone density and yak bone collagen peptide. The preparation method comprises the following steps: providing yak bone powder; mixing the yak bone powder with a phosphate buffer solution, adjusting the pH value to 5-5.5, and then mixing with a complex bacteria for demineralization treatment to obtain a demineralized product; mixing the demineralized product with a complex enzyme for enzymolysis reaction to obtain an enzymolysis product; and heating, centrifugal filtering, and freeze-drying the enzymolysis product to obtain the yak bone collagen peptide; wherein the complex enzyme comprises trypsin, alkaline protease, and flavor protease, and the complex bacteria comprises Lactobacillus plantarum and propionic acid bacteria.

[0006] Chinese patent application CN118830631A discloses a growth-promoting gamma-aminobutyric acid granule, which belongs to the technical field of gamma-aminobutyric acid. In each 100g ingredient ratio: DHA algal oil 0.01-0.1g, xylitol 50-80g, gamma-aminobutyric acid 1-10g, yam powder 0.1-1g, dried tangerine peel powder 0.1-1g, poria cocos powder 0.1-1g, licorice powder 0.1-1g, hawthorn powder 0.1-1g, whey protein powder 1-10g, zinc gluconate 0.01-0.15g, fructooligosaccharide 1-10g, galactooligosaccharide 1-10g, calcium aspartate 1-10g, taurine 0.01-1g, fruit powder 1-20g, and citric acid 0.1-5g. However, the above-mentioned patent does not have related experimental data to prove its effect of promoting children's bone growth, and does not involve the changes of body length, bone development, and growth hormone after taking.

[0007] Therefore, it is the research focus of researchers in the field to develop a preparation method of yak bone collagen peptide and its composition, which has better effects of promoting body length, bone development, and growth hormone secretion. SUMMARY

[0008] The present application provides a preparation method of yak bone collagen peptide and its composition, which has better effects of promoting body length, bone development, and growth hormone secretion.

[0009] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0010] In a first aspect, the present application provides a preparation method of yak bone collagen peptide, comprising the following steps:

[0011] S1: after ultrasonic pretreatment of yak bone, supercritical CO2 is used for defatting to obtain a defatted product;

[0012] S2: the defatted product is mixed with a deodorizing agent to obtain a deodorized product; the deodorizing agent is an aqueous solution of plant extract; the plant extract is at least one selected from Amomum tsao-ko extract, turmeric extract, green tea extract or momordica grosvenori extract;

[0013] S3: the deodorized product is subjected to enzymatic reaction by a fixed immobilized enzyme reactor; the complex enzyme in the fixed immobilized enzyme reactor is prolyl endopeptidase, papain and ficin; a crude product is obtained;

[0014] S4: the crude product is subjected to ultrafiltration by a filter membrane with a molecular weight cut-off of 1 kDa, and then subjected to macroporous resin adsorption to obtain a product.

[0015] Preferably, in step S1, the yak bone needs to be cleaned and broken.

[0016] Preferably, in step S1, the ultrasonic pretreatment is that the yak bone is mixed with water, the solid-liquid ratio is 1 kg: 5-8 L, the power is 450-550 W, and the time is 25-35 min.

[0017] Further preferably, in step S1, the ultrasonic pretreatment is that the yak bone is mixed with water, the solid-liquid ratio is 1 kg: 6 L, the power is 500 W, and the time is 30 min.

[0018] Preferably, in step S1, the supercritical CO2 defatting is that the pressure is 70-75 atm and the temperature is 30-40℃.

[0019] Further preferably, in step S1, the supercritical CO2 defatting is that the pressure is 72 atm and the temperature is 35℃.

[0020] Preferably, in step S2, the plant extract is composed of Amomum tsao-ko extract, turmeric extract, green tea extract and momordica grosvenori extract.

[0021] Preferably, the mass ratio of the Amomum tsao-ko extract, turmeric extract, green tea extract and momordica grosvenori extract is 1-3: 2-3: 2-3: 0.5-1.5.

