A method for extracting and purifying natural active collagen

Abstract

The present invention discloses a method for extracting and purifying natural active collagen, specifically a method for purifying type I collagen, belonging to the technical field of collagen production. The present invention adopts a purification scheme of salting out, ultrafiltration purification and liquid replacement, and can successfully obtain natural type I collagen with high purity and high activity, improving the problems of long purification cycle of collagen, high content of non-target proteins, resulting in low biological activity and low protein purity in traditional purification technologies. The method for purifying type I collagen mainly includes: S1: After the raw materials are pretreated, enzymatic hydrolysis technology is used for reaction, and then type I collagen hydrolysate is obtained by centrifugation; S2: Soluble miscellaneous proteins and impurities such as pepsin are preliminarily removed by salting out process; S3: The crude extract is redissolved, and the raw material liquid is obtained by filtration through a polypropylene pleated filter element. The raw material liquid is purified and concentrated by tangential flow ultrafiltration to obtain type I collagen purified liquid.

Description

Technical Field

[0001] The present invention discloses a method for extracting and purifying natural active collagen, belonging to the technical field of collagen production. Background Art

[0002] Collagen, as the most abundant and widely distributed protein in organisms in nature, has become a rich industrial raw material. The proportion of type I collagen content can be as high as 90% of the total collagen content. Type I collagen exists widely in the form of collagen fibers as tissue supports in skin, bones, tendons, ligaments, vertebrae, blood vessel walls, etc. And as the main component of the extracellular matrix, it not only endows each tissue with stable mechanical properties, but also endows it with the ability to promote cell growth and migration, and also plays an important role in the physiological and pathological processes of cells, tissues, and even the whole body. Due to its good biocompatibility, low immunogenicity, biodegradability, promoting cell proliferation, differentiation and migration, promoting platelet coagulation and wound healing and other functions, type I collagen has now been widely used clinically and marketed in products in many fields such as surgical sutures, hemostatic sponges, hemostatic fibers, hydrogels, dressings, artificial skin, artificial blood vessels, artificial esophagus, heart valves, tissue engineering repair, corneas, nerve repair and drug carriers. As the main protein component of human skin, type I collagen has functions such as defense, support, protection, and nutrition. With the increase of age, its regeneration ability will continuously decline, resulting in the loss of skin collagen, cross-linking and solidification inside the collagen fiber molecules, and the decrease of the content of extracellular matrix glycosaminoglycans, making the skin lose elasticity and luster, resulting in skin aging, losing elasticity and luster, and appearing spots and wrinkles. Therefore, type I collagen is extremely popular in many fields such as medical beauty, medicine, and cosmetics.

[0003] Ultrafiltration is a pressure membrane separation technology that utilizes the microporous structure of a semipermeable membrane. That is, by using an external / internal pressure as the driving force, only soluble substances or solutions such as proteins larger than a specific pore size can pass through the membrane, so that macromolecular soluble substances are retained and cannot pass through, thereby realizing the selective recovery, separation, fractionation, purification and concentration of substances by a membrane separation method. Ultrafiltration technology is easy to operate, has low energy consumption, is easy to control and maintain, has mild separation conditions and high efficiency. However, as the use time prolongs, the retained impurities will continuously accumulate on the inner membrane wall of the membrane, resulting in concentration polarization and a decrease in ultrafiltration efficiency. When the impurity concentration on the membrane surface reaches the limit, a gel layer will form on the membrane surface, causing a decrease in the membrane ultrafiltrate permeation volume and the end of the membrane life, resulting in an increase in input costs and limiting its application.

[0004] Tangential flow ultrafiltration technology is a membrane technology that uses tangential flow for concentration, dialysis, and separation. The molecular weight cutoff range is usually 1 to 1000 KDa. On the basis of retaining the advantages of traditional ultrafiltration technology, the installation, cleaning, storage, and replacement of tangential flow technology equipment are convenient and quick. Unlike conventional wound membrane direct current filtration, in tangential flow filtration, the liquid flows tangentially through the membrane surface. The transmembrane pressure difference generated by the flow presses part of the solution through the filter membrane, while the retained part circulates back in the system. The membrane package used in the production of this technology has a uniform pore size distribution, accurate interception accuracy, high concentration or dialysis liquid exchange efficiency, small working volume, and high interception efficiency, which can maximize the product yield. During the entire purification process, the liquid flows continuously through the membrane surface at a certain speed. Compared with traditional direct current ultrafiltration technology, tangential flow ultrafiltration also flushes the membrane surface, making it difficult to form a gel layer on the membrane surface, so that the particles in the feed liquid will not quickly block the filter membrane, maintain a stable filtration speed, increase the life of the membrane, and save production input costs.

[0005] At present, most of the type I collagen is produced by industrial dialysis to remove impurities and retain the purified collagen. However, the dialysis purification method requires huge equipment, consumes a lot of dialysis reagents, has a long purification cycle, and is prone to contamination during the purification process. Therefore, it is a considerable investment in production equipment, production site, production environment, or purification raw material costs. Summary of the invention

[0006] In view of the shortcomings of the prior art, such as easy clogging of membrane packages, large equipment footprint, long production cycle and low purification efficiency, the present invention provides a method for extracting and purifying natural active collagen. The method for purifying type I collagen has the advantages of short time consumption, simple operation and low production input cost. It has been verified that the obtained collagen retains the activity of natural collagen, has a considerable yield, a complete molecular structure and high purity.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for extracting and purifying natural active collagen, comprising the following steps:

[0008] S1: Select fresh animal tissues with animal quarantine certificates and rich in type I collagen, freeze and slice them, wash, sterilize and defatted, then homogenize and break them. The homogenized and broken tissues are defatted and desugared again, and then centrifuged at high speed to obtain Achilles tendon fibers. Then, a combination of acid and protease is used to extract collagen under mild enzymatic conditions. After the extraction, sodium hydroxide solution is used to adjust the pH of the enzymatic solution to 5. The precipitate is the Achilles tendon fibers that have not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then the enzymatic supernatant is obtained by high-speed centrifugation;

[0009] S2: Add a high-concentration salt solution to the enzymatic hydrolysate supernatant obtained in step S1 until the salt concentration of the salting-out system reaches the specified value, then perform low-temperature salting out for 12 hours, centrifuge to obtain the precipitated protein, and then wash the precipitate with pure water multiple times, centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysate and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen.

