Bovine collagen peptide, enzymolysis method and application of bovine collagen peptide in field of preparation of bone repair products

Abstract

The invention belongs to the technical field of collagen extraction, and particularly relates to bovine collagen peptide, an enzymolysis method and application in the field of bone repair product preparation. The invention provides an enzymolysis method of collagen peptide in cowhide, which comprises the following steps: degreasing fresh cowhide, removing impurities, fully crushing, firstly adding a deep eutectic solvent to assist extraction, then introducing a microbial fermentation enzyme to carry out enzymolysis, and carrying out ultrafiltration on an enzymolysis product to retain the part with the molecular weight of less than 3kDa, thereby obtaining the collagen peptide in the cowhide. Through verification, the collagen peptide prepared by the extraction process has good antioxidant activity and ACE inhibitory activity, has stem cell osteogenic differentiation induction activity, and is expected to be applied to bone repair activity research and product development.

Description

Technical Field

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

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Collagen is the most abundant structural protein in animals, widely distributed in skin, bones, and various connective tissues. It accounts for approximately 25%-30% of the body's total protein. Studies by Lin Lin et al. have found that collagen peptides can not only scavenge free radicals but also exert antioxidant effects by increasing the activity of glutathione peroxidase and superoxide dismutase. The effect is optimal when the molecular weight is less than 2 kDa. However, lower molecular weight and higher amino acid content do not necessarily mean stronger antioxidant activity; antioxidant activity is also related to peptide sequence, enzyme cleavage sites, and structure. With advancements in biotechnology, collagen extraction and high-value utilization have become new growth areas. Collagen is widely used in the food, pharmaceutical, and cosmetic industries, and can be used to prepare medical excipients, artificial skin, drug carriers, functional products, absorbable medical devices, health products, and beverages. Currently, the aforementioned high-value collagen products mainly come from fish skin, which has the advantages of high purity and simple extraction processes, but the disadvantages are high raw material costs and difficulty in obtaining them.

[0004] As a major consumer of beef, my country also ranks among the world's leading producers of cowhide. Currently, over 60% of the annual cowhide production goes to the traditional leather manufacturing industry, with only 20% used for collagen processing. Although collagen processing has high economic value, extracting collagen from cowhide presents numerous challenges. For example, cowhide contains a large number of pyridinoline cross-links and disulfide bonds, forming a dense cross-linked network. Introducing acids or alkalis can hydrolyze these cross-links, significantly reducing the mechanical strength of collagen and causing its degradation. Furthermore, the resulting wastewater is corrosive and requires strict treatment before discharge. In contrast, enzymatic extraction is gentler, but it requires thorough removal of the cross-linked structures; otherwise, the enzymatic efficiency will be significantly reduced. Additionally, the cost of biological enzymes is relatively high, and they are difficult to recycle.

[0005] Existing research has confirmed that bovine hide collagen has a much higher proline content than collagen from other animal sources, making it a high-density collagen raw material with better physiological activity. The School of Bioengineering at East China University of Science and Technology previously reported the isolation of a novel ACE-inhibiting peptide from bovine hide collagen. In light of the above research status, this invention proposes that bovine hide, as a high-density collagen extraction raw material, can be used to extract highly active collagen peptides, which would improve economic efficiency. However, existing acid / alkali / enzyme extraction processes have low extraction efficiency, leading to an increase in the amount of extracted components. Therefore, providing a cost-effective extraction method that can obtain highly active components will have promising application prospects. Summary of the Invention

[0006] In a first aspect, the present invention provides an enzymatic hydrolysis method for collagen peptides in bovine hide, comprising the following steps: defatting and removing impurities from fresh bovine hide and then fully crushing it; extracting it with a eutectic solvent and retaining the supernatant, which is denoted as crude protein solution; adding microbial fermentation enzymes for enzymatic hydrolysis; and ultrafiltration of the hydrolysis product to retain the portion with a molecular weight less than 3 kDa, thereby obtaining the product. The aforementioned eutectic solvent is obtained by mixing choline chloride, glycerol, and citric acid in a molar ratio of 0.8~1.2:0.8~1.2:0.1~0.3. The aforementioned microbial fermentation enzymes were obtained from Bacillus coagulans through gelatin-induced fermentation.

[0007] In previous studies of this invention, collagen peptides with good ACE inhibitory activity were prepared by using tilapia skin as raw material and undergoing enzymatic hydrolysis via gelatin-induced fermentation using microbial fermentation enzymes. This invention designs to extract highly active collagen peptide products from bovine hide. However, due to the denser texture of bovine hide, the extraction efficiency using only the aforementioned microbial fermentation enzyme hydrolysis is too low to meet production requirements.