[0022] Further preferably, the mass ratio of the Amomum tsao-ko extract, turmeric extract, green tea extract and momordica grosvenori extract is 2: 3: 3: 1.

[0023] Preferably, in step S2, the concentration of the plant extract in the aqueous solution of the plant extract is 3-5 wt%.

[0024] Preferably, in step S2, the ratio of the defatted product to the deodorant is 1 kg: 10-15 L. Further preferably, in step S2, the ratio of the defatted product to the deodorant is 1 kg: 12 L.

[0025] Preferably, in step S3, the mass ratio of the prolyl endopeptidase, papain and ficin is 1-3: 1-2: 1-2.

[0026] Further preferably, in step S3, the mass ratio of the prolyl endopeptidase, papain and ficin is 2: 1: 1.

[0027] Preferably, in step S3, the enzymatic reaction occurs through the immobilized complex enzyme reactor, and the enzymatic reaction is:

[0028] The complex enzyme is immobilized by a mesoporous silica carrier, the pH is 6.0-6.5, and the temperature is 35-50°C.

[0029] Further preferably, in step S3, the enzymatic reaction occurs through the immobilized complex enzyme reactor, and the enzymatic reaction is:

[0030] The complex enzyme is immobilized by a mesoporous silica carrier, the pH is 6.0, and the temperature is 40°C.

[0031] Preferably, the mass of the complex enzyme is 0.1-0.5% of the net content of the defatted product.

[0032] Preferably, in step S4, the filter membrane is an inorganic nanofiltration ceramic membrane.

[0033] Preferably, in step S4, the type of the macroporous resin is Sephadex G-15.

[0034] In a second aspect, the present application provides a composition for promoting bone development, comprising the yak bone collagen peptide as described above.

[0035] Preferably, the composition further comprises γ-aminobutyric acid, L-lysine, calcium citrate, a complex mineral, a complex vitamin, and an auxiliary material.

[0036] Preferably, the complex mineral is selected from at least one of magnesium oxide, ferrous fumarate, zinc oxide, or sodium selenite.

[0037] Further preferably, the complex mineral is magnesium oxide, ferrous fumarate, zinc oxide, and sodium selenite.

[0038] Preferably, the complex vitamin is at least one of vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6 or vitamin B 12 .

[0039] Further preferably, the complex vitamin is vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6 and vitamin B 12 .

[0040] Preferably, the adjuvant is arginine and leucine.

[0041] More preferably, the raw materials of the composition include yak bone collagen peptide, gamma-aminobutyric acid, L-lysine, calcium citrate, magnesium oxide, ferrous fumarate, zinc oxide, sodium selenite, vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B 12 , arginine and leucine.

[0042] Preferably, the raw materials include 1.0-5.0 parts of yak bone collagen peptide, 60-200 parts of gamma-aminobutyric acid, 32.0-98.0 parts of L-lysine, 20-55 parts of calcium citrate, 30-53 parts of magnesium oxide, 1-4 parts of ferrous fumarate, 1-4 parts of zinc oxide, 0.003-0.008 parts of sodium selenite, 0.0010-0.0035 parts of vitamin D3, 0.06-0.010 parts of vitamin A, 0.03-0.07 parts of folic acid, 1-3.5 parts of vitamin E, 0.1-0.5 parts of vitamin B1, 0.1-0.5 parts of vitamin B2, 0.1-0.5 parts of vitamin B6, 0.0002-0.0005 parts of vitamin B 12 , 0.1-0.35 parts of arginine and 0.1-0.35 parts of leucine.

