A bovine bone collagen peptide that promotes repair, its enzymatic hydrolysis method, and its application.

Abstract

This invention discloses a bovine bone collagen peptide that promotes bone repair, its enzymatic hydrolysis method, and its application, belonging to the field of collagen peptide extraction technology. Impurities on bovine bones are removed, the bones are crushed, dried, and then ground into bovine bone powder. The bovine bone powder is added to ethyl acetate and stirred to defatted, then filtered and washed. The bone powder is then stirred in hydrochloric acid solution to remove calcium, and after decalcification, filtered and washed. Water is added to the bovine bone powder and mixed well. The temperature is raised to 50-60℃, the pH is adjusted to 7-8, and trypsin is added for enzymatic hydrolysis. After enzymatic hydrolysis, enzyme inactivation is performed. The hydrolysate is cooled to 50-60℃, the pH is adjusted to 6-6.5, serine is added and stirred to dissolve, and neutral protease is added for enzymatic hydrolysis. After enzymatic hydrolysis, enzyme inactivation is performed. The hydrolysate is centrifuged to remove the precipitate, the supernatant is concentrated, and then freeze-dried to obtain the bovine bone collagen peptide that promotes bone repair. The bovine bone collagen peptide prepared by this invention has a molecular weight of 800-2000 Da and a content greater than 85%, which can better promote bone cell proliferation, prevent or treat osteoporosis and other related bone diseases, and improve bone health.

Description

Technical Field

[0001] This invention belongs to the field of collagen peptide extraction technology, specifically relating to a bovine bone collagen peptide that promotes repair, its enzymatic hydrolysis method, and its application. Background Technology

[0002] Bovine bones account for approximately 20% of a cow's body weight and are one of the main byproducts of the beef cattle industry. Bovine bones contain 16-25% protein, primarily type I collagen. Currently, most bovine bones are used to produce bone paste, bone meal, and bone glue, resulting in low added value. However, the collagen in bovine bones can be purified through enzymatic hydrolysis and separation to obtain bovine bone collagen peptides. These peptides are easily absorbed by the human body, quickly replenishing collagen nutrients, promoting the body's own collagen synthesis, effectively increasing skin elasticity, strengthening bones, and reducing osteoporosis. Using bovine bones to prepare collagen peptides is a low-cost, high-protein method that effectively improves the utilization rate of bovine bones.

[0003] Extensive research has been conducted on the extraction of collagen from bovine bones, commonly employing methods such as acid extraction, alkaline extraction, enzymatic extraction, hydrothermal extraction, and hot-press extraction. In practical applications, several methods are often combined to improve the extraction rate of collagen from bovine bones. Commonly used enzymatic hydrolysants for bovine bone collagen include trypsin, alkaline protease, neutral protease, and flavor protease. The hydrolysis efficiency is affected by temperature, protease ratio, and dosage. Utilizing multiple proteases acting simultaneously or sequentially on the protein substrate exhibits a synergistic effect, significantly improving the hydrolysis efficiency.

[0004] Studies have shown that bovine bone collagen peptides play a positive role in alleviating osteoporosis. In vitro experiments have demonstrated that bovine bone collagen peptides can promote the proliferation of human osteoblasts and promote bone repair. High molecular weight bovine bone collagen peptides are not easily absorbed by the body. Enzymatic hydrolysis can degrade bovine bone collagen into smaller peptides. Complex enzymes can better break down collagen chains, releasing more collagen peptides. The higher the degree of hydrolysis by the complex enzyme, the smaller the molecular weight of the resulting collagen peptides. However, small molecular weight collagen peptides are easily degraded and lost, making it difficult for them to exert their bone repair effects. Therefore, controlling the molecular weight of bone collagen peptides is of great significance for maximizing their bone repair effects. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a bovine bone collagen peptide that promotes bone repair, its enzymatic hydrolysis method, and its application. The bovine bone collagen peptides prepared by the enzymatic hydrolysis method of this invention have a molecular weight mostly concentrated between 800 and 2000 Da (accounting for more than 85%), which can better promote osteoblast proliferation and bone repair.