[0008] Eutectic solvents are non-volatile ionic liquids that are cost-effective, safe, and stable. They not only possess good biocompatibility but also allow for the targeted optimization of collagen peptides of specific molecular weights by adjusting the component ratios. Therefore, this invention proposes the use of eutectic solvents for the auxiliary extraction of collagen. By optimizing the component ratios, this invention provides a ternary eutectic solvent system of choline chloride-glycerol-citric acid, which not only effectively improves the extraction efficiency of collagen but also produces collagen peptide products with good antioxidant and ACE inhibitory activities. Furthermore, this invention discovers that the aforementioned eutectic solvent not only facilitates the release of collagen from the dense cross-linked structure of bovine hide but also helps improve the enzymatic hydrolysis efficiency of microbial fermentation enzymes. Therefore, the aforementioned microbial fermentation enzyme hydrolysis stage also uses a eutectic solvent as the reaction environment. Finally, the eutectic solvent is separated from the collagen product by dialysis, and the separated eutectic solvent can be reused after evaporation to remove moisture.

[0009] Furthermore, regarding the preparation method provided in the first aspect, the present invention also provides the following preferred technical solutions: The above-mentioned degreasing of fresh cowhide involves soaking in an organic reagent, such as acetone, anhydrous ethanol, or n-hexane. Impurity removal includes removing excess hair, tissue, and protein contaminants from the surface of the cowhide. Hair and tissue can be removed mechanically, while protein contaminants can be removed by soaking. Further, the specific degreasing and impurity removal process is as follows: the cowhide is soaked in an organic reagent at a material-to-liquid ratio of 1:4~6, then rinsed. Next, a sodium chloride solution (5%~7%) is added at a material-to-liquid ratio of 1:2~4 and continuously stirred for 11~14 hours to remove protein contaminants.

[0010] The preparation method of the above-mentioned eutectic solvent is as follows: choline chloride, glycerol and citric acid are mixed in the above molar ratio and stirred at 55~65℃ for 2~4h to obtain a transparent homogeneous liquid; the fully crushed cowhide is added to the eutectic solvent for extraction to obtain a crude protein solution, and the solid-liquid ratio of cowhide to eutectic solvent is 1:15~18; the above extraction method has a wide applicable temperature range, especially at lower temperatures, which helps to maintain the active structure of collagen. The preferred temperature range is 4℃~60℃, and the extraction time is 10min~6h.

[0011] Furthermore, in one embodiment of the present invention, the cowhide is added to a eutectic solvent and extracted by stirring at 20-37°C for 4-6 hours. In another embodiment, the cowhide is added to a eutectic solvent and extracted by microwave-assisted extraction at 25-37°C for 15 minutes with a microwave power of 350-450W.

[0012] The above-mentioned microbial fermentation enzyme is prepared as follows: Bacillus coagulans seed culture is inoculated into a fermenter at an inoculation rate of 5-7%, with a stirring speed of 220-270 r / min, an aeration rate of 2.5-3.5 L / min, a fermentation temperature of 30-38℃, and a fermentation time of 30-36 h. The fermentation medium includes the following components: 2.0% glucose, 0.5% peptone, 0.4-0.6% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, with the remainder being water, and a pH of 7.5. The fermentation product is then subjected to salting out to obtain the microbial fermentation enzyme. In a preferred embodiment of the present invention, the above-mentioned microbial fermentation enzyme also contains an activator, wherein the activator is CaCl2. 2+ The concentration is 4~6mM, Ca 2+ The preferred source is inorganic salts, such as calcium sulfate and calcium chloride.

[0013] The parameters for the above enzymatic hydrolysis are as follows: the amount of microbial fermentation enzyme added is 1000~1500U / mL, the pH of the reaction system is adjusted to 7.5~8, the enzymatic hydrolysis is carried out at 35~37℃ for 4~6h, and the enzymatic hydrolysis reaction is terminated by high temperature heating.

[0014] In a second aspect, bovine collagen peptides prepared by the method described in the first aspect are provided.

[0015] Thirdly, the application of the bovine collagen peptides described in the second aspect in the preparation of bone repair products is provided.

[0016] This invention utilizes the aforementioned collagen peptides to prepare a gel-type cell culture medium. During the research process, it was discovered that the bovine collagen peptides can induce osteogenic differentiation of spinal cord mesenchymal stem cells. Based on this activity, the bovine collagen peptides hold promise for application in the development of bone repair products.

[0017] The bone repair products include, but are not limited to, applications in food, beverages, health products, drugs, or cosmetics.

[0018] Examples of the food products include biscuits, pastries, powders, and granules; examples of the beverages include compound fruit juices, concentrated liquids, carbonated drinks, or tea drinks; examples of the health products include tablets, capsules, oral liquids, powders, and granules; and the dosage forms of the cosmetics include aqueous solutions, emulsions, creams, films, powders, oils, or suspensions. The above examples are not intended to be specific limitations; any product form acceptable in the fields of food, beverages, health products, and cosmetics is within the scope of protection of this invention. Possible dosage forms for the drugs include capsules, tablets, oral liquids, beverages, powders, granules, and gels.

