A method for extracting bovine collagen and its application

Abstract

The invention discloses a method for extracting bovine bone collagen and its application, belonging to the technical field of collagen extraction. The extraction method comprises 7 steps of pretreatment, defatting, decalcification, acid treatment, enzymolysis, crude product preparation and purification, wherein the best test conditions and reagents are selected for each step, and the bovine bone is pretreated by defatting and decalcifying in a specific method, effectively removing fat and inorganic mineral calcium salts, and protecting the activity of collagen components to the maximum extent, improving the extraction rate of collagen and product quality; in addition, the method of combining enzymolysis and acid treatment is adopted to adjust the best reaction conditions, and obtain highly active collagen to the maximum extent, and the prepared high-purity and high-quality bovine bone collagen can be further applied to the fields of health food and cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen preparation, and specifically relates to a method for extracting bovine bone collagen and application thereof. Background Art

[0002] my country is a major producer and consumer of livestock and poultry meat, with beef production accounting for 8-12% of the total livestock and poultry meat production in my country. In the production and consumption of beef, cattle bones, as the main by-product, account for about 20% to 30% of the total body weight of cattle. They are mainly composed of bone, periosteum and bone marrow, which are rich in protein, fat, chondroitin, as well as minerals such as calcium, iron, phosphorus, zinc and vitamins. Collagen is the main protein in cattle bones, with a content of more than 90%. Collagen has functional properties such as emulsification, gelation and foaming, and also has physiological functions such as promoting metabolism and delaying aging. In addition, it contains a variety of essential amino acids for the human body, which can meet the needs of different groups of people for amino acids. It is widely used in health products, biological drugs, cosmetics and food industries. With the continuous development of the beef cattle industry, my country's annual output of cattle bones exceeds 12 million tons. However, due to immature processing technology and poor handling methods, a large number of cattle bones are simply processed into feed or sold as primary products such as bone meal, resulting in waste of resources and increased processing costs. Therefore, how to stably and efficiently extract and deeply process bovine bone collagen has become a research hotspot in recent years.

[0003] At present, the commonly used collagen extraction methods mainly include hot water extraction, acid extraction, alkali extraction and enzyme extraction, but the yield of hot water extraction is relatively low; acid treatment under low temperature conditions can better retain the natural triple helix structure of collagen, but its disadvantage is that it will destroy tryptophan, and the acid solution will also corrode the equipment, which has certain limitations; alkaline extraction commonly uses lime, sodium hydroxide and sodium carbonate, etc., and its process steps are simple and the cost is low, but its application is not widespread, mainly because the collagen peptide chain is easily broken under alkaline conditions. Therefore, there are not many reports on the use of alkaline extraction alone; enzymatic hydrolysis can not only hydrolyze the terminal peptides of collagen fibril protein and increase the yield of collagen, but also will not destroy the triple helix structure of collagen, and can maintain its excellent physical and biochemical properties. Therefore, enzymatic hydrolysis is currently the most widely used method. Although the content of collagen in bovine bones is high, it also contains fatty acids and inorganic minerals (such as calcium phosphate, magnesium phosphate, calcium salts, etc.). Usually, before extracting collagen, the bovine bones need to be degreased and decalcified to remove impurities that affect the extraction of collagen. The method and effect of degreasing and decalcification directly affect the extraction efficiency and quality of collagen. Commonly used degreasing and decalcification methods use a variety of chemical reagents such as strong alkali, hydrochloric acid, acetone, n-hexane, and ether. However, the use of too many chemical reagents will have a certain impact on the structure and safety performance of collagen, and the removal efficiency is also unstable. The residual fat and calcium will generate insoluble fatty acid calcium in the subsequent process, which will be deposited on the surface of the bone and agglomerate, seriously hindering the enzymatic hydrolysis reaction and reducing the extraction efficiency and quality of collagen. Therefore, studying an efficient method for extracting bovine bone collagen has become a technical problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting bovine bone collagen, wherein the bovine bones are pre-treated with fat and calcium by a specific method, fat and inorganic mineral calcium salts are effectively removed, and the activity of collagen components can be protected to the maximum extent, thereby improving the extraction rate of collagen and product quality; in addition, a combination of enzymatic hydrolysis and acid treatment is used to regulate the optimal reaction conditions, thereby obtaining highly active collagen to the maximum extent.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0006] A method for extracting bovine collagen comprises the following steps:

[0007] A. Pretreatment: Take fresh cattle bones, clean them, dry them and grind them into powder, pass them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5-10 minutes, take them out and naturally cool them to room temperature to obtain pretreated cattle bone powder;

[0008] B. Degreasing: Take the pre-treated bovine bone powder and add it to the degreasing solvent, and treat it with ultrasonic stirring at room temperature for 30-45 minutes, centrifuge and separate the solid product to dry to obtain the defatted bovine bone powder;

[0009] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add the decalcification agent and stir at 40-45°C for 10-12h, filter, take the solid product and set aside;

[0010] D. Acid treatment: the solid product obtained in step C is mixed with 1 mol / L oxalic acid solution according to the solid-liquid ratio, and then an appropriate amount of Aspergillus niger fermentation liquid is added, and after mixing evenly, the mixture is shaken and fermented for 6-8 hours at 25-30° C. and 100-200 rpm to obtain a mixed fermentation liquid;

[0011] E. Enzymolysis: adjust the pH of the mixed fermentation liquid to 2.5-3.5, add 1wt% pepsin to the mixed liquid, perform enzymolysis at 30-40°C for 8-10h, and obtain enzymolysis liquid after inactivating the enzyme;

[0012] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 6-10h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, add NaCl to the supernatant for salting out, centrifuge after salting out, and take the precipitate as crude collagen;

[0013] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0014] Preferably, the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauramide propyl hydroxysulfobetaine in a mass ratio of 10:0.5-2; the mass ratio of the degreasing solvent to the pretreated bovine bone powder is 2:1.

[0015] Preferably, the decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomerized sodium decanol polyoxyethylene ether sulfate in a mass ratio of 10:5:0.2-0.5; the mass ratio of the decalcification agent to defatted bovine bone powder is 1:1.

[0016] Preferably, the solid-liquid ratio of the solid product obtained in step C to the oxalic acid solution is 1 g:10 ml.

[0017] Preferably, the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0018] Preferably, the Aspergillus niger fermentation broth is prepared as follows: 5 g of activated Aspergillus niger powder is transferred to a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation broth is obtained by fermenting at 37° C. for 72 h.

[0019] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0020] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / gram.

[0021] Preferably, the modified composite biochar is prepared by the following method:

[0022] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0023] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0024] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0025] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0026] Preferably, the modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, and then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0027] The bovine bone collagen prepared by the bovine bone collagen extraction method of the present invention is high-purity and has good stability, can be used for the preparation of health food and cosmetics, and has good market application prospects.

[0028] The present invention uses high pressure pretreatment on cattle bone powder, which can destroy the chemical bonds and structures in cattle bones, making them looser, more conducive to the penetration of degreasing agents and decalcifying agents, efficiently remove minerals such as fat and inorganic calcium salts, and also make collagen more conducive to extraction; the degreasing agent composed of 0.4mol / L hydrochloric acid solution and lauramide propyl hydroxysulfonyl betaine is used, on the one hand, 0.4mol / L hydrochloric acid solution can dissolve fatty acids, and the addition of an appropriate amount of lauramide propyl hydroxysulfonyl betaine promotes the dissolution of fatty acids, and the combination of the two can have an excellent removal effect on the fat in cattle bones. The decalcifying agent used is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA and isomeric decyl alcohol polyoxyethylene ether sodium sulfate, wherein the modified chitosan is in a nano-sheet structure, has a larger specific surface area and pore structure, can adsorb more metal ions such as calcium ions and magnesium ions, and can also enhance and promote the chelation between EDTA and metal ions such as calcium ions. Modified chitosan provides adsorption and chelating sites, EDTA forms a stable chelate, and the addition of an appropriate amount of isomeric decanol polyoxyethylene ether sodium sulfate improves the wettability and dispersibility of the solution. The three complement each other and together achieve excellent decalcification effects, and none of them can be missing.