[0010] S3: Filter the ultrafiltration stock solution of type I collagen obtained in step S2 through a pleated filter element at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration treatment. Use tangential flow ultrafiltration technology to purify and change the liquid at a pressure of 0.1 - 5 bar and a pump speed of 5 - 30 Hz. Continuously add the replacement liquid during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the completion of purification. Then use ultrapure water for ultrafiltration again until the pH value of the solution remains unchanged at 3.5, and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Freeze-dry the purified solution of type I collagen to obtain the collagen freeze-dried sponge.

[0011] Preferably, the protease is one or a combination of pepsin, ficin, papain, trypsin, pancreatin, collagenase.

[0012] Preferably, the acidic solution is any one of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, lactic acid.

[0013] Preferably, the mild enzymatic hydrolysis conditions are 4°C - 35°C and stirring for 96 h.

[0014] Preferably, the high-concentration salt solution is a 4.5 M NaCl aqueous solution; the salting-out system consists of the high-concentration salt solution and the enzymatic hydrolysate supernatant; the final salt concentration of the salting-out system is 0 mol / L - 2.5 mol / L.

[0015] Preferably, the concentration of the ultrafiltration stock solution of type I collagen is 1.5 mg / mL - 2.5 mg / mL, and the concentration of the purified solution of type I collagen is 3 mg / mL - 4 mg / mL.

[0016] Preferably, the pore size of the pleated filter element is 5 μm or 10 μm, the cut-off molecular weight of the ultrafiltration membrane in the pleated filter element is 10 kDa, the ultrafiltration membrane material is hydrophilic polyethersulfone or regenerated cellulose, and the ultrafiltration membrane is an internal pressure type ultrafiltration membrane or an external pressure type ultrafiltration membrane.

[0017] Preferably, the replacement liquid is hydrochloric acid or acetic acid.

[0018] The present invention provides a method for extracting and purifying natural active collagen, having the following beneficial effects:

[0019] 1. A method for purifying type I collagen by subjecting an enzymolysis solution to rough extraction, redissolving, and then ultrafiltration again. Compared with the current large-scale production technologies on the market, it can effectively shorten the purification time of type I collagen and improve the purity of collagen.

[0020] 2. The stepwise purification method adopted in the present invention enables the ultrafiltration purification to reach the required purity and concentration in only 5 to 8 hours. Compared with the existing purification technologies, it can greatly improve the purification efficiency, shorten the purification time, retain the protein activity, improve the production efficiency, and meet the requirements of industrialized large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic flow chart of a method for purifying natural active collagen of the present invention;

[0022] Figure 2 It is the viscosity test of enzymolysis solutions at different temperatures over time;

[0023] Figure 3 It is the change of SDS-PAGE gel electrophoresis light bands of enzymolysis solutions at different temperatures over time;

[0024] Figure 4 It is the circular dichroism (CD) spectrum analysis of collagen in enzymolysis systems at different temperatures;

[0025] Figure 5 It is the change of SDS-PAGE gel electrophoresis light bands of the influence of salting-out conditions on collagen purification;

[0026] Figure 6 It is the ultrafiltration purification experiment, where (a) is the number of liquid replacements and (b) is the purification time;

[0027] Figure 7 It is the collagen extraction rate;

[0028] Figure 8 It is the SDS-PAGE gel electrophoresis diagram for purity detection and process verification; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Referring to the attached Figure 1 , the present invention will be further described in conjunction with specific embodiments for better understanding of the present invention.

[0030] 1. Process flow design:

[0031] Example 1:

[0032] S1: Select fresh animal tissues with a qualified animal quarantine certificate and rich in type I collagen, freeze and slice them, and after washing, sterilizing, and degreasing, homogenize and crush them. The homogenized and crushed tissues are degreased and de-sugared again, and then tendon fibers are obtained by high-speed centrifugation. Then, a method combining acid and protease is used to extract collagen under mild enzymatic hydrolysis conditions. After extraction, the pH of the enzymatic hydrolysis solution is adjusted to 5 with sodium hydroxide solution. The precipitate is the tendon fibers that have not been completely homogenized, crushed, and enzymatically hydrolyzed. Then, the supernatant after enzymatic hydrolysis is obtained by high-speed centrifugation.

[0033] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1. After the salting-out system reaches the specified salt concentration, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times and centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis solution and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen;

[0034] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter element at a peristaltic pump speed of 160 r / min and then perform ultrafiltration treatment. Purify and change the solution using tangential flow ultrafiltration technology at a pressure of 0.1 - 5 bar and a pump speed of 5 - 30 Hz. During the purification process, continuously add the replacement solution and detect the change in the conductivity of the waste liquid at the permeate end of the replacement solution until the conductivity no longer changes, indicating the end of purification. Then, use ultrapure water for ultrafiltration again to keep the pH value of the solution at 3.5 without change and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Freeze-dry the purified solution of type I collagen to obtain a collagen freeze-dried sponge.

