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

Abstract

The invention provides a preparation method of yak bone collagen peptide, a composition for promoting skeletal development and application of the composition, and relates to the technical field of health food. The preparation method comprises the following steps: carrying out ultrasonic pretreatment on yak bones, and degreasing through supercritical CO2 to obtain a degreased product; mixing the degreased product with a fishy smell removing agent to obtain a fishy smell removing product; the fishy smell removing agent is an aqueous solution of a plant extract; the plant extract is selected from at least one of amomum tsao-ko extract, turmeric extract, green tea extract or momordica grosvenori extract; the fishy smell removal product is subjected to an enzymatic hydrolysis reaction through an immobilized compound enzyme reactor, wherein the compound enzyme is prolyl endopeptidase, papain and ficin; obtaining a crude product; carrying out ultrafiltration by adopting a filter membrane with the molecular weight cut-off of 1kDa, and adsorbing through macroporous resin. The yak bone collagen peptide and the composition prepared by the invention can obviously improve the body length, promote the skeletal development and promote the secretion of growth hormone, and meet the growth requirements of children.

Description

Technical Field

[0001] This invention relates to the field of health food technology, specifically to a method for preparing yak bone collagen peptides, a composition for promoting bone development, and their applications. Background Technology

[0002] Bones are a vital structural component of the human body, bearing multiple functions such as supporting the body, protecting organs, coordinating movement, and storing minerals. Infancy and childhood are critical periods for bone development, and bone health during this time has a decisive impact on an individual's long-term health. Firstly, bone density increases rapidly during this period, with bones continuously increasing in length and hardness; bone formation and mineral deposition primarily occur during this process. Adequate nutrition and moderate exercise during this time are crucial factors in promoting healthy bone development. Secondly, strong bone development during childhood can prevent bone diseases in adulthood, such as osteoporosis. Numerous studies have shown that peak bone mass (PBM) reached around puberty is a key indicator for predicting osteoporosis risk. PBM formation mainly occurs during childhood and adolescence. Therefore, optimizing bone development during this period helps increase peak bone mass, thereby reducing the risk of bone diseases in adulthood.

[0003] Collagen peptides are small-molecule proteins derived from collagen or gelatin through protease hydrolysis, typically with a molecular weight of approximately 10 Da to 10 kDa. Structurally, collagen peptides are a mixture of peptides in which the triple helix structure of collagen is completely opened, and the peptide chains are degraded into short-chain peptides. Collagen peptides are generally composed of a small number of amino acids, and are commonly found in the market as oligopeptides or small peptides due to their small molecular weight and easy absorption. Furthermore, collagen peptides possess several unique bioactive functions, such as immune regulation, antioxidation, antibacterial activity, antihypertensive effects, anti-aging properties, and prevention and treatment of osteoarthritis and osteoporosis. Yaks are a unique breed of cattle native to high-altitude regions and are a rare and precious semi-wild endemic species in my country and the world. Compared to ordinary cattle bones, yak bones have a higher bone density and superior quality, containing a larger amount of small collagen peptides and a higher amino acid content, making them an ideal source for extracting small-molecule bone collagen peptides. Yak bone collagen peptides are high-quality active collagen peptides extracted from the bones of yaks raised at altitudes above 3,000 meters in natural pastures. They are small molecule collagen peptides that are 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. It is an amino acid widely found in vertebrates, plants, and microorganisms. GABA is an important inhibitory neurotransmitter in the central nervous system, possessing good water solubility and thermal stability. As a low-molecular-weight non-protein amino acid, GABA has been proven safe for consumption and can be used in the production of beverages and other foods. Studies have shown that adequate GABA intake can promote children's growth; therefore, GABA is an important nutrient for children's growth.