[0006] This invention is achieved through the following technical solution:

[0007] This invention provides an enzymatic hydrolysis method for bovine bone collagen peptides that promote repair, comprising the following steps:

[0008] (1) Remove impurities from the cow bones, crush them to 5~20mm, dry them at 100~120℃, and then grind them into cow bone powder with a particle size of 60~120 mesh.

[0009] (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt it. After defatting, filter and wash.

[0010] (3) After washing the bovine bone powder in step (2), stir it in hydrochloric acid solution to remove calcium. After the decalcification is completed, filter and wash it.

[0011] (4) After washing the beef bone powder in step (3), add water and mix well. Heat the temperature to 50~60℃, adjust the pH value to 7~8, add trypsin for enzymatic hydrolysis, and perform enzyme inactivation treatment after the enzymatic hydrolysis is completed.

[0012] (5) Cool the enzyme hydrolysate treated in step (4) to 50~60℃, adjust the pH to 6~6.5, add 0.2~0.5% serine by weight of bovine bone powder, stir to dissolve, then add neutral protease for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, perform enzyme inactivation treatment;

[0013] (6) After the enzyme inactivation treatment in step (5), the enzymatic hydrolysate is centrifuged to remove the precipitate, and the supernatant is concentrated and freeze-dried to obtain bovine bone collagen peptides that promote repair.

[0014] Furthermore, the stirring and degreasing time in step (2) is 3~6h; in step (3), the hydrochloric acid solution is replaced every 30~60min until the calcium content in the hydrochloric acid solution is less than 0.05wt%.

[0015] Furthermore, the concentration of the hydrochloric acid solution mentioned in step (3) is 2~6wt%.

[0016] Furthermore, in step (4), the amount of trypsin added is 0.3-0.5% of the mass of bovine bone powder, and the enzymatic hydrolysis time is 2-4 hours.

[0017] Furthermore, in step (5), the amount of neutral protease added is 0.2-0.5% of the mass of bovine bone meal, and the enzymatic hydrolysis time is 0.5-2h.

[0018] Furthermore, the enzyme inactivation conditions in steps (4) and (5) are 95~100℃ and 20~60min; the centrifugation conditions in step (6) are 10000~15000r / min and 10~30min.

[0019] Furthermore, in step (4), the ratio of bovine bone powder to water is 1:8~15.

[0020] This invention also provides bovine bone collagen peptides that promote repair, prepared by the enzymatic hydrolysis method described above.

[0021] Furthermore, the proportion of the bovine bone collagen peptides that promote repair having a molecular weight of 1200~2000 Da is greater than 85%.

[0022] In this invention, the above-described bovine bone collagen peptides that promote repair are used in the preparation of drugs for the treatment or prevention of bone-related diseases.

[0023] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0024] (1) The enzymatic hydrolysis method for promoting the repair of bovine bone collagen peptides provided by the present invention uses only two enzymes for enzymatic hydrolysis, and the stepwise enzymatic hydrolysis operation is simple. First, trypsin is used for enzymatic hydrolysis. After enzymatic hydrolysis, the content of peptides with a molecular weight higher than 3000 Da is relatively high. Neutral protease further hydrolyzes it into small molecule peptides, so that most peptides have a molecular weight lower than 3000 Da. By removing calcium from bovine bones and adding serine during the neutral protease hydrolysis process, the neutral protease can inhibit the hydrolysis of bone collagen peptides into peptides with too small a molecular weight to a certain extent.

[0025] (2) The bovine bone collagen peptides prepared by the enzymatic hydrolysis method of the present invention have a molecular weight distribution of 800~2000 Da and a content of more than 85%. Protein peptides in this range can better promote bone cell proliferation, thereby preventing or treating osteoporosis and other related bone diseases and improving bone health. Attached Figure Description

[0026] Figure 1 The graph shows the proliferative activity of the bovine bone collagen peptides prepared in Example 1 at different concentrations on osteoblasts (MC3T3-E1).

[0027] Figure 2 This diagram illustrates the proliferative activity of different bovine bone collagen peptides on osteoblasts (MC3T3-E1). Detailed Implementation

[0028] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0031] In the following examples and comparative examples, the protein content of bovine bone was 21.6 wt%, of which the collagen content was 19.8 wt%; trypsin and neutral protease were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0032] Example 1

[0033] (1) Remove impurities from the cow bones, crush them to 5~20mm, dry them at 105±5℃, grind them after drying and pass them through an 80-mesh sieve to obtain cow bone powder;

[0034] (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt it for 5 hours. After defatting, filter and wash.