[0035] 2. Influence of extraction temperature on collagen extraction efficiency and activity:

[0036] Example 2:

[0037] S1: Select fresh bovine tendon tissues with a qualified animal quarantine certificate and rich in type I collagen, freeze and slice them, and after washing, sterilizing, and degreasing, homogenize and crush them. The homogenized and crushed tissues are degreased and de-sugared again, and then tendon fibers are obtained by high-speed centrifugation. Then, use 0.5 mol / L acetic acid solution and pepsin to stir at 5 °C for 96 hours for collagen extraction. After extraction, the pH of the enzymatic hydrolysis solution is adjusted to 5 with sodium hydroxide solution. The precipitate is the tendon fibers that have not been completely homogenized, crushed, and enzymatically hydrolyzed. Then, the supernatant after enzymatic hydrolysis is obtained by high-speed centrifugation;

[0038] S2: Add a high-concentration salt solution to the enzymatic hydrolysate supernatant obtained in S1 to make the NaCl concentration in the salting-out system 2.0 mol / L. Perform salting-out at low temperature for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water multiple times, centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen;

[0039] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 10 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration treatment. Use tangential flow ultrafiltration technology to purify and change the solution at a pressure of 1.5 bar and a pump speed of 25 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the completion of purification. Use ultrapure water to perform ultrafiltration again to keep the pH value of the solution at 3.5 without change, and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Lyophilize the purified solution of type I collagen to obtain a collagen lyophilized sponge.

[0040] Example 3:

[0041] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze it, slice it, and after cleaning, sterilization, and degreasing treatments, perform homogenization and crushing. The homogenized and crushed tissue is degreased and de-sugared again, and then Achilles tendon fibers are obtained by high-speed centrifugation. Then, use 0.5 mol / L acetic acid solution and pepsin to stir for 96 hours at 20 °C for collagen extraction. After the extraction is completed, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and crushed and not enzymatically hydrolyzed. Then, obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation;

[0042] S2: Add a high-concentration salt solution to the enzymatic hydrolysate supernatant obtained in S1 to make the NaCl concentration in the salting-out system 2.0 mol / L. Perform salting-out at low temperature for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water multiple times, centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen;

[0043] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 5 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration. Use tangential flow ultrafiltration technology to purify and change the solution at a pressure of 1.5 bar and a pump speed of 20 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the pH value of the solution remains unchanged at 3.5 after purification, and then perform concentration treatment to obtain the concentrated purified type I collagen solution. Freeze-dry the purified type I collagen solution to obtain a collagen freeze-dried sponge.

[0044] Example 4:

[0045] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze it, slice it, and after washing, sterilizing, and degreasing, perform homogenization and crushing. The homogenized and crushed tissue is degreased and de-sugared again, and then Achilles tendon fibers are obtained by high-speed centrifugation. Then, use 0.5 mol / L acetic acid solution and pepsin to stir at 35 °C for 96 hours for collagen extraction. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and not enzymatically hydrolyzed. Then, obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation;

[0046] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1 to make the NaCl concentration in the salting-out system 2.0 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times, centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis for stirring and re-dissolution to obtain the ultrafiltration stock solution of type I collagen;

[0047] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 5 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration. Use tangential flow ultrafiltration technology to purify and change the solution at a pressure of 2.5 bar and a pump speed of 17 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating that the purification is complete. Use ultrapure water for ultrafiltration again until the pH value of the solution remains unchanged at 3.5, and then perform concentration treatment to obtain the concentrated purified type I collagen solution. Freeze-dry the purified type I collagen solution to obtain a collagen freeze-dried sponge.

[0048] 3. Exploration of salting-out crude purification:

[0049] Example 5:

[0050] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen. Freeze and section it. After washing, sterilizing, and degreasing, homogenize and crush it. The homogenized and crushed tissue is degreased and de-sugared again. Then, obtain Achilles tendon fibers by high-speed centrifugation. Next, use 0.5 mol / L acetic acid solution and pepsin to stir for 96 hours at 20 °C for collagen extraction. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and not enzymatically hydrolyzed. Then, obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation;

[0051] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1 to make the NaCl concentration in the salting-out system 0 mol / L, and thus obtain the ultrafiltration stock solution of type I collagen;

[0052] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a 10-μm pore size pleated filter at a peristaltic pump speed of 160 r / min and then perform ultrafiltration treatment. Use tangential flow ultrafiltration technology to purify and change the solution at a pressure of 2.5 bar and a pump speed of 10 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the end of purification. Use ultrapure water to perform ultrafiltration again to keep the pH value of the solution at 3.5 without change and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Freeze-dry the purified solution of type I collagen to obtain a collagen freeze-dried sponge.

[0053] Example 6:

[0054] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen. Freeze and section it. After washing, sterilizing, and degreasing, homogenize and crush it. The homogenized and crushed tissue is degreased and de-sugared again. Then, obtain Achilles tendon fibers by high-speed centrifugation. Next, use 0.5 mol / L acetic acid solution and pepsin to stir for 96 hours at 20 °C for collagen extraction. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and not enzymatically hydrolyzed. Then, obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation;

[0055] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1 to make the NaCl concentration in the salting-out system 0.25 mol / L. Perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times and centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen;

[0056] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 5 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration. Purify and change the solution using tangential flow ultrafiltration technology at a pressure of 1.0 bar and a pump speed of 5 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the pH value of the solution remains unchanged at 3.5 after purification, and then perform concentration treatment to obtain the concentrated purified type I collagen solution. Freeze-dry the purified type I collagen solution to obtain a collagen freeze-dried sponge.