[0043] More preferably, the raw materials include 2.0-3.0 parts of yak bone collagen peptide, 65-195 parts of gamma-aminobutyric acid, 32.5-97.5 parts of L-lysine, 25-50 parts of calcium citrate, 31-52.5 parts of magnesium oxide, 2-3 parts of ferrous fumarate, 2-3 parts of zinc oxide, 0.004-0.006 parts of sodium selenite, 0.0015-0.003 parts of vitamin D3, 0.07-0.09 parts of vitamin A, 0.04-0.06 parts of folic acid, 1-3 parts of vitamin E, 0.2-0.4 parts of vitamin B1, 0.2-0.4 parts of vitamin B2, 0.2-0.4 parts of vitamin B6, 0.0003-0.0004 parts of vitamin B 12 , 0.1-0.3 parts of arginine and 0.1-0.3 parts of leucine.

[0044] In a third aspect, the present application provides use of the yak bone collagen peptide or the composition in the preparation of a health food for promoting growth and bone development.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] 1. The yak bone collagen peptide prepared by the specific method can significantly promote growth and bone development; the ultrasonic-super critical CO2 degreasing significantly improves the degreasing rate, facilitating the subsequent extraction of collagen peptide; the specific deodorizing agent can significantly improve the taste; the immobilized enzyme reactor can not only reduce the cost but also improve the enzymolysis efficiency; the specific complex enzyme can significantly improve the yield of small molecular peptides less than 1 kDa, which is helpful for human body absorption; and finally, the specific purification method can significantly improve the quality of the product.

[0047] 2. The yak bone collagen peptide prepared by the specific method is combined with gamma-aminobutyric acid, L-lysine, calcium citrate, complex minerals and complex vitamins, and the zebrafish efficacy experiment can prove that the prepared composition can significantly improve the body length, promote bone development and promote the secretion of growth hormone, and meet the growth needs of children. DETAILED DESCRIPTION

[0048] In order to make the technical means, creative characteristics and purposes achieved by the present application easy to understand, the following specific embodiments are further illustrated. The following embodiments are preferred embodiments of the present application, but not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. It is worth noting that the raw materials used in the present application are ordinary commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings generally understood by those skilled in the art to which the present application belongs.

[0049] Amomum villosum extract: purchased from Shaanxi Junhe Biotechnology Co., Ltd.;

[0050] Turmeric extract: purchased from Hebei Rencan Biotechnology Co., Ltd.;

[0051] Green tea extract: purchased from Shaanxi Dongshengshenghe Biotechnology;

[0052] Momordica grosvenori extract: purchased from Hunan Lvman Biotechnology Co., Ltd.;

[0053] Prolyl endopeptidase: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 500,000 U / g protein;

[0054] Papain: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 400,000 U / g protein;

[0055] Ficin: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 100,000 U / g protein.

[0056] Example 1

[0057] A yak bone collagen peptide, the preparation method is:

[0058] S1: After the yak bone is washed and broken, ultrasonic pretreatment is carried out by mixing with water: the ratio of material to liquid is 1 kg: 6 L, the power is 500 W, and the time is 30 min; then supercritical CO2 is used for degreasing, the pressure is 72 atm, and the temperature is 35℃, to obtain a degreasing product;

[0059] S2: The degreasing product is mixed with a deodorizing agent, the ratio of material to liquid is 1 kg: 12 L, to obtain a deodorizing product;

[0060] The deodorizing agent is a 4wt% aqueous solution of plant extract; the plant extract is a mixture of Amomum tsao-ko extract, turmeric extract, green tea extract and monk fruit extract with a mass ratio of 2:3:3:1;

[0061] S3: The deodorizing product is subjected to enzymatic reaction by a fixed immobilized complex enzyme reactor, the complex enzyme is immobilized by mesoporous silica carrier, the pH is 6.0, and the temperature is 40℃; a crude product is obtained; the mass of the complex enzyme is 0.3% of the net content of the degreasing product.

[0062] The complex enzyme in the fixed immobilized complex enzyme reactor is prolyl endopeptidase, papain and ficin with a mass ratio of 2:1:1;

[0063] S4: The crude product is subjected to ultrafiltration by an inorganic nanofiltration ceramic membrane with a molecular weight cut-off of 1kDa, and then subjected to adsorption by macroporous resin Sephadex G-15, to obtain a product.