[0035] (3) After washing the bovine bone powder in step (2), stir and decalcify it in a 4wt% hydrochloric acid solution. Change the hydrochloric acid solution every 40 minutes until the calcium content in the hydrochloric acid solution is less than 0.05wt%. After decalcification, filter and wash.

[0036] (4) After washing the beef bone powder in step (3), add water with 10 times the weight of beef bone powder and heat it to 55±2℃. Adjust the pH value to 7.2 and add 0.4% of trypsin by weight of beef bone powder for enzymatic hydrolysis. The enzymatic hydrolysis time is 3h. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment at 95℃ for 30min.

[0037] (5) The enzyme hydrolysate treated in step (4) was cooled to 55±2℃, the pH was adjusted to 6.4, 0.3% serine by weight of bovine bone powder was added, stirred and dissolved, and then 0.25% neutral protease by weight of bovine bone powder was added for enzymatic hydrolysis. The hydrolysis time was 1.5h. After the hydrolysis was completed, the enzyme was inactivated at 95℃ for 30min.

[0038] (6) After the enzyme inactivation treatment in step (5), the enzyme hydrolysate is centrifuged (12000r / min, 15min) to remove the precipitate. The supernatant is concentrated under reduced pressure and then freeze-dried to obtain bovine bone collagen peptides that promote repair.

[0039] Example 2

[0040] (1) Remove impurities from the cow bones, crush them to 55±2℃, dry them at 105±5℃, grind them after drying and pass them through an 80-mesh sieve to obtain cow bone powder;

[0041] (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt the mixture for 4 hours. After defatting, filter and wash the mixture.

[0042] (3) After washing the bovine bone powder in step (2), stir and decalcify it in a 3wt% hydrochloric acid solution. Change the hydrochloric acid solution every 30 minutes until the calcium content in the hydrochloric acid solution is less than 0.05wt%. After decalcification is completed, filter and wash.

[0043] (4) After washing the beef bone powder in step (3), add water at 12 times the weight of the beef bone powder and mix well. Heat the mixture to 55±2℃, adjust the pH value to 7.1, add 0.4% of the weight of the beef bone powder with trypsin for enzymatic hydrolysis, and the enzymatic hydrolysis time is 3h. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment at 95℃ for 30min.

[0044] (5) The enzyme hydrolysate treated in step (4) was cooled to 55±2℃, the pH was adjusted to 6.5, 0.2% serine by weight of bovine bone powder was added, stirred and dissolved, and then 0.25% neutral protease by weight of bovine bone powder was added for enzymatic hydrolysis. The hydrolysis time was 1.5h. After the hydrolysis was completed, the enzyme was inactivated at 95℃ for 30min.

[0045] (6) After the enzyme inactivation treatment in step (5), the enzyme hydrolysate is centrifuged (12000r / min, 15min) to remove the precipitate. The supernatant is concentrated under reduced pressure and then freeze-dried to obtain bovine bone collagen peptides that promote repair.

[0046] Example 3

[0047] (1) Remove impurities from the cow bones, crush them to 5~20mm, dry them at 105±5℃, grind them after drying and pass them through an 80-mesh sieve to obtain cow bone powder;

[0048] (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt it for 6 hours. After defatting, filter and wash.

[0049] (3) After washing the bovine bone powder in step (2), stir and decalcify it in a 4wt% hydrochloric acid solution. Change the hydrochloric acid solution every 40 minutes until the calcium content in the hydrochloric acid solution is less than 0.05wt%. After decalcification, filter and wash.

[0050] (4) After washing the beef bone powder in step (3), add water with 12 times the weight of beef bone powder and heat to 55±2℃. Adjust the pH value to 7.2 and add 0.5% of the weight of beef bone powder with trypsin for enzymatic hydrolysis. The enzymatic hydrolysis time is 2h. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment at 95℃ for 30min.

[0051] (5) The enzyme hydrolysate treated in step (4) was cooled to 55±2℃, the pH was adjusted to 6.5, 0.4% serine by weight of bovine bone powder was added, stirred and dissolved, and then 0.3% neutral protease by weight of bovine bone powder was added for enzymatic hydrolysis. The hydrolysis time was 2h, and after the hydrolysis was completed, the enzyme was inactivated at 95℃ for 30min.