[0057] Example 7:

[0058] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze it, slice it, and after washing, sterilizing, and degreasing, perform homogenization and fragmentation. The homogenized and fragmented tissue is degreased and de-sugared again, and then Achilles tendon fibers are obtained by high-speed centrifugation. Then, collagen is extracted by stirring with 0.5 mol / L acetic acid solution and pepsin at 20 °C for 96 hours. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and fragmented and not enzymatically hydrolyzed. Then, the supernatant after enzymatic hydrolysis is obtained by high-speed centrifugation;

[0059] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1 to make the NaCl concentration in the salting-out system 0.5 mol / L, perform low-temperature salting out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times and centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen;

[0060] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 5 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration. Purify and change the solution using tangential flow ultrafiltration technology at a pressure of 1.5 bar and a pump speed of 10 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating that the purification is complete. Use ultrapure water to perform ultrafiltration again until the pH value of the solution remains unchanged at 3.5, and then perform concentration treatment to obtain the concentrated purified type I collagen solution. Freeze-dry the purified type I collagen solution to obtain a collagen freeze-dried sponge.

[0061] Example 8:

[0062] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze and slice it, homogenize and crush it after washing, sterilizing and degreasing, degrease and desugar the homogenized and crushed tissue again, then obtain Achilles tendon fibers by high-speed centrifugation. Next, use 0.5 mol / L acetic acid solution and pepsin to stir for 96 hours at 20 °C for collagen extraction. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and crushed and not enzymatically hydrolyzed. Then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation;

[0063] S2: Add a high-concentration salt solution to the enzymatic hydrolysate supernatant obtained in S1 to make the NaCl concentration in the salting-out system 0.75 mol / L, perform low-temperature salting out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times, centrifuge and filter again, and add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis for stirring and re-dissolution to obtain the ultrafiltration stock solution of type I collagen;

[0064] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a 10-μm pore size pleated filter element at a peristaltic pump speed of 160 r / min and then perform ultrafiltration treatment. Use tangential flow ultrafiltration technology to purify and change the solution at a pressure of 2.3 bar and a pump speed of 20 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the purification is complete. Use ultrapure water to perform ultrafiltration again to keep the pH value of the solution at 3.5 without change and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Freeze-dry the purified solution of type I collagen to obtain a collagen freeze-dried sponge.

[0065] Example 9:

[0066] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze and slice it, homogenize and crush it after washing, sterilizing and degreasing, degrease and desugar the homogenized and crushed tissue again, then obtain Achilles tendon fibers by high-speed centrifugation. Next, use 0.5 mol / L acetic acid solution and pepsin to stir for 96 hours at 20 °C for collagen extraction. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and crushed and not enzymatically hydrolyzed. Then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation;

[0067] S2: Add a high-concentration salt solution to the enzymatic hydrolysate supernatant obtained in S1 to make the NaCl concentration in the salting-out system 1.0 mol / L, perform low-temperature salting out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times, centrifuge and filter again, and add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis for stirring and re-dissolution to obtain the ultrafiltration stock solution of type I collagen;

[0068] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 10 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration. Purify and change the solution using tangential flow ultrafiltration technology at a pressure of 2.5 bar and a pump speed of 25 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the completion of purification. Then, use ultrapure water for ultrafiltration again to keep the pH value of the solution at 3.5 without change, and then perform concentration treatment to obtain the concentrated purified type I collagen solution. Freeze-dry the purified type I collagen solution to obtain a collagen freeze-dried sponge.

[0069] Example 10:

[0070] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze it, slice it, and after cleaning, sterilization, and degreasing treatments, perform homogenization and crushing. The homogenized and crushed tissue is degreased and de-sugared again, and then Achilles tendon fibers are obtained by high-speed centrifugation. Then, use 0.5 mol / L acetic acid solution and pepsin to stir at 20 °C for 96 hours for collagen extraction. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and crushed and not enzymatically hydrolyzed. Then, obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation;

[0071] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1 to make the NaCl concentration in the salting-out system 1.25 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times and centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis for stirring and re-dissolution to obtain the ultrafiltration stock solution of type I collagen;

[0072] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 10 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration. Purify and change the solution using tangential flow ultrafiltration technology at a pressure of 2.5 bar and a pump speed of 25 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the completion of purification. Then, use ultrapure water for ultrafiltration again to keep the pH value of the solution at 3.5 without change, and then perform concentration treatment to obtain the concentrated purified type I collagen solution. Freeze-dry the purified type I collagen solution to obtain a collagen freeze-dried sponge.

[0073] Example 11:

[0074] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze it, section it, and after washing, sterilizing, and degreasing, homogenize and crush it. The homogenized and crushed tissue is degreased and de-sugared again, and then Achilles tendon fibers are obtained by high-speed centrifugation. Then, 0.5 mol / L acetic acid solution and pepsin are used to stir for 96 hours at 20 °C for collagen extraction. After extraction, sodium hydroxide solution is used to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and crushed and not enzymatically hydrolyzed. Then, the supernatant after enzymatic hydrolysis is obtained by high-speed centrifugation;

[0075] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1 to make the NaCl concentration in the salting-out system 1.5 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then wash the precipitate with pure water multiple times and centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis for stirring and re-dissolution to obtain the ultrafiltration stock solution of type I collagen;

[0076] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a 10-μm pore size pleated filter element at a peristaltic pump speed of 160 r / min and then perform ultrafiltration treatment. Use tangential flow ultrafiltration technology to purify and change the solution at a pressure of 2.5 bar and a pump speed of 25 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the end of purification. Then, use ultrapure water for ultrafiltration again to keep the pH value of the solution at 3.5 without change and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Freeze-dry the purified solution of type I collagen to obtain a collagen freeze-dried sponge.