[0029] The present invention uses oxalic acid acidification treatment at the collagen extraction stage, and then adds Aspergillus niger fermentation liquid to continue fermentation for a period of time. The Aspergillus niger fermentation liquid contains multiple enzymes such as pectinase and acid protease and citric acid components. Oxalic acid and citric acid are both organic acids. They work together to destroy the connection between collagen and other tissue components in cattle bones by lowering the pH value of the solution, so that it is easier to be released, thereby improving the extraction efficiency of collagen. The multiple enzymes contained in the fermentation liquid can work together with pepsin to efficiently hydrolyze the protein in cattle bones, thereby improving the yield of collagen; and the addition of modified composite biochar to the enzymatic hydrolyzate can further adsorb and remove residual organic impurities, thereby improving the color and quality of the collagen product.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: the method for extracting collagen from bovine bones of the present invention has a high yield, the collagen structure is complete, the product has excellent color and quality, is safe and environmentally friendly, and has good promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1It is a graph of the electrophoresis analysis results of the present invention; wherein M is a standard protein Marker, 0 is the sample of Example 1, 1 is the sample of Comparative Example 1, 2 is the sample of Comparative Example 2, 3 is the sample of Comparative Example 3, 4 is the sample of Comparative Example 4, and 5 is the sample of Comparative Example 5.

[0032] Figure 2 This is the appearance of the bovine collagen products obtained in Example 1 and Comparative Examples 1-5 of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0034] Embodiment 1, a method for extracting bovine collagen, comprising the following steps:

[0035] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0036] B. The pretreated bovine bone meal was added to 2 kg of degreasing solvent, and ultrasonic stirring was performed at room temperature for 30 min. The solid product was centrifuged and dried to obtain defatted bovine bone meal; the degreasing solvent was composed of 0.4 mol / L hydrochloric acid solution and lauroamidopropyl hydroxysulfobetaine in a mass ratio of 10:0.5;

[0037] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add a decalcification agent and stir for 10 hours at 40°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomeric decyl alcohol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.2.

[0038] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 8 hours at 25°C and 100 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0039] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 2.5, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 30°C for 8h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0040] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 6h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, add NaCl to the supernatant for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0041] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0042] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0043] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0044] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / g; and pepsin was purchased from Nanjing Yien Biotechnology Co., Ltd., with an enzyme activity of 300,000 u / g.

[0045] The modified composite biochar is prepared by the following method:

[0046] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0047] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0048] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0049] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0050] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0051] Embodiment 2, a method for extracting bovine collagen, comprising the following steps:

[0052] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 10 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0053] B. adding the pretreated bovine bone powder to 2 kg of degreasing solvent, subjecting the mixture to ultrasonic stirring treatment for 45 min at room temperature, centrifuging and drying the solid product to obtain the defatted bovine bone powder; the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauroamidopropyl hydroxysulfobetaine in a mass ratio of 10:2;

[0054] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add the decalcification agent and stir for 12 hours at 45°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomerized decanol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.5.

[0055] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 8 hours at 30°C and 200 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0056] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 3.5, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 40°C for 10h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0057] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 10h at room temperature under natural light, then filter with a filter, centrifuge the filtrate at high speed, take the supernatant and add NaCl for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0058] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0059] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0060] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0061] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / g; pepsin was purchased from Nanjing Yien Biotechnology Co., Ltd., with an enzyme activity of 300,000 u / g.