[0019] Furthermore, the bone repair products can be applied to diseases or symptoms including but not limited to fracture repair, bone defect repair, osteoporosis, spinal fusion, osteonecrosis, periodontal defects, dental implants, craniofacial bone repair, or other bone metabolic diseases.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1. Currently, the extraction of collagen from bovine hide still mainly relies on traditional acid / alkali / enzymatic hydrolysis methods, which have low extraction efficiency. This invention proposes to introduce a eutectic reagent to optimize the pretreatment process of bovine hide, effectively eliminating the limitation of the dense cross-linked structure in bovine hide on collagen extraction and significantly improving the yield.

[0021] 2. This invention also found that eutectic solvents, as solvents for enzymatic hydrolysis, can effectively improve the efficiency of enzymatic hydrolysis. On the one hand, hydrogen bond donors in eutectic solvents encapsulate enzyme molecules through a hydrogen bond network, effectively maintaining the integrity of the enzyme's active site and inhibiting enzyme folding. On the other hand, eutectic solvents have good solubility for the substrate (collagen), making it easier for the enzyme to reach the substrate's action site. Eutectic solvents can be separated from the product by dialysis, and after concentration to remove excess water, they can be reused for the pretreatment of bovine hides, effectively reducing reagent costs in the pretreatment stage.

[0022] 3. In the applicant's prior research, a highly efficient microbial fermentation enzyme for decomposing collagen was obtained by inducing Bacillus licheniformis to coagulate with gelatin, and the enzymatic hydrolysate exhibited good ACE inhibitory activity. This invention applies this enzyme to the enzymatic hydrolysis of collagen in bovine hide, and the resulting product still exhibits good ACE inhibitory activity, making it a high-value peptide product. Furthermore, this invention discovers that introducing an appropriate amount of calcium ions into the aforementioned microbial fermentation enzyme can further improve the enzymatic hydrolysis effect.

[0023] 4. The present invention also provides the application of the above-mentioned collagen peptide products in the field of bone repair. It has been verified that the above-mentioned collagen peptides can effectively promote the expression of osteogenic-related markers in spinal cord mesenchymal stem cells and promote cell mineralization. Attached Figure Description

[0024] Figure 1 The expression of OPN and Runx2 in collagen peptide-induced cultured stem cells after 7 days in Example 1; in, Figure 1 Image A shows the bands of OPN and Runx2 expression detected by Western blot. Figure 1 B represents the quantitative results of OPN expression; Figure 1 C represents the quantitative results of Runx2 expression.

[0025] Figure 2 The results of ALP activity detection were obtained from collagen peptide-induced stem cell culture for 7 days in Example 1. in, Figure 2 Image A shows a staining photograph of cells in a culture dish; Figure 2 B represents the quantitative results of ALP activity. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] In the context of this specification, the word "comprising" is considered to mean "especially including". It should not be interpreted as "consisting of only".

[0029] As described in the background section, the extraction of active collagen from bovine hide usually requires the introduction of acid or alkali. In order to solve the above-mentioned technical problems, this invention proposes an enzymatic hydrolysis method for collagen peptides from bovine hide.

[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 and comparative examples.

[0031] Example 1 This embodiment provides an enzymatic hydrolysis method for collagen peptides in bovine hide, comprising the following steps: (1) Pretreatment: Take fresh cowhide, remove surface hair and residual tissue, wash it clean, and cut the cowhide into small pieces of 3×3cm using a low temperature (4℃) cutting method. Add acetone at a material-to-liquid ratio of 1:5 and soak for 4 hours. Take it out and rinse repeatedly until there is no odor. Add 5% NaCl solution at a material-to-liquid ratio of 1:3 and stir continuously for 12 hours to remove impurities and proteins. Take out the cowhide, chop it thoroughly, and dry it for later use.

[0032] (2) Preparation of eutectic solvent: choline chloride, glycerol and citric acid are mixed in a molar ratio of 1:1:0.2 and placed in a flask. The mixture is stirred at 60°C for 3 hours until the components in the flask are converted into a transparent homogeneous liquid.

[0033] (3) The cowhide treated in step (1) was added to the eutectic solvent prepared in step (2) at a solid-liquid ratio of 1:17. The mixed reaction system was extracted by microwave-assisted extraction with a microwave power of 400W, a reaction temperature of 37℃, and a reaction time of 15min. After the temperature of the reaction system dropped to room temperature, it was centrifuged at 8000r / min for 15min, and the supernatant was retained and recorded as crude protein solution.