[0077] Example 12:

[0078] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze it, section it, and after washing, sterilizing, and degreasing, homogenize and crush it. The homogenized and crushed tissue is degreased and de-sugared again, and then Achilles tendon fibers are obtained by high-speed centrifugation. Then, 0.5 mol / L acetic acid solution and pepsin are used to stir for 96 hours at 20 °C for collagen extraction. After extraction, sodium hydroxide solution is used to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and crushed and not enzymatically hydrolyzed. Then, the supernatant after enzymatic hydrolysis is obtained by high-speed centrifugation;

[0079] S2: Add a high-concentration salt solution to the enzymatic hydrolysate supernatant obtained in S1 to make the NaCl concentration in the salting-out system 1.75 mol / L. Perform salting-out at low temperature for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water multiple times, centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen.

[0080] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 10 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration treatment. Use tangential flow ultrafiltration technology to purify and change the solution at a pressure of 2.5 bar and a pump speed of 25 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process and detect the change in the conductivity of the waste liquid at the permeate end of the replacement liquid until the conductivity no longer changes, indicating the completion of purification. Use ultrapure water to perform ultrafiltration again until the pH value of the solution remains unchanged at 3.5, and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Freeze-dry the purified solution of type I collagen to obtain a collagen freeze-dried sponge.

[0081] Example 13:

[0082] S1: Select fresh bovine Achilles tendon tissue with an animal quarantine certificate and rich in type I collagen, freeze it, slice it, and perform homogenization and fragmentation after washing, sterilization, and degreasing. The homogenized and fragmented tissue is degreased and de-sugared again, and then Achilles tendon fibers are obtained by high-speed centrifugation. Then, use 0.5 mol / L acetic acid solution and pepsin to stir at 20 °C for 96 hours for collagen extraction. After extraction, use sodium hydroxide solution to adjust the pH of the enzymatic hydrolysate to 5. The precipitate is the Achilles tendon fibers that were not completely homogenized and fragmented and not enzymatically hydrolyzed. Then, obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation.

[0083] S2: Add a high-concentration salt solution to the enzymatic hydrolysate supernatant obtained in S1 to make the NaCl concentration in the salting-out system 1.0 mol / L. Perform salting-out at low temperature for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water multiple times, centrifuge and filter again. Add the centrifuged flocculent protein to the same acidic solution as the enzymatic hydrolysis and stir to redissolve it to obtain the ultrafiltration stock solution of type I collagen.

[0084] S3: Filter the ultrafiltration stock solution of type I collagen obtained in S2 through a pleated filter with a pore size of 10 μm at a peristaltic pump speed of 160 r / min, and then perform ultrafiltration. Purify and change the solution using tangential flow ultrafiltration technology at a pressure of 2.5 bar and a pump speed of 10 Hz. Continuously add 0.05 mol / L acetic acid solution during the purification process until the same number of purification cycles as in Example 9 is reached, indicating the completion of purification. Ultrafilter again with ultrapure water until the pH value of the solution remains unchanged at 3.5, and then perform concentration treatment to obtain the concentrated purified solution of type I collagen. Freeze-dry the purified solution of type I collagen to obtain a collagen freeze-dried sponge.

[0085] 4. Viscosity test of enzymolysis solutions at different temperatures over time:

[0086] Test the viscosity changes of the enzymolysis solutions at each time point during the enzymolysis process in Examples 2 to 4. The samples are all enzymolysis solutions in the enzymolysis system, and the enzymolysis sampling points are 12, 24, 36, 48, 60, 72, 84, and 96 hours respectively. Perform rheological tests using a rheometer (Discover DHR-2). The diameter of the plate is 20 mm, the spacing is 1 mm, the fixed strain γ = 1%, and the scanning angular frequency range ω = 0.001 - 100 rad / s. The test results are shown in the appendix Figure 2 . On the basis of the same pre-treatment of enzymolysis and acid-enzyme ratio, explore different enzymolysis temperature systems. By adjusting the enzymolysis temperature, three systems, namely Example 2, Example 3, and Example 4, are obtained respectively. Measure the viscosities of the enzymolysis solutions at each time point under three different temperature conditions, and take a viscosity measurement every 12 hours. Use the viscosity at a shear rate of 0.01 as the standard for comparison and verification. Figure 2The results showed that as the temperature increased, compared with the enzymatic hydrolysis systems of Example 1 and Example 2, in the enzymatic hydrolysis system of Example 4, the rapid acid absorption and swelling viscosity of Achilles tendon fibers reached 47211.8 Pa·s within 12 hours at 35°C, and the enzymatic hydrolysis reaction proceeded rapidly with the extension of time. The telopeptides of collagen were rapidly excised. At the same time, the continuously hydrolyzed collagen in the swollen Achilles tendon had an increasing solubility in acetic acid, resulting in a continuous decrease in viscosity until the viscosity decreased to 48.374 Pa·s after 72 hours and basically no longer changed with the extension of time. In Example 2 and Example 3, the maximum acid absorption and swelling values were reached at 36 hours and 48 hours respectively, which were 101752 Pa·s and 89692.9 Pa·s respectively. Compared with the enzymatic hydrolysis system of Example 2, the 20°C enzymatic hydrolysis system of Example 3 underwent rapid enzymatic hydrolysis while absorbing acid and swelling, resulting in a delay in the swelling time. The enzymatic activity of the 5°C enzymatic hydrolysis system of Example 2 was relatively low, so it quickly reached the acid absorption and swelling value. As the enzymatic hydrolysis time extended, the soluble collagen in both systems continuously dissolved, leading to a slow decrease in their viscosities. Moreover, compared with the 5°C enzymatic hydrolysis system, the viscosity decrease trend was more obvious, while compared with the 35°C enzymatic hydrolysis system, the decrease trend was more gentle and controllable. In summary, among the three enzymatic hydrolysis systems, the 35°C enzymatic hydrolysis system had the most intense enzymatic hydrolysis conditions and could complete the enzymatic hydrolysis in a short time, but the enzymatic hydrolysis reaction was too intense to be controlled in the actual production process. The 5°C enzymatic hydrolysis system had the slowest enzymatic hydrolysis rate and a long reaction cycle, making it difficult to achieve mass production in the actual production process. The 20°C enzymatic hydrolysis system had mild enzymatic hydrolysis conditions and a suitable enzymatic hydrolysis cycle. After comparison, the 20°C enzymatic hydrolysis reaction conditions of Example 3 were selected for subsequent process exploration.