[0062] The modified composite biochar is prepared by the following method:

[0063] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0064] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0065] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0066] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0067] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0068] Embodiment 3, a method for extracting bovine collagen, comprising the following steps:

[0069] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 8 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0070] B. adding the pretreated bovine bone powder to 2 kg of degreasing solvent, subjecting the mixture to ultrasonic stirring treatment for 40 min at room temperature, centrifuging and separating the solid product, and drying the solid product to obtain the defatted bovine bone powder; the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauroamidopropyl hydroxysulfobetaine in a mass ratio of 10:1;

[0071] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add the decalcification agent and stir for 11 hours at 40°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomerized decanol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.3.

[0072] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 7 hours at 28° C. and 150 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0073] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 3.0, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 35°C for 10h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0074] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 8h under natural light at room temperature, then filter with a filter screen, centrifuge the filtrate at high speed, take the supernatant and add NaCl for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0075] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0076] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0077] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0078] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / gram.

[0079] The modified composite biochar is prepared by the following method:

[0080] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0081] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0082] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0083] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0084] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0085] Embodiment 4, a method for extracting bovine collagen, comprising the following steps:

[0086] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 10 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0087] B. adding the pretreated bovine bone powder to 2 kg of degreasing solvent, subjecting the mixture to ultrasonic stirring treatment for 40 min at room temperature, centrifuging and drying the solid product to obtain the defatted bovine bone powder; the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauroamidopropyl hydroxysulfobetaine in a mass ratio of 10:1.5;

[0088] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add the decalcification agent and stir for 10 hours at 42°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomerized decanol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.4.

[0089] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 7 hours at 30°C and 200 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0090] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 3.0, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 37°C for 10h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0091] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 7h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, take the supernatant and add NaCl for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0092] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0093] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0094] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0095] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / gram.

[0096] The modified composite biochar is prepared by the following method:

[0097] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0098] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0099] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0100] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0101] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0102] Comparative Example 1, a method for extracting bovine collagen, comprising the following steps:

[0103] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0104] B. Add the pretreated bovine bone powder to 2 kg of degreasing solvent, stir ultrasonically at room temperature for 30 minutes, centrifuge to separate the solid product, and dry to obtain the defatted bovine bone powder; the degreasing solvent is 0.4 mol / L hydrochloric acid solution.

[0105] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add a decalcification agent and stir for 10 hours at 40°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomeric decyl alcohol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.2.

[0106] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 8 hours at 25°C and 100 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0107] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 2.5, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 30°C for 8h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0108] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 6h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, add NaCl to the supernatant for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0109] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0110] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0111] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0112] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / g; pepsin was purchased from Nanjing Yien Biotechnology Co., Ltd., with an enzyme activity of 300,000 u / g.

[0113] The modified composite biochar is prepared by the following method:

[0114] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0115] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0116] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0117] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0118] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0119] This comparative example is basically the same as Example 1, except that the degreasing agent is a 0.4 mol / L hydrochloric acid solution.

[0120] Comparative Example 2, a method for extracting bovine collagen, comprising the following steps:

[0121] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0122] B. adding the pretreated bovine bone powder to 2 kg of degreasing solvent, subjecting the mixture to ultrasonic stirring for 30 min at room temperature, centrifuging and drying the solid product to obtain the defatted bovine bone powder; the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauroamidopropyl hydroxysulfobetaine in a mass ratio of 10:0.5;

[0123] C. Decalcification: add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add a decalcification agent, stir at 40°C for 10 hours, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1; the decalcification agent is 0.2 mol / L EDTA;

[0124] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 8 hours at 25°C and 100 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0125] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 2.5, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 30°C for 8h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0126] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 6h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, add NaCl to the supernatant for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0127] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0128] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0129] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0130] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / g; and pepsin was purchased from Nanjing Yien Biotechnology Co., Ltd., with an enzyme activity of 300,000 u / g.

[0131] The modified composite biochar is prepared by the following method:

[0132] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0133] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0134] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0135] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0136] This comparative example is basically the same as Example 1, except that the decalcifying agent is only 0.2 mol / L EDTA.