[0064] Example 2

[0065] A yak bone collagen peptide, the preparation method is:

[0066] S1: After the yak bone is washed and broken, ultrasonic pretreatment is carried out by mixing with water: the ratio of material to liquid is 1 kg: 5 L, the power is 550 W, and the time is 25 min; then supercritical CO2 is used for degreasing, the pressure is 75 atm, and the temperature is 40℃, to obtain a degreasing product;

[0067] S2: The degreasing product is mixed with a deodorizing agent, the ratio of material to liquid is 1 kg: 10 L, to obtain a deodorizing product;

[0068] The deodorant is a 4wt% aqueous solution of plant extracts; the plant extracts are a mixture of Amomum tsao-ko extract, turmeric extract, green tea extract and Momordica grosvenori extract at a mass ratio of 1:2:2:0.5;

[0069] S3: The deodorized product is subjected to enzymatic reaction by a fixed complex enzyme reactor, the complex enzyme is immobilized by mesoporous silica carrier, the pH is 6.2 and the temperature is 50℃; a crude product is obtained; the mass of the complex enzyme is 0.1% of the net content of the defatted product.

[0070] The complex enzyme in the fixed complex enzyme reactor is prolyl endopeptidase, papain and ficin at a mass ratio of 1:1:1;

[0071] S4: The crude product is subjected to ultrafiltration by an inorganic nanofiltration ceramic membrane with a molecular weight cut-off of 1kDa, and then is subjected to adsorption by a macroporous resin Sephadex G-15, to obtain a product.

[0072] Example 3

[0073] A yak bone collagen peptide, a preparation method thereof is:

[0074] S1: After the yak bone is cleaned and broken, the yak bone is mixed with water for ultrasonic pretreatment: the solid-liquid ratio is 1kg:8L, the power is 450W, and the time is 35min; then the yak bone is defatted by supercritical CO2, the pressure is 70atm, and the temperature is 40℃, to obtain a defatted product;

[0075] S2: The defatted product is mixed with a deodorant, and the solid-liquid ratio is 1kg:15L, to obtain a deodorized product;

[0076] The deodorant is a 4wt% aqueous solution of plant extracts; the plant extracts are a mixture of Amomum tsao-ko extract, turmeric extract, green tea extract and Momordica grosvenori extract at a mass ratio of 3:3:3:1.5;

[0077] S3: The deodorized product is subjected to enzymatic reaction by a fixed complex enzyme reactor, the complex enzyme is immobilized by mesoporous silica carrier, the pH is 6.5 and the temperature is 35℃; a crude product is obtained; the mass of the complex enzyme is 0.5% of the net content of the defatted product.

[0078] The complex enzyme in the fixed complex enzyme reactor is prolyl endopeptidase, papain and ficin at a mass ratio of 3:2:2;

[0079] S4: The crude product is subjected to ultrafiltration by an inorganic nanofiltration ceramic membrane with a molecular weight cut-off of 1kDa, and then is subjected to adsorption by a macroporous resin Sephadex G-15, to obtain a product.

[0080] Example 4

[0081] A composition for promoting bone development, comprising the following raw materials in terms of mass fraction: 2.0 parts of yak bone collagen peptide prepared in Example 1, 65 parts of γ-aminobutyric acid, 32.5 parts of L-lysine, 25 parts of calcium citrate, 31 parts of magnesium oxide, 2 parts of ferrous fumarate, 2 parts of zinc oxide, 0.004 parts of sodium selenite, 0.0015 parts of vitamin D3, 0.07 parts of vitamin A, 0.04 parts of folic acid, 1 part of vitamin E, 0.2 parts of vitamin B1, 0.2 parts of vitamin B2, 0.2 parts of vitamin B6, 0.0003 parts of vitamin B 12 , 0.1 parts of arginine and 0.1 parts of leucine.