[0087] 5. SDS-PAGE gel electrophoresis light band changes of enzymatic hydrolysis solutions at different temperatures over time:

[0088] Test the collagen light band changes in the solution system at different time points during the enzymatic hydrolysis in Examples 2 to 4. The samples were all enzymatic hydrolysis solutions of the enzymatic hydrolysis systems, and the enzymatic hydrolysis sampling points were 24, 48, 72, 84, and 96 hours respectively. The light band changes were tested by SDS-PAGE gel electrophoresis. The thickness of the gel plate was 1.5 mm, the mass fraction of the stacking gel was 5%, the mass fraction of the separating gel was 8%, and the collagen loading concentration was 1 mg / mL. In the early stage, constant voltage electrophoresis was carried out at 80 V for about 40 min, and in the later stage, constant voltage electrophoresis was carried out for about 80 min until the bromophenol blue dye reached the bottom of the gel and the electrophoresis was stopped. The stacking gel was cut, and the separating gel was stained in Coomassie Brilliant Blue Fast Staining Solution P0017 for 10 min, decolorized with distilled water until the bands were clear, and the decolorized film was analyzed with the Bio-Rad Gel Imaging System Image Lab to record the band optical density. The test results are shown in the appendix Figure 3 。

[0089] On the basis of the same pre-treatment of enzymatic hydrolysis and acid-enzyme ratio, different enzymatic hydrolysis temperature systems were explored. By adjusting the enzymatic hydrolysis temperature, three systems of Examples 2, 3, and 4 were obtained. The enzymatic hydrolysates at each time point under 3 different temperature conditions were weighed, diluted with acid solution in the same ratio, and the changes in the light bands of collagen were analyzed. The same time points were selected for the comparison and verification of the changes in the bands and the optical density of the bands. Usually, type I collagen is a heterotrimer [ɑ1(Ⅰ)]2ɑ2(Ⅰ) composed of two different ɑ-chains, ɑ1(Ⅰ) and ɑ2(Ⅰ), with a molecular weight of about 300-400KDa, and the collagen after enzymatic digestion forms a third ɑ-chain ɑ3. The dimer peptide chain formed by two of the three ɑ-peptide chains of the type I collagen molecule is called the β-peptide chain, indicating that the triple helix structure of type I collagen is complete. Figure 3 The results showed that with the extension of time, the light bands at 100KDa - 130KDa in Example 2 and Example 3 changed from three to two, and the light brightness of the bands was also continuously increasing, indicating that the enzymatic hydrolysis process was ongoing. After comparison, in Example 3 compared with Example 2, the bands had become two within 72 hours, and the increase in optical density was more obvious, indicating a faster enzymatic hydrolysis rate. In Example 4, the bands had become two at 48 hours, and the optical density of the light bands hardly changed with the extension of time, and the optical density of the light bands at 96 hours was lower than that of Example 2 and Example 3, indicating that the enzymatic hydrolysis in Example 3 was faster, but the whole enzymatic hydrolysis process was too fast to control and there was a risk of over-enzymatic hydrolysis. To sum up, the changes in the light bands and density changes in the three enzymatic hydrolysis systems were consistent with the viscosity test results. After comparison, the enzymatic hydrolysis reaction conditions of 20°C in Example 3 were selected for subsequent process exploration.

[0090] 6. Analysis of the circular dichroism (CD) spectra of collagen in different temperature enzymatic hydrolysis systems:

[0091] The changes in the CD spectra of the freeze-dried sponges of collagen in the enzymatic hydrolysis systems of Examples 2 - 4 were tested. The samples were all freeze-dried products after 96 hours of enzymatic hydrolysis in each enzymatic hydrolysis system. The secondary structure of type I collagen was analyzed by comparison using a circular dichroism spectrometer (JASCO - 1700). Type I collagen with a concentration of 0.1mg / mL was prepared and scanned in the wavelength range of 190 - 250nm. The parameters were set as follows: cell thickness 1mm, scanning speed 50nm / min, scanning at room temperature, and slit width 1nm. The test results are shown in the appendix Figure 4 。