[0137] Comparative Example 3, a method for extracting bovine collagen, comprising the following steps:

[0138] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0139] B. adding the pretreated bovine bone powder to 2 kg of degreasing solvent, subjecting the mixture to ultrasonic stirring for 30 min at room temperature, centrifuging and drying the solid product to obtain the defatted bovine bone powder; the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauroamidopropyl hydroxysulfobetaine in a mass ratio of 10:0.5;

[0140] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add a decalcification agent and stir for 10 hours at 40°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomeric decyl alcohol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.2.

[0141] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 8 hours at 25°C and 100 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0142] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 2.5, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 30°C for 8h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0143] F. Preparation of crude product: Add 3wt% coconut shell activated carbon to the enzymatic hydrolyzate, stir for 6h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, add NaCl to the supernatant for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0144] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0145] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0146] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water, 103.4 kPa, and poured onto a plate after sterilization for 21 min.

[0147] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / g; and pepsin was purchased from Nanjing Yien Biotechnology Co., Ltd., with an enzyme activity of 300,000 u / g.

[0148] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0149] This comparative example is basically the same as Example 1, except that coconut shell activated carbon is used for adsorption and impurity removal in the preparation of the crude product.

[0150] Comparative Example 4, a method for extracting bovine collagen, comprising the following steps:

[0151] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0152] B. Add the pretreated bovine bone meal to 2 kg of degreasing solvent, treat with ultrasonic stirring at room temperature for 30 min, centrifuge and dry the solid product to obtain defatted bovine bone meal; the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and sodium cocoyl glycinate in a mass ratio of 10:0.5;

[0153] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add a decalcification agent and stir for 10 hours at 40°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomeric decyl alcohol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.2.

[0154] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 8 hours at 25°C and 100 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0155] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 2.5, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 30°C for 8h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0156] F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 6h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, add NaCl to the supernatant for salting out, the final concentration of NaCl is 0.9mol / L, centrifuge after salting out, and take the precipitate as crude collagen;

[0157] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0158] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0159] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0160] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / g; pepsin was purchased from Nanjing Yien Biotechnology Co., Ltd., with an enzyme activity of 300,000 u / g.

[0161] The modified composite biochar is prepared by the following method:

[0162] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0163] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0164] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0165] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0166] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0167] This comparative example is basically the same as Example 1, except that the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and sodium cocoyl glycinate in a mass ratio of 10:0.5.

[0168] Comparative Example 5, a method for extracting bovine collagen, comprising the following steps:

[0169] A. Take 1 kg of fresh cattle bones, clean them, dry them and grind them into powder, sieve them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5 minutes, take them out and cool them naturally to room temperature to obtain pretreated cattle bone powder;

[0170] B. adding the pretreated bovine bone powder to 2 kg of degreasing solvent, subjecting the mixture to ultrasonic stirring for 30 min at room temperature, centrifuging and drying the solid product to obtain the defatted bovine bone powder; the degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauroamidopropyl hydroxysulfobetaine in a mass ratio of 10:0.5;

[0171] C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add a decalcification agent and stir for 10 hours at 40°C, filter, take the solid product and set aside; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1. The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomeric decyl alcohol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.2.

[0172] D. Acid treatment: The solid product obtained in step C is mixed with 1 mol / L oxalic acid solution at a solid-liquid ratio of 1 g:10 ml, and then Aspergillus niger fermentation broth is added. After mixing evenly, the mixture is shaken and fermented for 8 hours at 25°C and 100 rpm to obtain a mixed fermentation broth; the volume ratio of the Aspergillus niger fermentation broth to the oxalic acid solution is 2:1.