[0082] The preparation method is to mix the above raw materials uniformly.

[0083] Example 5

[0084] A composition for promoting bone development, comprising the following raw materials in terms of mass fraction: 3.0 parts of yak bone collagen peptide prepared in Example 2, 195 parts of γ-aminobutyric acid, 97.5 parts of L-lysine, 50 parts of calcium citrate, 52.5 parts of magnesium oxide, 3 parts of ferrous fumarate, 3 parts of zinc oxide, 0.006 parts of sodium selenite, 0.003 parts of vitamin D3, 0.09 parts of vitamin A, 0.06 parts of folic acid, 3 parts of vitamin E, 0.4 parts of vitamin B1, 0.4 parts of vitamin B2, 0.4 parts of vitamin B6, 0.0004 parts of vitamin B 12 , 0.3 parts of arginine and 0.3 parts of leucine.

[0085] The preparation method is to mix the above raw materials uniformly.

[0086] Example 6

[0087] A composition for promoting bone development, comprising the following raw materials in terms of mass fraction: 2.5 parts of yak bone collagen peptide prepared in Example 3, 65 parts of γ-aminobutyric acid, 32.5 parts of L-lysine, 25 parts of calcium citrate, 31 parts of magnesium oxide, 2 parts of ferrous fumarate, 2 parts of zinc oxide, 0.004 parts of sodium selenite, 0.0015 parts of vitamin D3, 0.07 parts of vitamin A, 0.04 parts of folic acid, 1 part of vitamin E, 0.2 parts of vitamin B1, 0.2 parts of vitamin B2, 0.2 parts of vitamin B6, 0.0003 parts of vitamin B 12 , 0.1 parts of arginine and 0.1 parts of leucine.

[0088] The preparation method is to mix the above raw materials uniformly.

[0089] Comparative Example 1

[0090] A yak bone collagen peptide is prepared according to the method described in Example 1 of Chinese Patent CN119753072A.

[0091] Comparative Example 2

[0092] A yak bone collagen peptide, the preparation method is:

[0093] S1: after yak bone cleaning and crushing, mixed with water, the ratio of material to liquid is 1kg:6L, high temperature cooking at 100-125℃ for 4-6h, get the cooking liquid, after cooling to room temperature, the cooking liquid is subjected to oil-water separation, remove the oil, get the defatted product;

[0094] S2: the defatted product is mixed with a deodorizing agent, the ratio of material to liquid is 1kg:12L, get the deodorizing product;

[0095] The deodorizing agent is prepared by mixing citric acid, malic acid, gluconic acid and mint leaf extract at a ratio of 1:1:1:2;

[0096] The remaining operations are consistent with example 1.

[0097] Comparative Example 3

[0098] A yak bone collagen peptide, the preparation method is:

[0099] S1-S2 is consistent with example 1.

[0100] S3: the deodorizing product is subjected to enzymatic reaction by immobilized complex enzyme reactor, the complex enzyme is immobilized by mesoporous silica carrier, the pH is 6.0, the temperature is 40℃; get the crude product; the mass of the complex enzyme is 0.3% of the net content of the defatted product.

[0101] The complex enzyme in the immobilized complex enzyme reactor is alkaline protease, neutral protease and flavor protease with a mass ratio of 2:1:1;

[0102] S4: the crude product is subjected to ultrafiltration by inorganic nanofiltration ceramic membrane with a molecular weight cut-off of 1kDa, and then adsorbed by SP Sephadex C25 through macroporous resin, get the product.

[0103] Comparative Example 4

[0104] A composition for promoting bone development, which is different from example 4 in that the yak bone collagen peptide is prepared by comparative example 1, and the rest is consistent with example 4.

[0105] Comparative Example 5

[0106] A composition for promoting bone development, which is different from example 4 in that the yak bone collagen peptide is prepared by comparative example 2, and the rest is consistent with example 4.