[0005] Chinese patent application CN119753072A discloses a method for preparing yak bone collagen peptides that increase bone density, and the yak bone collagen peptides themselves. The preparation method includes the following steps: providing yak bone powder; mixing the yak bone powder with a phosphate buffer solution, adjusting the pH to 5-5.5, and then mixing it with a complex of bacteria for demineralization treatment to obtain a demineralized product; mixing the demineralized product with a complex enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolysate; heating, centrifuging, filtering, and freeze-drying the enzymatic hydrolysate to produce yak bone collagen peptides; wherein the complex enzymes include trypsin, alkaline protease, and flavor protease, and the complex bacteria include *Lactobacillus plantarum* and *Propionibacterium acnes*.

[0006] Chinese patent application CN118830631A discloses a growth-promoting γ-aminobutyric acid (GABA) granule, relating to the field of GABA technology. The composition per 100g is as follows: DHA algal oil 0.01-0.1g, xylitol 50-80g, GABA 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, fructooligosaccharides 1-10g, galactooligosaccharides 1-10g, calcium aspartate 1-10g, taurine 0.01-1g, fruit powder 1-20g, and citric acid 0.1-5g. However, none of the aforementioned patents contain relevant experimental data to prove their ability to promote children's bone growth, nor do they involve changes in body length, bone development, or growth hormone levels after consumption.

[0007] Therefore, developing a method for preparing yak bone collagen peptides and its composition, which can better promote body length, bone development and growth hormone secretion, is a research focus of researchers in this field. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for preparing yak bone collagen peptides and a composition thereof, which also has better effects on promoting body length, bone development, and growth hormone secretion.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing yak bone collagen peptides, comprising the following steps: S1: Yak bone is pretreated with ultrasound and then defatted with supercritical CO2 to obtain the defatted product; S2: The defatting product is mixed with a deodorizing agent to obtain a deodorizing product; the deodorizing agent is an aqueous solution of a plant extract; the plant extract is selected from at least one of cardamom extract, turmeric extract, green tea extract or monk fruit extract; S3: The deodorized product undergoes enzymatic hydrolysis in an immobilized complex enzyme reactor, wherein the complex enzymes in the immobilized complex enzyme reactor are prolyl endopeptidase, papain, and fig protease; to obtain the crude product; S4: The crude product is obtained by ultrafiltration using a filter membrane with a molecular weight cutoff of 1 kDa, followed by adsorption through a macroporous resin.

[0010] Preferably, in step S1, the yak bones need to be cleaned and crushed.

[0011] Preferably, in step S1, the ultrasonic pretreatment is as follows: yak bone is mixed with water at a material-to-liquid ratio of 1kg:5-8L, the power is 450-550W, and the time is 25-35min.

[0012] More preferably, in step S1, the ultrasonic pretreatment is as follows: yak bone is mixed with water at a material-to-liquid ratio of 1 kg: 6 L, the power is 500 W, and the time is 30 min.

[0013] Preferably, in step S1, the supercritical CO2 degreasing is performed at a pressure of 70-75 atm and a temperature of 30-40°C. More preferably, in step S1, the supercritical CO2 degreasing is performed at a pressure of 72 atm and a temperature of 35°C.

[0014] Preferably, in step S2, the plant extract consists of cardamom extract, turmeric extract, green tea extract, and monk fruit extract.

[0015] Preferably, the mass ratio of the cardamom extract, turmeric extract, green tea extract, and monk fruit extract is 1-3:2-3:2-3:0.5-1.5.

[0016] More preferably, the mass ratio of the cardamom extract, turmeric extract, green tea extract, and monk fruit extract is 2:3:3:1.

[0017] Preferably, in step S2, the concentration of the plant extract in the aqueous solution of the plant extract is 3-5 wt%.

[0018] Preferably, in step S2, the ratio of the degreasing product to the deodorizing agent is 1 kg: 10-15 L. More preferably, in step S2, the ratio of the degreasing product to the deodorizing agent is 1 kg: 12 L.

[0019] Preferably, in step S3, the mass ratio of prolyl endopeptidase, papain, and fig protease is 1-3:1-2:1-2.