[0034] (4) Inducing microbial fermentation to obtain enzymatic hydrolysate: Bacillus coagulans was inoculated into seed culture medium (1% beef extract, 2% peptone, 0.5% NaCl, balance water, pH 7.5), fermented at 38℃, and cultured in shake flasks for 24 h. The culture was then transferred to a fermenter (2.0% glucose, 0.5% peptone, 0.4% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, balance water, pH 7.5), with an inoculum size of 6%. The stirring speed was 250 r / min, the aeration rate was 3 L / min, the fermentation temperature was 30℃, and the fermentation time was 36 h. After fermentation, the fermentation broth was centrifuged at 6500 r / min and 4℃ for 15 min, and the supernatant was retained. While stirring, add ammonium sulfate to the supernatant until saturated. Let stand overnight at 4°C to allow collagenase in the fermentation product to fully precipitate. Centrifuge and retain the precipitate. Transfer it to 0.2M phosphate buffer (pH 7.8) for reconstitution. Dialyze with water 1-3 times to obtain the microbial fermentation enzyme for later use.

[0035] (5) Enzymatic hydrolysis: Add the microbial fermentation enzyme obtained in step (4) to the crude protein solution in step (3) at a concentration of 1000 U / mL, adjust the pH of the reaction system to 7.5-8, and hydrolyze at 37℃ for 5 h. Heat the reaction system to 100℃ for 6 min to terminate the enzymatic hydrolysis reaction.

[0036] (6) Ultrafiltration: The enzymatic hydrolysis product was resuspended in an appropriate PBS solution, and an ultrafiltration membrane with a molecular weight cutoff of 3kDa was selected for ultrafiltration. The portion with a molecular weight less than 3kDa was retained and recorded as the extract. The extract was transferred to a dialysis bag with a molecular weight cutoff of 8kDa and dialyzed with water for 72 hours (water was changed every 12 hours) to obtain collagen peptides.

[0037] Example 2 In this embodiment, another enzymatic hydrolysis method for collagen peptides in bovine hide is provided. The difference from Example 1 is that in step (3), the bovine hide treated in step (1) is added to the eutectic solvent prepared in step (2) at a solid-liquid ratio of 1:15~18. The mixed reaction system is stirred at 37°C for 6 hours. After the temperature of the reaction system drops to room temperature, it is centrifuged at 8000r / min for 15 minutes, and the supernatant is retained. The rest of the settings are the same as in Example 1.

[0038] Example 3 In this embodiment, another enzymatic hydrolysis method for collagen peptides in bovine hide is provided. The difference from Example 1 is that the microbial fermentation enzyme prepared in step (4) is added with calcium sulfate to a concentration of 0.5 mM and set aside. The rest of the settings are the same as in Example 1.

[0039] Example 4 This embodiment provides an enzymatic hydrolysis method for collagen peptides in bovine hide, comprising the following steps: (1) Pretreatment: Take fresh cowhide, remove surface hair and residual tissue, wash it clean, and cut the cowhide into small pieces of 3×3cm using a low temperature (4℃) cutting method. Add acetone at a material-to-liquid ratio of 1:4 and soak for 4 hours. Take it out and rinse repeatedly until there is no odor. Add 7% NaCl solution at a material-to-liquid ratio of 1:2 and stir continuously for 11 hours to remove impurities and proteins. Take out the cowhide, chop it thoroughly, and dry it for later use.

[0040] (2) Preparation of eutectic solvent: Choline chloride, glycerol and citric acid were mixed in a molar ratio of 0.8:0.8:0.1 and placed in a flask. The mixture was stirred at 65°C for 2 hours until the components in the flask were converted into a transparent homogeneous liquid.

[0041] (3) Add the cowhide treated in step (1) to the eutectic solvent prepared in step (2) at a solid-liquid ratio of 1:15. The mixed reaction system is extracted by microwave-assisted extraction with a microwave power of 350W, a reaction temperature of 37℃, and a reaction time of 15min. After the temperature of the reaction system drops to room temperature, centrifuge at 8000r / min for 15min, retain the supernatant, and record it as crude protein solution.

[0042] (4) Inducing microbial fermentation to obtain enzymatic hydrolysate: Bacillus coagulans was inoculated into seed culture medium (1% beef extract, 2% peptone, 0.5% NaCl, balance water, pH 7.5), fermented at 38℃, and cultured in shake flasks for 24 h. The culture was then transferred to a fermenter (2.0% glucose, 0.5% peptone, 0.4% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, balance water, pH 7.5), with an inoculum size of 6%. The stirring speed was 250 r / min, the aeration rate was 3 L / min, the fermentation temperature was 30℃, and the fermentation time was 36 h. After fermentation, the fermentation broth was centrifuged at 6500 r / min and 4℃ for 15 min, and the supernatant was retained. While stirring, add ammonium sulfate to the supernatant until saturated. Let stand overnight at 4°C to allow collagenase in the fermentation product to fully precipitate. Centrifuge and retain the precipitate. Transfer it to 0.2M phosphate buffer (pH 7.8) for reconstitution. Dialyze with water 1-3 times to obtain the microbial fermentation enzyme for later use.