[0052] (6) After the enzyme inactivation treatment in step (5), the enzyme hydrolysate is centrifuged (12000r / min, 15min) to remove the precipitate. The supernatant is concentrated under reduced pressure and then freeze-dried to obtain bovine bone collagen peptides that promote repair.

[0053] Example 4

[0054] (1) Remove impurities from the cow bones, crush them to 5~20mm, dry them at 105±5℃, grind them after drying and pass them through an 80-mesh sieve to obtain cow bone powder;

[0055] (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt it for 5 hours. After defatting, filter and wash.

[0056] (3) After washing the bovine bone powder in step (2), stir and decalcify it in a 4wt% hydrochloric acid solution. Change the hydrochloric acid solution every 40 minutes until the calcium content in the hydrochloric acid solution is less than 0.25wt%. After decalcification, filter and wash.

[0057] (4) After washing the beef bone powder in step (3), add water at 15 times the weight of the beef bone powder and mix well. Heat the mixture to 55±2℃, adjust the pH value to 7.4, add 0.3% of the weight of the beef bone powder with trypsin for enzymatic hydrolysis, and the enzymatic hydrolysis time is 4h. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment at 95℃ for 30min.

[0058] (5) The enzyme hydrolysate treated in step (4) was cooled to 55±2℃, the pH was adjusted to 6.2, 0.2% serine by weight of bovine bone powder was added, stirred and dissolved, and then 0.2% neutral protease by weight of bovine bone powder was added for enzymatic hydrolysis. The enzymatic hydrolysis time was 2h, and after the enzymatic hydrolysis was completed, the enzyme was inactivated at 95℃ for 30min.

[0059] (6) After the enzyme inactivation treatment in step (5), the enzyme hydrolysate is centrifuged (12000r / min, 15min) to remove the precipitate. The supernatant is concentrated under reduced pressure and then freeze-dried to obtain bovine bone collagen peptides that promote repair.

[0060] Example 5

[0061] (1) Remove impurities from the cow bones, crush them to 5~20mm, dry them at 105±5℃, grind them after drying and pass them through an 80-mesh sieve to obtain cow bone powder;

[0062] (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt the mixture for 4 hours. After defatting, filter and wash the mixture.

[0063] (3) After washing the bovine bone powder in step (2), stir and decalcify it in a 4wt% hydrochloric acid solution. Change the hydrochloric acid solution every 40 minutes until the calcium content in the hydrochloric acid solution is less than 0.05wt%. After decalcification, filter and wash.

[0064] (4) After washing the beef bone powder in step (3), add water with 10 times the weight of beef bone powder and heat to 55±2℃. Adjust the pH value to 7.4 and add 0.4% of trypsin by weight of beef bone powder for enzymatic hydrolysis. The enzymatic hydrolysis time is 3h. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment at 95℃ for 30min.

[0065] (5) The enzyme hydrolysate treated in step (4) was cooled to 55±2℃, the pH was adjusted to 6.4, 0.5% serine by weight of bovine bone meal was added, stirred and dissolved, and then 0.4% neutral protease by weight of bovine bone meal was added for enzymatic hydrolysis. The hydrolysis time was 1h, and after the hydrolysis was completed, the enzyme was inactivated at 95℃ for 30min.

[0066] (6) After the enzyme inactivation treatment in step (5), the enzyme hydrolysate is centrifuged (12000r / min, 15min) to remove the precipitate. The supernatant is concentrated under reduced pressure and then freeze-dried to obtain bovine bone collagen peptides that promote repair.

[0067] Comparative Example 1

[0068] Compared with Example 1, no serine was added in step (5) of Comparative Example 1, and the rest of the operation was the same as in Example 1, to obtain bovine bone collagen peptides that promote repair.

[0069] Comparative Example 2

[0070] (1) Remove impurities from the cow bones, crush them to 5~20mm, dry them at 105±5℃, grind them after drying and pass them through an 80-mesh sieve to obtain cow bone powder;

[0071] (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt it for 5 hours. After defatting, filter and wash.