[0020] More preferably, in step S3, the mass ratio of prolyl endopeptidase, papain, and fig protease is 2:1:1.

[0021] Preferably, in step S3, the enzymatic hydrolysis reaction occurring in the immobilized composite enzyme reactor is as follows: The complex enzyme was immobilized on a mesoporous silica carrier at a pH of 6.0-6.5 and a temperature of 35-50℃.

[0022] More preferably, in step S3, the enzymatic hydrolysis reaction occurring in the immobilized composite enzyme reactor is as follows: The complex enzyme was immobilized on a mesoporous silica carrier at pH 6.0 and temperature 40°C.

[0023] Preferably, the mass of the compound enzyme is 0.1-0.5% of the net content of the defatted product.

[0024] Preferably, in step S4, the filter membrane is an inorganic nanofiltration ceramic membrane.

[0025] Preferably, in step S4, the macroporous resin is Sephadex G-15.

[0026] Secondly, the present invention provides a composition for promoting bone development, comprising the yak bone collagen peptides described above.

[0027] Preferably, the composition further includes γ-aminobutyric acid, L-lysine, calcium citrate, complex minerals, complex vitamins, and excipients.

[0028] Preferably, the composite mineral is selected from at least one of magnesium oxide, ferrous fumarate, zinc oxide, or sodium selenite.

[0029] More preferably, the composite mineral is magnesium oxide, ferrous fumarate, zinc oxide, and sodium selenite.

[0030] Preferably, the complex vitamin is selected from vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6, or vitamin B1. 12 At least one of them.

[0031] More preferably, the complex vitamins are vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6, and vitamin B1. 12 .

[0032] Preferably, the excipients are arginine and leucine.

[0033] More preferably, the raw materials of the composition include: yak bone collagen peptides, γ-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, and vitamin B1. 12 Arginine and leucine.

[0034] Preferably, the raw materials, by weight, include the following: 1.0-5.0 parts yak bone collagen peptide, 60-200 parts γ-aminobutyric acid, 32.0-98.0 parts L-lysine, 20-55 parts calcium citrate, 30-53 parts magnesium oxide, 1-4 parts ferrous fumarate, 1-4 parts zinc oxide, 0.003-0.008 parts sodium selenite, 0.0010-0.0035 parts vitamin D3, 0.06-0.010 parts vitamin A, 0.03-0.07 parts folic acid, 1-3.5 parts vitamin E, 0.1-0.5 parts vitamin B1, 0.1-0.5 parts vitamin B2, 0.1-0.5 parts vitamin B6, and 0.0002-0.0005 parts vitamin B1. 12 0.1-0.35 parts arginine and 0.1-0.35 parts leucine.

[0035] More preferably, by weight parts, it includes the following raw materials: 2.0-3.0 parts yak bone collagen peptide, 65-195 parts γ-aminobutyric acid, 32.5-97.5 parts L-lysine, 25-50 parts calcium citrate, 31-52.5 parts magnesium oxide, 2-3 parts ferrous fumarate, 2-3 parts zinc oxide, 0.004-0.006 parts sodium selenite, 0.0015-0.003 parts vitamin D3, 0.07-0.09 parts vitamin A, 0.04-0.06 parts folic acid, 1-3 parts vitamin E, 0.2-0.4 parts vitamin B1, 0.2-0.4 parts vitamin B2, 0.2-0.4 parts vitamin B6, and 0.0003-0.0004 parts vitamin B1. 12 0.1-0.3 parts arginine and 0.1-0.3 parts leucine.

[0036] Thirdly, the present invention provides the application of the above-mentioned yak bone collagen peptides or compositions in the preparation of health foods that promote growth and bone development.

[0037] Compared with the prior art, the present invention has the following beneficial effects: 1. The yak bone collagen peptides prepared by this invention through a specific method can significantly promote growth and bone development; ultrasonic-supercritical CO2 degreasing significantly improves the degreasing rate, facilitating subsequent collagen peptide extraction; a specific deodorizing agent significantly improves the taste; enzymatic hydrolysis using an immobilized composite enzyme reactor not only reduces costs but also improves hydrolysis efficiency; a specific composite enzyme can significantly increase the yield of small molecule peptides less than 1 kDa, which is beneficial for human absorption; finally, a specific purification method significantly improves the quality of the product.