[0043] (5) Enzymatic hydrolysis: Add the microbial fermentation enzyme obtained in step (4) to the crude protein solution in step (3) at a concentration of 1500 U / mL, adjust the pH of the reaction system to 7.5-8, and hydrolyze at 35℃ for 6 hours. Heat the reaction system to 100℃ for 6 minutes to terminate the enzymatic hydrolysis reaction.

[0044] (6) Ultrafiltration: The enzymatic hydrolysis product was resuspended in an appropriate PBS solution, and an ultrafiltration membrane with a molecular weight cutoff of 3kDa was selected for ultrafiltration. The portion with a molecular weight less than 3kDa was retained and recorded as the extract. The extract was transferred to a dialysis bag with a molecular weight cutoff of 8kDa and dialyzed with water for 72 hours (water was changed every 12 hours) to obtain collagen peptides.

[0045] Example 5 This embodiment provides an enzymatic hydrolysis method for collagen peptides in bovine hide, comprising the following steps: (1) Pretreatment: Take fresh cowhide, remove surface hair and residual tissue, wash it clean, and cut the cowhide into small pieces of 3×3cm using a low temperature (4℃) cutting method. Add acetone at a material-to-liquid ratio of 1:6 and soak for 4 hours. Take it out and rinse repeatedly until there is no odor. Add 5% NaCl solution at a material-to-liquid ratio of 1:4 and stir continuously for 14 hours to remove impurities and proteins. Take out the cowhide, chop it thoroughly, and dry it for later use.

[0046] (2) Preparation of eutectic solvent: Choline chloride, glycerol and citric acid were mixed in a molar ratio of 1.2:1.2:0.3 and placed in a flask. The mixture was stirred at 55°C for 4 hours until the components in the flask were converted into a transparent homogeneous liquid.

[0047] (3) The cowhide treated in step (1) was added to the eutectic solvent prepared in step (2) at a solid-liquid ratio of 1:18. The mixed reaction system was extracted by microwave-assisted extraction with a microwave power of 450W, a reaction temperature of 25℃, and a reaction time of 15min. After the temperature of the reaction system dropped to room temperature, it was centrifuged at 8000r / min for 15min, and the supernatant was retained and recorded as crude protein solution.

[0048] (4) Inducing microbial fermentation to obtain enzymatic hydrolysate: Bacillus coagulans was inoculated into seed culture medium (1% beef extract, 2% peptone, 0.5% NaCl, balance water, pH 7.5), fermented at 38℃, and cultured in shake flasks for 24 h. The culture was then transferred to a fermenter (2.0% glucose, 0.5% peptone, 0.4% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, balance water, pH 7.5), with an inoculum size of 6%. The stirring speed was 250 r / min, the aeration rate was 3 L / min, the fermentation temperature was 30℃, and the fermentation time was 36 h. After fermentation, the fermentation broth was centrifuged at 6500 r / min and 4℃ for 15 min, and the supernatant was retained. While stirring, add ammonium sulfate to the supernatant until saturated. Let stand overnight at 4°C to allow collagenase in the fermentation product to fully precipitate. Centrifuge and retain the precipitate. Transfer it to 0.2M phosphate buffer (pH 7.8) for reconstitution. Dialyze with water 1-3 times to obtain the microbial fermentation enzyme for later use.

[0049] (5) Enzymatic hydrolysis: Add the microbial fermentation enzyme obtained in step (4) to the crude protein solution in step (3) at a concentration of 1000 U / mL, adjust the pH of the reaction system to 7.5-8, and hydrolyze at 37℃ for 4 hours. Heat the reaction system to 100℃ for 6 minutes to terminate the enzymatic hydrolysis reaction.

[0050] (6) Ultrafiltration: The enzymatic hydrolysis product was resuspended in an appropriate PBS solution, and an ultrafiltration membrane with a molecular weight cutoff of 3kDa was selected for ultrafiltration. The portion with a molecular weight less than 3kDa was retained and recorded as the extract. The extract was transferred to a dialysis bag with a molecular weight cutoff of 8kDa and dialyzed with water for 72 hours (water was changed every 12 hours) to obtain collagen peptides.

[0051] The above embodiments 4-5 have basically the same technical effects as embodiments 1-3.