[0107] Comparative Example 6

[0108] A composition for promoting bone development, which differs from Example 4 in that the yak bone collagen peptide is the yak bone collagen peptide prepared in Comparative Example 3, while the rest is the same as Example 4.

[0109] Test Example 1

[0110] The yak bone collagen peptides prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to the following tests, and the test results are shown in Table 1:

[0111] (1) Bone mineral density measurement:

[0112] Refer to the national method for assessing bone density function in health food products.

[0113] Thirty healthy female Sprague-Dawley (SD) rats (60.0±10.0g, 3 weeks old) were purchased from Harbin Medical University, China. The rats were housed in accordance with animal management and usage guidelines, with three rats per cage. Standard cages allowed free access to food and water. The ambient temperature was maintained between 22±2℃ and 45±5℃, and the light-out / darkness cycle was 12 hours. Rats were randomly divided into 6 groups, each group being fed with the collagen peptides of Examples 1-3 and Comparative Examples 1-3 at a dose of 500 mg / kg BW for 9 weeks. During this period, the rats' activity was observed. After the last feeding, the rats were sacrificed, and the left and right femurs were quickly dissected. The femurs were then soaked in physiological saline and stored at -40°C for later testing. For the measurement, the left femur was thawed and the surface moisture was removed. It was then evenly arranged on a muscle simulation plate, and the entire left femur was scanned using a LUNAR Prodigy dual-energy X-ray absorptiometry (DXA) bone densitometer. The average bone mineral density was used to represent the bone mineral density. The data were analyzed using GE Lunar software and are shown in Table 1.

[0114] (2) Determination of bone calcium content:

[0115] The determination method was carried out according to the national standard GB / T5009.92-2003 using a SpectrAA-220FS atomic absorption spectrophotometer, as detailed in Table 1.

[0116] Table 1. Bone mineral density

[0117]

[0118] As shown in Table 1, compared with the comparative example, the yak bone collagen peptides prepared by the specific method in this embodiment of the invention can significantly improve bone density and increase bone calcium content.

[0119] Test Example 2

[0120] Determination of the content and corresponding molecular weight of yak bone collagen peptides

[0121] The detection method for the content of yak bone collagen peptides prepared in Examples 1-3 and Comparative Examples 1-3 was carried out in accordance with the determination method for the content of yak bone collagen peptides described in Appendix B of GB / T 22492-2008 and GB / T 22729-2008; the determination method for the relative molecular weight distribution of the yak bone collagen peptides was carried out in accordance with Appendix A of GB / T 22492-2008, and the test results are shown in Table 2:

[0122] Table 2. Content and corresponding molecular weight of yak bone collagen peptides

[0123]

[0124] As shown in Table 2, compared with the comparative example, the collagen peptide content in the yak bone collagen peptide prepared by the specific method of the present invention is significantly increased, and its molecular weight is concentrated in 500-1000 Da, which is conducive to human absorption.

[0125] Test Example 3

[0126] Taste test:

[0127] Test Method: The products prepared in Examples 1-3 and Comparative Examples 1-3 were used as the research subjects. The irritation, bitterness, and acceptability after trial were scored. In this trial, Examples 1-3 and Comparative Examples 1-3 were divided into 6 groups, with 5 people selected from each group for testing. The participants were healthy individuals aged 18-45 years with no history of smoking or alcohol abuse. The scoring criteria are shown in Table 3. Irritation was scored from 1 to 10, with higher scores indicating stronger irritation; bitterness was scored from 1 to 10, with higher scores indicating stronger bitterness; and acceptability was scored from 1 to 10, with higher scores indicating stronger acceptability. The average score was calculated, and the final trial results are shown in Table 4.