[0038] 2. This invention combines yak bone collagen peptides prepared by a specific method with γ-aminobutyric acid, L-lysine, calcium citrate, compound minerals, and compound vitamins. Through zebrafish efficacy experiments, it can be demonstrated that the composition prepared by this invention can significantly increase body length, promote bone development, and promote the secretion of growth hormone, thus meeting the growth needs of children. Detailed Implementation

[0039] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0040] Cardamom extract: purchased from Shaanxi Junhe Biotechnology Co., Ltd.; Turmeric extract: purchased from Hebei Rencan Biotechnology Co., Ltd.; Green tea extract: purchased from Shaanxi Dongshengshenghe Biotechnology Co., Ltd. Monk fruit extract: purchased from Hunan Greenvine Biotechnology Co., Ltd.; Prolyl endopeptidase: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 500,000 U / g protein; Papain: Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 400,000 U / g protein. Fig protease: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 100,000 U / g protein.

[0041] Example 1 A yak bone collagen peptide, prepared by the following method: S1: Yak bones are cleaned, crushed, and mixed with water for ultrasonic pretreatment: the material-to-liquid ratio is 1kg:6L, the power is 500W, and the time is 30min; then, they are degreased by supercritical CO2 at a pressure of 72atm and a temperature of 35℃ to obtain the degreased product. S2: The degreasing product is mixed with the deodorizing agent at a ratio of 1kg:12L to obtain the deodorizing product; The deodorizing agent is an aqueous solution of 4 wt% plant extracts; the plant extracts are a mixture of cardamom extract, turmeric extract, green tea extract and monk fruit extract in a mass ratio of 2:3:3:1. S3: The deodorized product undergoes enzymatic hydrolysis in an immobilized complex enzyme reactor. The complex enzyme is immobilized on a mesoporous silica carrier at pH 6.0 and temperature 40°C to obtain the crude product. The mass of the complex enzyme is 0.3% of the net content of the degreased product.

[0042] The immobilized complex enzyme reactor contains prolyl endopeptidase, papain, and fig protease in a mass ratio of 2:1:1. S4: The crude product is ultrafiltered using an inorganic nanofiltration ceramic membrane with a molecular weight cutoff of 1 kDa, and then adsorbed through the macroporous resin Sephadex G-15 to obtain the product.

[0043] Example 2 A yak bone collagen peptide, prepared by the following method: S1: Yak bones are cleaned, crushed, and mixed with water for ultrasonic pretreatment: the material-to-liquid ratio is 1kg:5L, the power is 550W, and the time is 25min; then, they are degreased by supercritical CO2 at a pressure of 75atm and a temperature of 40℃ to obtain the degreased product. S2: The degreasing product is mixed with the deodorizing agent at a ratio of 1kg:10L to obtain the deodorizing product; The deodorizing agent is an aqueous solution of 4 wt% plant extracts; the plant extracts are a mixture of cardamom extract, turmeric extract, green tea extract and monk fruit extract in a mass ratio of 1:2:2:0.5. S3: The deodorized product undergoes enzymatic hydrolysis in an immobilized complex enzyme reactor. The complex enzyme is immobilized on a mesoporous silica carrier at pH 6.2 and temperature 50°C to obtain the crude product. The mass of the complex enzyme is 0.1% of the net content of the degreased product.

[0044] The immobilized complex enzyme reactor contains prolyl endopeptidase, papain, and fig protease in a mass ratio of 1:1:1. S4: The crude product is ultrafiltered using an inorganic nanofiltration ceramic membrane with a molecular weight cutoff of 1 kDa, and then adsorbed through the macroporous resin Sephadex G-15 to obtain the product.