[0052] Comparative Example 1 In this embodiment, another enzymatic hydrolysis method for collagen peptides in bovine hide is provided. The difference from Example 1 is that this embodiment does not use eutectic solvent-assisted treatment. After drying in step (1), the bovine hide is chopped and resuspended in 5% NaCl solution at a solid-liquid ratio of 1:15. Microbial fermentation enzyme prepared in step (4) is added at 1500 U / g. Other settings are the same as in Example 1.

[0053] Comparative Example 2 In this embodiment, another enzymatic hydrolysis method for collagen peptides in bovine hide is provided. The difference from Example 1 is that the preparation method of the eutectic solvent in step (2) of this embodiment is as follows: choline chloride and urea are mixed at a molar ratio of 1:2 and placed in a flask. The mixture is stirred at 60°C for 3 hours until the components in the flask are converted into a transparent homogeneous liquid. During stirring, a small amount of deionized water is added to assist dispersion. The total water content in the eutectic solvent prepared does not exceed 20%.

[0054] Comparative Example 3 In this embodiment, another enzymatic hydrolysis method for collagen peptides in bovine hide is provided. The difference from Example 1 is that this embodiment does not use eutectic solvent-assisted treatment. After drying the bovine hide in step (1), it is chopped and 0.5 mol / L citric acid solution is added at a solid-liquid ratio of 1:5 below 20°C. The mixture is stirred at 25°C for 48 hours. 1M NaOH is added to adjust the pH to 7.0 to terminate the acid hydrolysis process. Then, the microbial fermentation enzyme prepared in step (4) is added at 1500 U / g. Other settings are the same as in Example 1.

[0055] Comparative Example 4 In this embodiment, another enzymatic hydrolysis method for collagen peptides in bovine hide is provided. The difference from Example 1 is that in this embodiment, the crude protein solution obtained in step (3) is transferred to a dialysis bag with a molecular weight cutoff of 8 kDa for dialysis with water for 48 hours (water is changed every 12 hours). The crude protein in the dialysis bag is retained, dissolved in PBS, and then enzymatically hydrolyzed with microbial fermentation enzymes.

[0056] Performance verification I. Research Methods 1. Collagen extraction rate Hydroxyproline assay: (1) Acid hydrolysis of the sample to be tested: Weigh 1.0 g of the sample to be tested, add 10 mL of 6 M HCl, and boil for 3 min. (2) Establishment of standard curve: Established using L-hydroxyproline standard (0-10 μg / mL); (3) Colorimetric determination: The absorbance was measured at 558 nm. The dried cowhide from step (1) of Example 1 and the ultrafiltration product obtained from step (6) were measured using the above method, and the extraction rate was calculated according to the following formula: .

[0057] 2. Antioxidant activity (1) DPPH free radical scavenging method The DPPH free radical scavenging method was used for determination. DPPH stock solution (0.1 mM) was mixed with the sample at a 1:1 volume ratio and stored in the dark for 30 min. The reaction product was centrifuged at 12000 rpm for 5 min, and the supernatant was measured at 517 nm. The preparation details for each experimental group are as follows: Sample group: 2 mL of extract + 2 mL of DPPH stock solution; Blank group: 2 mL PBS + 2 mL DPPH stock solution; Control group: 2 mL of extract + 2 mL of anhydrous ethanol.

[0058] The formula for calculating the clearance rate is as follows: .

[0059] (2) ABTS free radical scavenging rate Preparation of ABTS working solution: Mix 7 mM ABTS with 2.45 mM K2S2O8 in equal volumes, react in the dark for 12-16 hours (25℃), and dilute with PBS to a 734 nm absorbance (A0) of 0.70 ± 0.02.

[0060] Sample group: Dissolve the collagen peptides obtained in the above examples and comparative examples in PBS (1 mg / mL), centrifuge (8000 rpm, 10 min) and collect the supernatant. Take 100 μL of supernatant and add 1 mL of ABTS working solution.

[0061] Blank group: 100μL PBS + 1mL ABTS working solution; Control group: Vortex mix thoroughly, react at 25°C in the dark for 10 minutes, and immediately measure the absorbance at 734 nm (A1). The clearance rate is calculated as follows: .

[0062] (3) Scavenging activity of superoxide anion free radicals Blank group: 2.8 mL Tris-HCl + 0.2 mL deionized water, preheated at 25°C for 5 minutes. Experimental group: 2.8 mL Tris-HCl + 0.1 mL sample solution + 0.1 mL deionized water Control group: Sample solution replaced with SOD (same concentration) Quickly add 0.1 mL of 3 mM pyrogallol to start the reaction, immediately start timing, and record the absorbance (At) at 325 nm every 30 seconds for 4 minutes. The absorbance change rate (%) = ∆A / t, i.e. (A4-A0) / 4 min. The clearance rate is calculated as follows: .