[0128] Table 3. Scoring Criteria

[0129]

[0130] Table 4. Taste Test Results (Scores)

[0131]

[0132] As shown in Table 4, compared with the comparative example, the yak bone collagen peptides prepared by the specific method of the present invention have significantly improved taste, are non-irritating and bitter, and have higher acceptability.

[0133] Test Example 4

[0134] (1) Experimental reagents:

[0135] Test sample: The compositions prepared in Examples 4-6 and Comparative Examples 4-6 were dissolved in water for use;

[0136] Positive control: SWISSE® Calcium & Vitamin D tablets, batch number 33152A, were dissolved in water and diluted to a concentration of 500 μg / mL for use.

[0137] (2) Experimental animals:

[0138] The zebrafish were all raised in fish water at 28°C (water quality: 200 mg of instant sea salt was added to 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; the pH was 6.5-8.5; the hardness was 50-100 mg / L CaCO3), and were provided by the fish breeding center of the Hengtai Biotechnology Innovation Center, with the experimental animal use license number being SYXK (Zhe) 2022-0004. The feeding and management met the requirements of the international AAALAC certification (certification number: 001458), and the IACUC ethical review number was IACUC-2025-12427-01.

[0139] (3) Experimental method:

[0140] 1.1. Maximum detection concentration (MTC) determination

[0141] Thirty wild-type AB strain zebrafish at 3 days post-fertilization (3 dpf) were randomly selected in a beaker, and 30 zebrafish were treated in each beaker (experimental group). The composition prepared in Example 4 was given in water (at a concentration of 2000 μg / mL), and the positive control Swisse “Calcium & Vitamin D” was given at a concentration of 500 μg / mL. A normal control group was also set up, and each beaker had a capacity of 20 mL. After 3 days of treatment at 28°C, the MTC of the sample on normal zebrafish was determined.

[0142] 1.2. Evaluation of growth and development promotion efficacy

[0143] Thirty wild-type AB strain zebrafish at 3 dpf were randomly selected in a beaker, and 30 zebrafish were treated in each beaker (experimental group). The test sample solutions prepared in Examples 4-6 and Comparative Examples 4-6 were given in the experimental groups 1-6 (at a concentration of 2000 μg / mL), and the positive control solution was given in the positive control group (at a concentration of 500 μg / mL). A normal control group was also set up, and water was given, and each beaker had a capacity of 20 mL.

[0144] After treatment at 28℃ for 3 days, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. ImageJ advanced image processing software was used for analysis and data acquisition to analyze the zebrafish body length. Ten zebrafish were randomly selected for alizarin red staining and photographed under a fluorescence microscope. NIS-ElementsD3.20 advanced image processing software was used for analysis and data acquisition to analyze the fluorescence intensity of the zebrafish skull. The remaining 10 zebrafish samples were collected according to the zebrafish growth hormone (GH) kit instructions, and data were acquired using a multi-functional microplate reader to analyze the GH content in the zebrafish. The growth-promoting efficacy of the samples was evaluated based on the statistical analysis results of the above indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0145] (4) Experimental results:

[0146] 2.1 MTC Results

[0147] Under the experimental conditions, the maximum detectable concentration (MTC) results are shown in Table 5. It can be seen that the composition prepared in Example 4 of this invention has an MTC of 2000 μg / mL for promoting growth and development, and has high safety.

[0148] Table 5. Results of MTC for promoting growth and development (n = 30)

[0149]

[0150] 2.2 Evaluation of its efficacy in promoting growth and development

[0151] The evaluation results of the growth-promoting efficacy of the above-mentioned samples are shown in Table 6.

[0152] Table 6. Evaluation results of the efficacy in promoting growth and development (n = 10)

[0153]

[0154] Note: Compared with the normal control group, # p<0.05, ## p<0.01, ### p<0.001; compared with Example 4, & p<0.05, && p<0.01, &&& p<0.001.

[0155] As can be seen from Table 6, the composition prepared in the embodiments of the present invention, under specific raw materials and proportions, achieves a significant improvement in its efficacy in promoting bone development.