[0045] Example 3 A yak bone collagen peptide, prepared by the following method: S1: Yak bones are cleaned, crushed, and mixed with water for ultrasonic pretreatment: the material-to-liquid ratio is 1kg:8L, the power is 450W, and the time is 35min; then, they are degreased by supercritical CO2 at a pressure of 70atm and a temperature of 40℃ to obtain the degreased product. S2: The degreasing product is mixed with the deodorizing agent at a ratio of 1kg:15L to obtain the deodorizing product; The deodorizing agent is an aqueous solution of 4 wt% plant extracts; the plant extracts are a mixture of cardamom extract, turmeric extract, green tea extract and monk fruit extract in a mass ratio of 3:3:3:1.5. S3: The deodorized product undergoes enzymatic hydrolysis in an immobilized complex enzyme reactor. The complex enzyme is immobilized on a mesoporous silica carrier at pH 6.5 and temperature 35°C to obtain the crude product. The mass of the complex enzyme is 0.5% of the net content of the degreased product.

[0046] The immobilized complex enzyme reactor contains prolyl endopeptidase, papain, and fig protease in a mass ratio of 3:2:2. S4: The crude product is ultrafiltered using an inorganic nanofiltration ceramic membrane with a molecular weight cutoff of 1 kDa, and then adsorbed through the macroporous resin Sephadex G-15 to obtain the product.

[0047] Example 4 A composition for promoting bone development, comprising, by weight parts, the following ingredients: 2.0 parts yak bone collagen peptide prepared in Example 1, 65 parts γ-aminobutyric acid, 32.5 parts L-lysine, 25 parts calcium citrate, 31 parts magnesium oxide, 2 parts ferrous fumarate, zinc oxide, 0.004 parts sodium selenite, 0.0015 parts vitamin D3, 0.07 parts vitamin A, 0.04 parts folic acid, 1 part vitamin E, 0.2 parts vitamin B1, 0.2 parts vitamin B2, 0.2 parts vitamin B6, and 0.0003 parts vitamin B1. 12 0.1 parts arginine and 0.1 parts leucine.

[0048] The preparation method is as follows: mix the above raw materials evenly.

[0049] Example 5 A composition for promoting bone development, comprising, by weight parts, the following ingredients: 3.0 parts yak bone collagen peptide prepared in Example 2, 195 parts γ-aminobutyric acid, 97.5 parts L-lysine, 50 parts calcium citrate, 52.5 parts magnesium oxide, 3 parts ferrous fumarate, zinc oxide, 0.006 parts sodium selenite, 0.003 parts vitamin D3, 0.09 parts vitamin A, 0.06 parts folic acid, 3 parts vitamin E, 0.4 parts vitamin B1, 0.4 parts vitamin B2, 0.4 parts vitamin B6, and 0.0004 parts vitamin B1. 12 0.3 parts arginine and 0.3 parts leucine.

[0050] The preparation method is as follows: mix the above raw materials evenly.

[0051] Example 6 A composition for promoting bone development, comprising, by weight parts, the following ingredients: 2.5 parts yak bone collagen peptide prepared in Example 3, 65 parts γ-aminobutyric acid, 32.5 parts L-lysine, 25 parts calcium citrate, 31 parts magnesium oxide, 2 parts ferrous fumarate, zinc oxide, 0.004 parts sodium selenite, 0.0015 parts vitamin D3, 0.07 parts vitamin A, 0.04 parts folic acid, 1 part vitamin E, 0.2 parts vitamin B1, 0.2 parts vitamin B2, 0.2 parts vitamin B6, and 0.0003 parts vitamin B1. 12 0.1 parts arginine and 0.1 parts leucine.

[0052] The preparation method is as follows: mix the above raw materials evenly.

[0053] Comparative Example 1 A yak bone collagen peptide was prepared according to the method described in Example 1 of Chinese Patent CN119753072A.