[0072] (3) After washing the bovine bone powder in step (2), stir and decalcify it in a 4wt% hydrochloric acid solution. Change the hydrochloric acid solution every 40 minutes until the calcium content in the hydrochloric acid solution is less than 0.05wt%. After decalcification, filter and wash.

[0073] (4) After washing the beef bone powder in step (3), add water at 10 times the weight of the beef bone powder and mix well. Heat to 55±2℃, adjust the pH value to 6.8, add 0.4% of the weight of the beef bone powder with trypsin for enzymatic hydrolysis and 0.25% of the weight of the beef bone powder with neutral protease. The enzymatic hydrolysis time is 3h. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment at 95℃ for 30min.

[0074] (5) After the enzyme inactivation treatment in step (4), the enzyme hydrolysate is centrifuged (12000r / min, 15min) to remove the precipitate. The supernatant is concentrated under reduced pressure and then freeze-dried to obtain bovine bone collagen peptides that promote repair.

[0075] Comparative Example 3

[0076] Compared with Example 1, the order of steps (4) and (5) in Comparative Example 3 was swapped, and the remaining operations were the same as in Example 1, to obtain bovine bone collagen peptides that promote repair.

[0077] Comparative Example 4

[0078] Compared with Example 1, Comparative Example 4 omits step (5), and the remaining operations are the same as in Example 1, to obtain bovine bone collagen peptides that promote repair.

[0079] Comparative Example 5

[0080] Compared with Example 1, step (4) was omitted in Comparative Example 5, and the remaining operations were the same as in Example 1, to obtain bovine bone collagen peptides that promote repair.

[0081] Comparative Example 6

[0082] Compared with Example 1, Comparative Example 5 omits step (3), and the remaining operations are the same as in Example 1, to obtain bovine bone collagen peptides that promote repair.

[0083] Characterization and activity testing of bovine bone collagen peptides that promote repair

[0084] Molecular weight distribution test

[0085] The molecular weight distribution of the bovine bone collagen peptides that promote repair prepared in Examples 1-5 and Comparative Examples 1-6 was analyzed, and the results are shown in Table 1 below:

[0086] Table 1. Molecular weight distribution of bovine bone collagen peptides

[0087]

[0088] Assay of the osteocyte proliferation activity of bovine bone collagen peptides that promote repair:

[0089] (1) Determination of the activity of different concentrations of bovine bone collagen peptides on osteoblast proliferation

[0090] MC3T3-E1 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare 3×10⁻⁶ cells. 4 Cells were seeded in 96-well plates with a cell suspension of 10 cells / mL and cultured for 24 h. MC3T3-E1 cells were then incubated for 20 h with different concentrations of bovine bone collagen peptide solutions (0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL). MTT solution was added to each well, and incubation continued for 4 h. The culture was then terminated, and the culture supernatant was carefully aspirated from each well. 150 μL of DMSO was added to each well, and the plates were shaken for 10 minutes. The absorbance of each well was measured at 570 nm using an ELISA reader, and the cell proliferation rate was calculated. The results are shown below. Figure 1 As shown, by Figure 1 It can be seen that the bovine bone collagen peptides prepared by the enzymatic hydrolysis method of the present invention, when used to treat MC3T3-E1 cells, resulted in good cell growth and proliferation promotion, exhibiting a certain concentration dependence.

[0091] (2) Determination of the activity of bovine bone collagen peptides on osteoblast proliferation in different embodiments and comparative examples

[0092] MC3T3-E1 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare 3×10⁻⁶ cells. 4 Cells were seeded in a 96-well plate with a cell suspension of 10 cells / mL and cultured for 24 h. After incubating MC3T3-E1 cells for 20 h with bovine bone collagen peptide solutions (80 μg / mL) prepared in Examples 1-5 and Comparative Examples 1-5, MTT solution was added to each well, and incubation continued for 4 h. The culture was then terminated, and the culture supernatant was carefully aspirated from the wells. 150 μL of DMSO was added to each well, and the plate was shaken for 10 minutes. The absorbance of each well was measured at 570 nm using an ELISA reader, and the cell proliferation rate was calculated. The results are as follows: Figure 2 As shown;