[0173] E. Enzymatic hydrolysis: adjust the pH of the mixed fermentation liquid to 2.5, add 1wt% pepsin to the mixed liquid, perform enzymatic hydrolysis at 30°C for 8h, and obtain enzymatic hydrolyzate after inactivating the enzyme;

[0174] F. Preparation of crude product: centrifuge the hydrolyzate at high speed, take the supernatant and add NaCl for salting out, the final concentration of NaCl is 0.9 mol / L, centrifuge again after salting out, and take the precipitate as crude collagen;

[0175] G. Purification: Dissolve the crude collagen in 0.5 mol / L acetic acid solution, then dialyze in 0.1 mol / L disodium hydrogen phosphate solution for 24 hours, then transfer to distilled water for dialyzing for 12 hours. After the dialysis is completed, the product is freeze-dried to obtain the final bovine collagen product.

[0176] The Aspergillus niger fermentation liquid is prepared as follows: 5 g of activated Aspergillus niger powder is transferred into a 5 L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

[0177] The PDA medium consists of: 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar. The container is shaken until the solute is dissolved, and the volume is fixed to 1 L with ionized water. After sterilization at 103.4 kPa for 21 min, the medium is poured onto a plate.

[0178] The Aspergillus niger used in the present invention was purchased from Shandong Changtai Biotechnology Co., Ltd., with a spore count of 5 billion / g; pepsin was purchased from Nanjing Yien Biotechnology Co., Ltd., with an enzyme activity of 300,000 u / g.

[0179] The modified composite biochar is prepared by the following method:

[0180] (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A.

[0181] (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A.

[0182] (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol.

[0183] (4) Take 0.5 g of the product A obtained in step (1) and immerse it in 20 mL of the sol obtained in step (3), perform hydrothermal reaction at 120 °C for 6 h, dry it at 100 °C, and grind it into powder to obtain modified composite biochar.

[0184] The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

[0185] This comparative example is basically the same as Example 1, except that in step F, the enzymatic hydrolysate is not treated with modified composite biochar, but is directly centrifuged and then subjected to subsequent treatments such as salting out.

[0186] Test Example 1

[0187] Polyacrylamide gel electrophoresis analysis of collagen obtained from different treatment groups

[0188] The collagen powder prepared in Example 1 and Comparative Examples 1-5 was used to prepare 4 mg / ml collagen solution as test samples. The test sample was mixed with 2x sample buffer at a ratio of 1:1, and then boiled in a water bath for 3-5 min. After cooling, centrifugation was performed to take 10 μL of the supernatant and load it with 10% concentrated gel and 5% separation gel. A direct voltage constant current power supply was used, with a concentrated gel voltage of 120 V and a separation gel voltage of 160 V. After the end, it was stained with Coomassie Brilliant Blue R-250 for 40 min. After decolorization to transparency, it was observed and analyzed with a gel imager. The results are as follows Figure 1 shown.

[0189] like Figure 1 As shown in , the sample of Example 1 has a β band at about 250 kDa and two electrophoretic bands at about 130 kDa, which are α1 and α2 chains, respectively, and have a typical collagen trimer structure. The collagen products prepared in Comparative Examples 1-5 also have the above three typical electrophoretic bands, but compared with Example 1, the bands tend to be thinner and lighter in color, which indicates that their collagen content is significantly lower than that of the sample of Example.

[0190] Test Example 2

[0191] Collagen yield determination

[0192] The quality of collagen obtained by different extraction methods in Examples 1-4 and Comparative Examples 1-5 was measured, and the collagen yield was calculated. The collagen yield calculation formula is as follows:

[0193] Collagen yield (%) = crude collagen mass / raw bone mass × 100.