[0054] Comparative Example 2 A yak bone collagen peptide, prepared by the following method: S1: After cleaning and crushing yak bones, mix them with water at a ratio of 1kg:6L. Cook the mixture at 100-125℃ for 4-6 hours to obtain cooking liquid. After cooling the cooking liquid to room temperature, separate the oil and water in the cooking liquid to remove the grease and obtain a defatted product. S2: The degreasing product is mixed with the deodorizing agent at a ratio of 1kg:12L to obtain the deodorizing product; The deodorizing agent is prepared by mixing citric acid, malic acid, gluconic acid and peppermint leaf extract in a ratio of 1:1:1:2. The remaining operations are the same as in Example 1.

[0055] Comparative Example 3 A yak bone collagen peptide, prepared by the following method: S1-S2 are the same as in Example 1.

[0056] S3: The deodorized product undergoes enzymatic hydrolysis in an immobilized complex enzyme reactor. The complex enzyme is immobilized on a mesoporous silica carrier at pH 6.0 and temperature 40°C to obtain the crude product. The mass of the complex enzyme is 0.3% of the net content of the degreased product.

[0057] The immobilized complex enzyme reactor contains an alkaline protease, a neutral protease, and a flavor protease in a mass ratio of 2:1:1. S4: The crude product is ultrafiltered using an inorganic nanofiltration ceramic membrane with a molecular weight cutoff of 1 kDa, and then adsorbed through a macroporous resin using SP Sephadex C25 to obtain the product.

[0058] Comparative Example 4 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 1, while the rest is the same as Example 4.

[0059] Comparative Example 5 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 2, while the rest is the same as Example 4.

[0060] Comparative Example 6 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.

[0061] Test Example 1 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: (1) Bone mineral density measurement: Refer to the national method for assessing bone density function in health food products.

[0062] 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.

[0063] (2) Determination of bone calcium content: 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.

[0064] Table 1. Bone mineral density

[0065] 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.

[0066] Test Example 2 Determination of the content and corresponding molecular weight of yak bone collagen peptides 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: Table 2. Content and corresponding molecular weight of yak bone collagen peptides

[0067] 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.

[0068] Test Example 3 Taste test: 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. Table 3. Scoring Criteria

[0069] Table 4. Taste Test Results (Scores)

[0070] 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.

[0071] Test Example 4 (1) Experimental reagents: Samples to be tested: The compositions prepared in Examples 4-6 and Comparative Examples 4-6, dissolved in water for later use; Positive control: SWISSE® Calcium Vitamin D tablets, batch number 33152A, dissolved in water and diluted to a concentration of 500 μg / mL, for later use.

[0072] (2) Laboratory animals: Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3), bred and provided by the Aquarium Breeding Center of Huante Bio-Innovation Experimental Center. The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The breeding and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2025-12427-01.

[0073] (3) Experimental methods: 1.1. Determination of Maximum Detectable Concentration (MTC) Wild-type AB strain zebrafish, 3 days post-fertilization (3 dpf), were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). The composition prepared in Example 4 (concentration 2000 μg / mL) was administered in water, along with a positive control of Swisse "Calcium & Vitamin D" at a concentration of 500 μg / mL. A normal control group was also included. Each beaker had a volume of 20 mL. After treatment at 28°C for 3 days, the MTC of the samples relative to normal zebrafish was measured.

[0074] 1.2. Evaluation of its efficacy in promoting growth and development Wild-type AB strain zebrafish with a 3-day pf growth rate were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Experimental groups 1-6 were given the test sample solutions (concentration of 2000 μg / mL) prepared in Examples 4-6 and Comparative Examples 4-6, while the positive control group was given the positive control solution (concentration of 500 μg / mL). A normal control group was also set up, which was given water. The volume of each beaker was 20 mL.

[0075] 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.

[0076] (4) Experimental results: 2.1 MTC Results 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.

[0077] Table 5. Results of MTC for promoting growth and development (n = 30)

[0078] 2.2 Evaluation of its efficacy in promoting growth and development The evaluation results of the growth-promoting efficacy of the above-mentioned samples are shown in Table 6.