[0093] Depend on Figure 2It is known that molecular weight distribution affects the proliferative activity of bovine bone collagen peptides on MC3T3-E1 cells. Bovine bone collagen peptides with a molecular weight distribution of 800-2000 Da can better promote osteoblast proliferation. This invention uses a combination of trypsin and neutral protease for stepwise enzymatic hydrolysis, which can cleave collagen peptides at different positions, facilitating the extraction of small peptide fragments. Using only one enzyme results in low hydrolysis levels and a high content of large peptide fragments, which is detrimental to its bone repair function. Adding serine during neutral protease hydrolysis inhibits the hydrolytic activity of neutral protease to some extent, reducing the generation of excessively small molecular weight collagen peptides. The different cleavage positions of trypsin and neutral protease, and the hydrolysis sequence, affect the molecular weight distribution of collagen peptides, thus causing differences in their proliferative activity on MC3T3-E1 cells. Bovine bone contains a large amount of calcium, which activates neutral protease; therefore, calcium is removed from the bovine bone before enzymatic hydrolysis to avoid the generation of excessive small peptides. The bovine bone collagen peptide prepared by this invention has a repair function and can promote bone cell proliferation, thereby preventing or treating osteoporosis and other related bone diseases and improving bone health.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for enzymatic hydrolysis of bovine bone collagen peptides that promote repair, characterized in that, Includes the following steps: (1) Remove impurities from the cow bones, crush them to 5~20mm, dry them at 100~120℃, and then grind them into cow bone powder with a particle size of 60~120 mesh. (2) Add the beef bone powder from step (1) to ethyl acetate and stir to defatt it. After defatting, filter and wash. (3) After washing the bovine bone powder in step (2), stir it in hydrochloric acid solution to remove calcium. After the decalcification is completed, filter and wash it. (4) After washing the beef bone powder in step (3), add water and mix well. Heat the temperature to 50~60℃, adjust the pH value to 7~8, add trypsin for enzymatic hydrolysis, and perform enzyme inactivation treatment after the enzymatic hydrolysis is completed. (5) Cool the enzyme hydrolysate treated in step (4) to 50~60℃, adjust the pH to 6~6.5, add 0.2~0.5% serine by weight of bovine bone powder, stir to dissolve, then add neutral protease for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, perform enzyme inactivation treatment; (6) After the enzyme inactivation treatment in step (5), the enzyme hydrolysate is centrifuged to remove the precipitate, the supernatant is concentrated and then freeze-dried to obtain bovine bone collagen peptides that promote repair. In step (4), the amount of trypsin added is 0.3-0.5% of the mass of bovine bone meal, and the enzymatic hydrolysis time is 2-4 hours. In step (5), the amount of neutral protease added is 0.2-0.5% of the mass of bovine bone meal, and the enzymatic hydrolysis time is 0.5-2 hours.

2. The enzymatic hydrolysis method for promoting bovine bone collagen peptides according to claim 1, characterized in that, In step (2), the stirring and degreasing time is 3-6 hours; in step (3), the hydrochloric acid solution is replaced every 30-60 minutes until the calcium content in the hydrochloric acid solution is less than 0.05 wt%.

3. The enzymatic hydrolysis method for promoting bovine bone collagen peptides for repair according to claim 1, characterized in that, The concentration of the hydrochloric acid solution mentioned in step (3) is 2~6wt%.

4. The enzymatic hydrolysis method for promoting bovine bone collagen peptides for repair according to claim 1, characterized in that, The enzyme inactivation conditions in steps (4) and (5) are 95~100℃ and 20~60min; the centrifugation conditions in step (6) are 10000~15000r / min and 10~30min.

5. The enzymatic hydrolysis method for promoting bovine bone collagen peptides according to claim 1, characterized in that, In step (4), the ratio of beef bone powder to water is 1:8~15.

6. A bovine bone collagen peptide that promotes repair, prepared by the enzymatic hydrolysis method according to any one of claims 1 to 5.

7. The bovine bone collagen peptide for promoting repair according to claim 6, characterized in that, The bovine bone collagen peptides that promote repair account for more than 85% of the total, with a molecular weight of 1200-2000 Da.

8. The use of the bovine bone collagen peptide of claim 6 or 7 that promotes repair in the preparation of a medicament for treating or preventing osteoporosis.

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