[0194] Table 1 Statistical results of collagen yields obtained by different extraction methods

[0195]

[0196] As can be seen from the results of Table 1 above, the bovine collagen yield extracted by the method of the present invention is 76.7% on average, which is significantly higher than the collagen yield prepared by the methods of Comparative Examples 1-5. As can be seen from the data of Comparative Examples 1 and 4, without the use of lauramide propyl hydroxysulfobetaine, the degreasing effect of a single 0.4 mol / L hydrochloric acid solution deteriorates, and the residual fatty acid affects the dissolution and extraction of collagen, thereby reducing the extraction yield of bovine collagen; and in Comparative Example 4, where the degreasing agent composition is changed, the collagen yield is not improved when the 0.4 mol / L hydrochloric acid solution and sodium cocoyl glycinate are used in combination, which shows that the degreasing agent combination used in the present invention is indispensable. The data of Comparative Example 2 show that the effect of promoting the chelation of calcium ions between the modified chitosan solution, EDTA, and isomerized decanol polyoxyethylene ether sodium sulfate disappears, and the calcium ion removal effect deteriorates, thereby resulting in a reduced yield. The data of Comparative Examples 3 and 5 show that the effects of using different adsorbents to remove impurities in the hydrolyzate (incompletely hydrolyzed raw material fragments, the enzyme itself, and other soluble substances) are completely different. The impurity removal effect of ordinary activated carbon is much lower than that of the modified composite biochar used in the present invention. Therefore, changing the amount and proportion of any raw material or step in the extraction method of the present invention will not achieve the above effect.

[0197] Test Example 3

[0198] Protease residue determination: The protease enzyme-linked immunosorbent assay reagent was used to determine the pepsin residue in the collagen solid samples prepared in Examples 1-4 and Comparative Examples 1-5, and the detection limit was ng / ml. The results are shown in Table 2.

[0199] Table 2 Protease residue determination results

[0200]

[0201] Test Example 4

[0202] The contents of characteristic amino acids (Gly, Lys, Pro) in collagen obtained from different treatment groups were determined

[0203] Weigh 100 mg of the collagen sample prepared in Examples 1-4 and Comparative Examples 1-5 and place it in a test tube, then add 10 mL of 6 mol / L HCl solution, fill with nitrogen for 1 min to exhaust the air in the tube, seal the test tube and place it in a drying oven at 110°C for 24 h of acid hydrolysis; after the acid hydrolysis is complete, mix and dilute to 50 mL; take 1 mL of the dilution and continue to blow nitrogen to dryness, then add 5 mL of 0.02 mol / L HCl solution to dilute, shake and mix, filter with a 0.20 μm filter membrane, and the filtrate is measured by an amino acid automatic detector. The specific results are shown in Table 3.

[0204] Table 3 Determination results of characteristic amino acids (Gly, Lys, Pro) in collagen products of different treatment groups (mg / g)

[0205]

[0206] From Table 3 above, it can be seen that the characteristic amino acid content of the collagen samples prepared in the examples of the present invention is significantly higher than that of the products prepared in Comparative Examples 1-5. This shows that the bovine bone collagen extracted by the method of the present invention has a high yield and high purity, and the product has a uniform white or slightly white color (such as Figure 2 ), the product quality is obviously better than the product obtained in the comparative example.