[0079] Table 6. Evaluation results of the efficacy in promoting growth and development (n = 10)

[0080] 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.

[0081] 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.

[0082] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing yak bone collagen peptides, characterized in that, Includes the following steps: S1: Yak bone is pretreated with ultrasound and then defatted with supercritical CO2 to obtain the defatted product; S2: The defatting product is mixed with a deodorizing agent to obtain a deodorizing product; the deodorizing agent is an aqueous solution of a plant extract; the plant extract is selected from at least one of cardamom extract, turmeric extract, green tea extract or monk fruit extract; S3: The deodorized product undergoes enzymatic hydrolysis in an immobilized complex enzyme reactor, wherein the complex enzymes in the immobilized complex enzyme reactor are prolyl endopeptidase, papain, and fig protease; to obtain the crude product; S4: The crude product is obtained by ultrafiltration using a filter membrane with a molecular weight cutoff of 1 kDa, followed by adsorption through a macroporous resin.

2. The preparation method according to claim 1, characterized in that, In step S1, the ultrasonic pretreatment is as follows: yak bone is mixed with water at a material-to-liquid ratio of 1kg:5-8L, power is 450-550W, and time is 25-35min; the supercritical CO2 degreasing is performed at a pressure of 70-75atm and a temperature of 30-40℃.

3. The preparation method according to claim 1, characterized in that, In step S2, the plant extract is composed of cardamom extract, turmeric extract, green tea extract and monk fruit extract in a mass ratio of 1-3:2-3:2-3:0.5-1.5; the concentration of the plant extract in the aqueous solution is 3-5 wt%.

4. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of prolyl endopeptidase, papain, and fig protease is 1-3:1-2:1-2.

5. The preparation method according to claim 1, characterized in that, In step S3, the enzymatic hydrolysis reaction occurring in the immobilized composite enzyme reactor specifically involves: The complex enzyme was immobilized on a mesoporous silica carrier at a pH of 6.0-6.5 and a temperature of 35-50℃.

6. A composition for promoting bone development, characterized in that, Includes the yak bone collagen peptides as described in any one of claims 1-5.

7. The composition according to claim 6, characterized in that, The composition further includes γ-aminobutyric acid, L-lysine, calcium citrate, complex minerals, complex vitamins, and excipients; the complex minerals are selected from at least one of magnesium oxide, ferrous fumarate, zinc oxide, or sodium selenite; the complex vitamins are selected from vitamin D3, vitamin A, folic acid, vitamin E, vitamin B1, vitamin B2, vitamin B6, or vitamin B1. 12 At least one of the following: the excipients are arginine and leucine.

8. The composition according to claim 7, characterized in that, The raw materials of the composition include: yak bone collagen peptides, γ-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, and vitamin B1. 12 Arginine and leucine.

9. The composition according to claim 8, characterized in that, By weight, it includes the following raw materials: 1.0-5.0 parts yak bone collagen peptides, 60-200 parts γ-aminobutyric acid, 32.0-98.0 parts L-lysine, 20-55 parts calcium citrate, 30-53 parts magnesium oxide, 1-4 parts ferrous fumarate, 1-4 parts zinc oxide, 0.003-0.008 parts sodium selenite, 0.0010-0.0035 parts vitamin D3, 0.06-0.010 parts vitamin A, 0.03-0.07 parts folic acid, 1-3.5 parts vitamin E, 0.1-0.5 parts vitamin B1, 0.1-0.5 parts vitamin B2, 0.1-0.5 parts vitamin B6, 0.0002-0.0005 parts vitamin B1 12 0.1-0.35 parts arginine and 0.1-0.35 parts leucine.

10. The use of yak bone collagen peptides prepared by the preparation method according to any one of claims 1-5 in the preparation of health foods that promote growth and bone development.

11. The use of the composition according to any one of claims 6-9 in the preparation of health foods that promote growth and bone development.

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