[0063] 3. ACE inhibitory activity Prepare a 6.5 mmol / L HHL solution (solvent: 0.3 mol / L borate buffer, pH 8.0). Add 20 μL of the sample to 50 μL of HHL solution, incubate, and then add ACE enzyme. Stop the reaction after 1.5 h. Extract the reaction product with ethyl acetate and measure the absorbance at 228 nm. The control group uses an equal amount of solvent instead of the sample solution, and the blank group does not contain any sample solution. The ACE inhibition rate is calculated using the following formula: .

[0064] In the above formula, A 样品 A represents the OD value of the sample to be tested. 空白样品 A represents the OD value of the blank sample group. 对照 The OD value of the control group, A 空白对照 This represents the OD value of the blank control group.

[0065] 4. Verification of bone repair activity The collagen peptides from Example 1 were added to α-MEM culture medium to prepare induction media with final collagen peptide concentrations of 10, 50, and 100 μM. MC3T3-E1 cells in logarithmic growth phase were selected and cultured at 1×10⁻⁶ cells / day. 5 Cells were seeded at a concentration of 100 cells / mL in 6-well plates, 2 mL per well, and cultured for 48 h. When cell confluence reached 80%–100%, the experimental group was inducing differentiation using the same induction medium, 2 mL per well. On day 7 of induction culture, the expression levels of osteogenic-related proteins Runx2 and OPN were analyzed by Western blot. After 14 days of induction culture, ALP activity was measured according to the alkaline phosphatase kit instructions.

[0066] II. Research Results 1. Collagen extraction rate Table 1. Collagen extraction rate and activity in Examples 1-3 and Comparative Examples 1-4 Comparing Example 1 and Example 2, Example 2 added a small amount of Ca to the microbial fermentation enzyme. 2+ As an activator, the results in Table 1 show that the extraction rate, antioxidant activity and ACE inhibitory activity of Example 2 are better than those of Example 1 and Example 3, proving that the introduction of the activator helps to improve the enzymatic hydrolysis effect.

[0067] In Example 3, microwave-assisted extraction was not used; instead, the extraction time was extended by mixing the eutectic solvent with the cowhide. Comparing Example 1 and Example 3, the extraction rates and collagen product activity were essentially the same, with no substantial difference.

[0068] Comparing the results of Examples 1-3 and Comparative Example 1, the collagen peptide yield of Comparative Example 2, where the bovine hide was directly treated with microbial fermentation enzymes after impurity removal, was significantly lower than that of Examples 1-3. This indicates that the microbial fermentation enzymes are unable to directly break down the dense cross-linked structure in the bovine hide, thus failing to fully expose the collagen. This also confirms that the introduction of a eutectic solvent system can effectively improve the collagen extraction rate.

[0069] Comparing the results of Examples 1-3 and Comparative Example 2, the collagen extraction effect of the binary eutectic system composed of choline chloride and urea in Comparative Example 1 was similar to that of Examples 1-3. However, the antioxidant properties of the collagen peptides obtained by the method of Comparative Example 1 were significantly reduced compared to Examples 1-3, and the ACE inhibition rate was also significantly lower than that of Examples 1-3. This proves that the antioxidant activity of the collagen peptides extracted by the method of Comparative Example 1 decreased to a certain extent during the extraction process. It is speculated that the strong hydrogen bonding of urea disrupts the triple helix structure of collagen, causing the antioxidant active sites (such as the phenolic hydroxyl groups of hydroxyproline and tyrosine) that were originally embedded inside to be overexposed, making the generated collagen peptides more susceptible to oxidative degradation.

[0070] During the research process of this invention, the traditional acid hydrolysis pretreatment method was also verified. The specific implementation method is Comparative Example 3. After removing impurities, the cowhide was first added to citric acid solution for acid hydrolysis, and then microbial fermentation enzymes were added to the acid hydrolysis product. Compared with Comparative Example 1, Comparative Example 3 showed a significant improvement in extraction rate, antioxidant activity and ACE inhibition activity, indicating that citric acid hydrolysis helps to improve the dissolution effect of collagen. However, it still has obvious shortcomings compared with Examples 1-3.

[0071] In addition, during the research process of this invention, we attempted to purify the crude protein extracted by the eutectic solvent and then mix it with microbial fermentation enzymes for enzymatic hydrolysis. The results are shown in Comparative Example 4. The extraction rate and the bioactivity of collagen peptides were significantly lower than those in Examples 1-3. This indicates that the eutectic solvent not only helps collagen to dissolve from the cross-linked structure of bovine hide, but also helps to improve the enzyme activity of microbial enzymes.