[0092] On the basis of the same enzymatic hydrolysis pretreatment and acid-enzyme ratio, different enzymatic hydrolysis temperature systems were explored. By adjusting the enzymatic hydrolysis temperature, three systems of Examples 2, 3, and 4 were obtained respectively. After the freeze-dried sponges of collagen in Example I of each group were redissolved with acetic acid solution, CD analysis was carried out, and the collagen concentration of each example group was the same. The special repetitive sequence structure of collagen results in its optical activity, and a CD characteristic map similar to the polyproline type II helix conformation of proteins will appear, that is, there is a weak positive Cotton effect between 225 nm and 250 nm, and a negative Cotton effect between 180 nm and 225 nm. When collagen is completely denatured, the ellipticity value at 225 nm - 250 nm decreases and the CD curve becomes smoother. Secondly, through the CD spectrum, the proportions of α-helix, β-sheet, β-turn and disordered folding in the secondary structure of collagen and standard type I collagen were obtained by DichroWeb - Online Circular Dichroism Analysis in Example 2 and Example 3 (Tables 1, 2, 3, and 4 are Example 2, Example 3, Example 4, and standard type I collagen respectively). Figure 4 The results show that with the continuous increase of temperature, obvious positive and negative Cotton effects appeared at 225 nm - 250 nm and 180 nm - 225 nm respectively in Example 2, Example 3, and Example 4, indicating that the native helical structure of the freeze-dried sponges of collagen obtained in each example was not damaged, and the enzymatic hydrolysis process well maintained the native conformation of collagen. But from Figure 3 we can see that with the continuous increase of temperature, the CD curve obviously shifts upward and becomes smoother, indicating that with the increase of temperature, although collagen maintains the native helical conformation, the stability of the helical conformation is also decreasing. Further analysis of the secondary structure ratio by DichroWeb - Online Circular Dichroism Analysis shows that the results in Tables 1 - 4 indicate that with the continuous increase of temperature, the secondary structure ratio decreases continuously, and compared with the standard type I collagen, the results of Example 3 are closer to those of the standard type I collagen in terms of both the CD curve and the total secondary structure content. To sum up, the CD curve measurement results of the three enzymatic hydrolysis systems are consistent with the viscosity test and SDS gel electrophoresis test results. After comparison, the enzymatic hydrolysis reaction conditions of 20 °C in Example 3 were selected for subsequent process exploration.

[0093] Table 1 Analysis data of the secondary results of the CD spectrum in Example 2

[0094] Result Helix 1 Helix 2 Fold 1 Fold 2 Corner Disorder Total Speculation 0.006 0.086 0.299 0.161 0.109 0.339 1 Singular Value Decomposition 0.048 0.113 0.185 0.103 0.025 0.312 0.786 Convergence 0.007 0.087 0.297 0.160 0.107 0.346 1.004 Stage 2 0.007 0.087 0.297 0.160 0.107 0.346 1.003 Final 0.007 0.087 0.292 0.160 0.107 0.346 1.005

[0095] Table 2 Analysis data of the secondary results of the CD spectrum in Example 3

[0096] Result Helix 1 Helix 2 Fold 1 Fold 2 Corner Disorder Total Speculation 0.006 0.086 0.299 0.161 0.109 0.339 1 Singular Value Decomposition 0.059 0.119 0.157 0.088 0.004 0.309 0.736 Convergence 0.008 0.087 0.294 0.159 0.106 0.343 0.997 Stage 2 0.007 0.086 0.294 0.159 0.105 0.341 0.993 Final 0.007 0.087 0.294 0.159 0.105 0.341 0.994

[0097] Table 3 Analysis data of the secondary CD spectrum results in Example 4

[0098] Result Helix 1 Helix 2 Fold 1 Fold 2 Corner Disorder Total Speculation 0.006 0.086 0.299 0.161 0.109 0.339 1 Singular Value Decomposition 0.053 0.116 0.178 0.099 0.020 0.319 0.785 Convergence 0.007 0.087 0.296 0.160 0.107 0.342 0.999 Stage 2 0.007 0.087 0.295 0.160 0.107 0.342 0.997 Final 0.011 0.089 0.287 0.155 0.099 0.337 0.978

[0099] Table 4 Analysis data of the secondary CD spectrum results of standard type I collagen

[0100] Result Helix 1 Helix 2 Fold 1 Fold 2 Corner Disorder Total Speculation 0.006 0.086 0.299 0.161 0.109 0.339 1 Singular Value Decomposition 0.099 0.142 0.034 0.024 -0.019 0.279 0.559 Convergence 0.009 0.087 0.292 0.158 0.102 0.342 0.99 Stage 2 0.009 0.087 0.292 0.157 0.102 0.341 0.989 Final 0.009 0.087 0.292 0.157 0.102 0.341 0.989

[0101] 7. Analysis of the influence of salting-out conditions on collagen purification:

[0102] Test the influence of salting-out concentration on the extraction rate of collagen in Examples 5 - 12. The samples are the supernatants after salting out with equal volume but different concentrations in each salting-out system. Test the change of protein light bands in the supernatant by SDS-PAGE gel electrophoresis. The thickness of the gel plate is 1.5 mm, the mass fraction of the stacking gel is 5%, the mass fraction of the separating gel is 10%. Dilute the centrifuged supernatant three times for sample preparation. Carry out constant voltage electrophoresis at 80 V for about 40 min in the early stage and about 80 min in the later stage until the bromophenol blue dye reaches the bottom of the gel to stop electrophoresis. Cut the stacking gel, stain the separating gel in the Coomassie brilliant blue rapid staining solution P0017 for 10 min, decolorize with distilled water until the bands are clear, and analyze the decolorized film with the Bio-Rad gel imaging system Image Lab to record the optical density of the bands. The test results are shown in the appendix Figure 5 。