[0156] Finally, it should be noted that the above is only to illustrate the technical solutions of the present application, and is not a limitation on the scope of protection of the present application, and simple modifications or equivalent replacements of the technical solutions of the present application by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing a yak bone collagen peptide, characterized by, It comprises the following steps: S1: After ultrasonic pretreatment of yak bone, defatting is carried out by supercritical CO2 to obtain a defatted product; the ultrasonic pretreatment is as follows: yak bone is mixed with water, the solid-liquid ratio is 1 kg: 5-8 L, the power is 450-550 W, and the time is 25-35 min; the supercritical CO2 defatting is as follows: the pressure is 70-75 atm, and the temperature is 30-40℃; S2: The defatted product is mixed with a deodorizing agent to obtain a deodorized product; the deodorizing agent is an aqueous solution of plant extract; the plant extract is composed of Amomum tsao-ko extract, turmeric extract, green tea extract and Siraitia grosvenorii extract with a mass ratio of 1-3: 2-3: 2-3: 0.5-1.5; S3: The deodorized product is subjected to enzymatic reaction by a fixed-bed enzyme reactor, the complex enzyme in the fixed-bed enzyme reactor is prolyl endopeptidase, papain and ficin; a crude product is obtained; the mass ratio of prolyl endopeptidase, papain and ficin is 1-3: 1-2: 1-2; the enzymatic reaction by the fixed-bed enzyme reactor is as follows: The complex enzyme is immobilized by a mesoporous silica carrier, the pH is 6.0-6.5, and the temperature is 35-50℃; S4: The crude product is subjected to ultrafiltration by a filter membrane with a molecular weight cut-off of 1 kDa, and then subjected to adsorption by a macroporous resin to obtain a product.

2. The production method according to claim 1, characterized by, In step S2, the concentration of the plant extract in the aqueous solution of the plant extract is 3-5 wt%.

3. A composition for promoting bone development, characterized by, Yak bone collagen peptide prepared by the preparation method of any one of claims 1-2.

4. The composition of claim 3, wherein, The composition further comprises gamma-aminobutyric acid, L-lysine, calcium citrate, a complex mineral, a complex vitamin, and an auxiliary material; the complex mineral is at least one selected from magnesium oxide, ferrous fumarate, zinc oxide, or sodium selenite; the complex vitamin is at least one selected from vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6, or vitamin B 12 ; and the auxiliary material is arginine and leucine.

5. The composition of claim 4, wherein, The raw materials of the composition include: yak bone collagen peptide, gamma-aminobutyric acid, L-lysine, calcium citrate, magnesium oxide, ferrous fumarate, zinc oxide, sodium selenite, vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B 12 , arginine and leucine.

6. The composition of claim 5, wherein, According to the mass fraction, it comprises the following raw materials: 1.0-5.0 parts of yak bone collagen peptide, 60-200 parts of gamma-aminobutyric acid, 32.0-98.0 parts of L-lysine, 20-55 parts of calcium citrate, 30-53 parts of magnesium oxide, 1-4 parts of ferrous fumarate, 1-4 parts of zinc oxide, 0.003-0.008 parts of sodium selenite, 0.0010-0.0035 parts of vitamin D3, 0.06-0.010 parts of vitamin A, 0.03-0.07 parts of folic acid, 1-3.5 parts of vitamin E, 0.1-0.5 parts of vitamin B1, 0.1-0.5 parts of vitamin B2, 0.1-0.5 parts of vitamin B6, 0.0002-0.0005 parts of vitamin B 12 , 0.1-0.35 parts of arginine, and 0.1-0.35 parts of leucine.

7. The application of yak bone collagen peptide prepared by the preparation method of any one of claims 1-2 in the preparation of health food for promoting growth and / or promoting bone development.

8. The application of the composition of any one of claims 3-6 in the preparation of health food for promoting growth and / or promoting bone development.

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