[0072] 2. Results of bone repair activity verification Western blot quantitative analysis results are as follows: Figure 1 As shown, the expression levels of osteogenic markers (OPN, Runx2) increased in stem cells cultured with collagen peptides, and the trend was consistent with the concentration of collagen peptides. Runx2 is a core transcription factor regulating osteoblast differentiation; its increased expression signifies the formal initiation of the osteogenic differentiation process and promotes the expression of downstream bone-related genes (such as OPN, OCN, and COL1A1). OPN (ostepontin) is a marker of late-stage differentiation in osteoblasts and participates in extracellular matrix mineralization and bone remodeling. Figure 1 The results confirmed that the collagen peptides prepared in this invention can effectively promote osteogenic differentiation of stem cells.

[0073] ALP activity analysis results are as follows: Figure 2 As shown, as the concentration of collagen peptides increases, the crystal deposition in the culture dish also increases significantly, proving that pre-mineralization preparation occurs after stem cell osteogenic differentiation.

[0074] The above research results confirm that the collagen peptides provided by this invention can potentially promote bone trauma repair and improve symptoms of osteoporosis and other diseases by activating osteogenic differentiation of bone marrow mesenchymal stem cells.

[0075] 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 collagen peptides in bovine hide, characterized in that, The process includes the following steps: fresh cowhide is defatted and impurities are removed, then it is thoroughly crushed, extracted with a eutectic solvent and the supernatant is retained, which is recorded as crude protein solution; microbial fermentation enzymes are added for enzymatic hydrolysis, and the hydrolysate is ultrafiltered to retain the portion with a molecular weight less than 3kDa, which is the final product; The eutectic solvent is obtained by mixing choline chloride, glycerol, and citric acid in a molar ratio of 0.8~1.2:0.8~1.2:0.4~0.

6. The microbial fermentation enzyme is obtained by gelatin-induced fermentation of Bacillus coagulans.

2. The enzymatic hydrolysis method for collagen peptides in bovine hide as described in claim 1, characterized in that, The specific degreasing and impurity removal process is as follows: Fresh cowhide is soaked in an organic reagent at a material-to-liquid ratio of 1:4~6, then taken out and washed; it is then added to a sodium chloride solution at a material-to-liquid ratio of 1:2~4 and stirred continuously for 11~14 hours to remove impurities and proteins.

3. The enzymatic hydrolysis method for collagen peptides in bovine hide as described in claim 1, characterized in that, The preparation method of the eutectic solvent is as follows: choline chloride, glycerol and citric acid are mixed in the molar ratio described above and stirred at 55~65℃ for 2~4h to obtain a transparent homogeneous liquid; the fully crushed cowhide is added to the eutectic solvent for extraction to obtain a crude protein solution, and the solid-liquid ratio of cowhide to eutectic solvent is 1:15~18; the temperature range of the extraction process is 4℃~60℃, and the extraction time is 10min~6h.

4. The enzymatic hydrolysis method for collagen peptides in bovine hide as described in claim 3, characterized in that, The extraction method is as follows: the cowhide is added to a eutectic solvent and extracted by stirring at 20~37℃ for 4~6 hours.

5. The enzymatic hydrolysis method for collagen peptides in bovine hide as described in claim 3, characterized in that, The extraction method is as follows: after adding the cowhide to a eutectic solvent, it is extracted with microwave assistance at a power of 350~450W and at 25~37℃ for 15 minutes.

6. The enzymatic hydrolysis method for collagen peptides in bovine hide as described in claim 1, characterized in that, The microbial fermentation enzyme is prepared as follows: Bacillus coagulans seed culture is inoculated into a fermenter at an inoculation rate of 5-7%, with a stirring speed of 220-270 r / min, an aeration rate of 2.5-3.5 L / min, a fermentation temperature of 30-38℃, and a fermentation time of 30-36 h. The fermentation medium contains the following components: 2.0% glucose, 0.5% peptone, 0.4-0.6% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, with the remainder being water, and a pH of 7.

5. The fermentation product is then subjected to salting out to obtain the microbial fermentation enzyme.

7. The enzymatic hydrolysis method for collagen peptides in bovine hide as described in claim 6, characterized in that, The microbial fermentation enzyme also contains an activator, which is Ca. 2+ The concentration is 4~6mM.

8. The enzymatic hydrolysis method for collagen peptides in bovine hide as described in claim 1, characterized in that, The parameters for the enzymatic hydrolysis are as follows: the amount of microbial fermentation enzyme added is 1000~1500U / mL, the pH of the reaction system is adjusted to 7.5~8, the enzymatic hydrolysis is carried out at 35~37℃ for 4~6h, and the enzymatic hydrolysis reaction is terminated by high temperature heating.

9. Bovine collagen peptides prepared by the method according to any one of claims 1-8.

10. The application of the bovine collagen peptide of claim 9 in the preparation of bone repair products, characterized in that, The bone repair products are selected from food, beverages, health products, drugs, or cosmetics.

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