[0207] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A method for extracting bovine collagen, characterized in that: It includes the following steps: A. Pretreatment: Take fresh cattle bones, clean them, dry them and grind them into powder, pass them through a 100-mesh sieve to obtain cattle bone powder, transfer the cattle bone powder into a high-pressure reactor at 0.1 MPa and 110°C, react for 5-10 minutes, take them out and naturally cool them to room temperature to obtain pretreated cattle bone powder; B. Degreasing: Take the pre-treated bovine bone powder and add it to the degreasing solvent, and treat it with ultrasonic stirring at room temperature for 30-45 minutes, centrifuge and separate the solid product to dry to obtain the defatted bovine bone powder; C. Decalcification: Add 20 times the amount of deionized water to the defatted bovine bone powder, mix well, then add the decalcification agent and stir at 40-45°C for 10-12h, filter, take the solid product and set aside; D. Acid treatment: the solid product obtained in step C is mixed with 1 mol / L oxalic acid solution according to the solid-liquid ratio, and then an appropriate amount of Aspergillus niger fermentation liquid is added, and after mixing evenly, the mixture is shaken and fermented for 6-8 hours at 25-30° C. and 100-200 rpm to obtain a mixed fermentation liquid; E. Enzymolysis: adjust the pH of the mixed fermentation liquid to 2.5-3.5, add 1wt% pepsin to the mixed liquid, perform enzymolysis at 30-40°C for 8-10h, and obtain the enzymolysis liquid after inactivating the enzyme; F. Preparation of crude product: Add 3wt% modified composite biochar to the enzymatic hydrolyzate, stir for 6-10h under natural light at room temperature, then filter with a filter, centrifuge the filtrate at high speed, add NaCl to the supernatant for salting out, centrifuge after salting out, and take the precipitate as crude collagen; G. Purification: The crude collagen was dissolved in 0.5 mol / L acetic acid solution, then dialyzed in 0.1 mol / L disodium hydrogen phosphate solution for 24 h, and then dialyzed in distilled water for 12 h. After the dialysis, the product was freeze-dried to obtain the final bovine collagen product; The degreasing solvent is composed of 0.4 mol / L hydrochloric acid solution and lauramide propyl hydroxysulfobetaine in a mass ratio of 10:0.5-2; the mass ratio of the degreasing solvent to the pretreated bovine bone powder is 2:1; The decalcification agent is composed of 5wt% modified chitosan solution, 0.2mol / L EDTA, and isomeric decanol polyoxyethylene ether sodium sulfate in a mass ratio of 10:5:0.2-0.5; the mass ratio of the decalcification agent to the defatted bovine bone powder is 1:1; The solid product obtained in step C and the oxalic acid solution have a solid-liquid ratio of 1 g:10 ml; The modified composite biochar is prepared by the following method: (1) The straw was cleaned and dried to constant weight, and then crushed to obtain straw powder. The straw powder was then added to a 2wt% K2CO3 solution at a material-liquid ratio of 1g:30ml, stirred at room temperature for 30-40min, and then dried. The mixture was transferred to a tubular furnace and heated to 800°C under nitrogen protection for 3h. The mixture was washed with 5wt% hydrochloric acid solution and deionized water until neutral, and dried to obtain product A. (2) Mix 5 mL of glacial acetic acid and 30 mL of anhydrous ethanol, then add 5 mL of tetrabutyl titanate and stir for 30 min to obtain solution A. (3) Mix 15 mL of anhydrous ethanol with 5 mL of water and adjust the pH to 2-3 to obtain solution B. Under vigorous magnetic stirring, slowly add solution B to solution A at a rate of 2 seconds per drop to obtain a uniform and transparent sol. (4) 0.5 g of the product A obtained in step (1) was immersed in 20 mL of the sol obtained in step (3), subjected to hydrothermal reaction at 120 °C for 6 h, dried at 100 °C, and ground into powder to obtain modified composite biochar; The modified chitosan is prepared by the following method: 0.5 g of chitosan is dissolved in 50 ml of 1% acetic acid solution, stirred at 45° C. for 30 min, then 0.5 ml of hydrogen peroxide is added, and the mixture is reacted in a 50-55° C. constant temperature water bath oscillator for 5 h to obtain a chitosan acetic acid solution, and then 10 ml of 0.5 mol / L sodium tripolyphosphate solution is slowly added dropwise to the chitosan acetic acid solution, reacted at 40-45° C. for 30 min, then washed with deionized water until neutral, and freeze-dried in a freeze dryer to obtain the modified chitosan.

2. The method for extracting bovine collagen according to claim 1, characterized in that: In the step D, the volume ratio of the Aspergillus niger fermentation liquid to the oxalic acid solution is 2:

1.

3. The method for extracting bovine collagen according to claim 1, characterized in that: The preparation method of the Aspergillus niger fermentation liquid is as follows: 5 g of activated Aspergillus niger powder is transferred to a 5L sealed plastic bottle, 25 g of PDA culture medium, 100 g of brown sugar and 5 kg of water are added, and the Aspergillus niger fermentation liquid is obtained by fermenting at 37° C. for 72 hours.

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