[0103] On the basis of the previous exploration of temperature extraction, the influence of different salting-out concentrations on the precipitation efficiency of type I collagen in this process system was carried out. Nine systems including Example 3 and Examples 5 - 12 were obtained by adjusting the same volume with different salting-out concentrations. Take the supernatants after centrifugation of the salting-out solutions of 9 different salting-out systems with equal volume, dilute them with acid solution in equal volume, analyze the change of the total light bands, and compare and verify the total light band density of each group (3, 5 - 12 in the figure represent Example 3 and Examples 5 - 12 respectively). Figure 5The results showed that under the same extraction process conditions, as the salting-out concentration continued to increase, collagen was continuously precipitated. When the salting-out concentration reached 0 M to 0.75 M, multiple protein bands could be seen on the colloid, and as the salting-out concentration increased, the total light band density in the supernatant of the salting-out solution continuously decreased. When the concentration reached 1.0 M, the protein light bands became three, and as the salting-out concentration increased further, the number of bands no longer changed, nor did the light intensity. This result indicated that as the salt concentration continued to increase, the amount of protein precipitation continuously increased, and when the salt concentration in the salting-out system reached 1.0 M, the protein reached the maximum precipitation amount and no longer changed as the salting-out concentration continued to increase. In summary, among the 9 salting-out systems, when the salting-out concentration reached 1.0 M in Example 10, the amount of protein precipitation in the salting-out system was the largest. In the actual production process, the input amount of salt raw materials was lower than the commonly used 2.0 M and 4.0 M in the traditional process, which could effectively reduce the time required for later purification and desalination and shorten the process cycle.

[0104] 8. Ultrafiltration purification test:

[0105] During the ultrafiltration process of the type I collagen ultrafiltrate in Examples 2 to 12, the liquid replacement multiple, the conductivity of the replaced liquid, and the purification time were recorded, and the optimal extraction and purification examples were obtained by comparing among Examples 2 to 12. The ultrafiltration membrane material was hydrophilic polyethersulfone, the ultrafiltration system was tangential flow ultrafiltration, the pressure of the membrane package during the ultrafiltration process was 0.1 - 5 bar, and the feed pump speed frequency was 5 - 30 Hz. The ultrafiltration exchange liquid was 0.05 mol / L acetic acid solution and ultrapure water. The conductivity of the permeate end of the replacement liquid was measured once for each one-fold volume replacement, and the actual liquid replacement multiple time was recorded according to the material state and purification efficiency. The actual production efficiency of the purified product was calculated based on the total protein content of the Achilles tendon. The test results are shown in the appendix Figure 6 、 Figure 7 。

[0106] Based on various previous detection and analysis, the purification efficiencies of the type I collagen raw material solutions in 11 groups from Examples 2 to 12 were compared. Figure 6 , Figure 7The results show that during the exploration of the extraction temperature in Examples 2 to 4, the purification efficiency of the type I collagen raw material solution is related to the extraction rate of collagen. The higher the extraction rate of collagen, the more times of purification liquid replacement, the longer the purification time, and the higher the yield. During the exploration of salting-out crude purification in Examples 5 to 12, the purification efficiency of the type I collagen raw material solution is related to the precipitation amount of collagen. In the salt concentration range of 0.25 to 0.75 mol / L, the lower the salt concentration, the lower the precipitation rate of collagen, the lower the number of purification liquid replacements, the shorter the purification time, and the lower the collagen yield. When the salt concentration reaches 0.75 to 2.0 mol / L, the precipitation of collagen reaches the maximum and does not change with the increase of salt concentration. Within this range, the protein yield does not change, while the purification exchange liquid multiple and purification time increase with the increase of salt concentration. In summary, the extraction efficiency and salting-out recovery efficiency of collagen in the 11 groups of examples have a greater impact on the number of purification liquid replacements and purification liquid replacement time in the later stage, that is, the purification efficiency. Therefore, considering comprehensively, Example 10 is the optimal example among all the extraction example groups.

[0107] 9. Purity detection and process verification:

[0108] Test the purity of the freeze-dried collagen product obtained in Example 10. The sample is the freeze-dried collagen sponge obtained in Example 10. According to the pharmaceutical industry standard of the People's Republic of China, "YY / T 1453-2016, Characterization Method of Type I Collagen for Tissue Engineering Medical Devices", SDS gel electrophoresis was used to determine the purity of type I collagen. The thickness of the gel plate is 1.5 mm, the mass fraction of the stacking gel is 4%, and the mass fraction of the separating gel is 7%. The freeze-dried collagen sponge was used to prepare a protein concentration of 1 mg / mL (sample 2), the collagenase digestion solution of the same concentration of collagen (sample 4), and the enzyme digestion solution with the same enzyme concentration as the digestion solution (sample 5). The concentrations of bovine serum albumin (BSA) are 0.025 mg / mL, 0.015 mg / mL, 0.005 mg / mL, and 0.001 mg / mL (samples 6, 7, 8, 9) respectively. 20 μm of each sample was loaded per well. In the early stage, constant voltage electrophoresis was carried out at 80 V for about 40 min, and in the later stage, constant voltage electrophoresis was carried out for about 80 min until the bromophenol blue dye reached the bottom of the gel and electrophoresis was stopped. Cut the stacking gel, stain the separating gel in the Coomassie brilliant blue rapid staining solution P0017 for 10 min, decolorize with distilled water until the bands are clear, and analyze the decolorized film with the Bio-Rad gel imaging system Image Lab imaging analysis system to record the optical density of the bands. The test results are shown in the appendix Figure 8 .

[0109] Taking advantage of the triple-helix structure unique to collagen among other proteins, combined with the action of specific collagenase, the total amount of contaminating proteins in a collagen sample was detected by determining the staining limit of Coomassie Brilliant Blue for bovine serum albumin (BSA). The purity of the collagen freeze-dried sponge obtained by the process of Example 9 was detected and the process was verified. Sample preparation was carried out according to "YY / T 1453-2016: Characterization Methods for Type I Collagen in Tissue Engineering Medical Device Products", where Sample 2 was standard Type I collagen (1 mg / mL), and Sample 4 was the collagen freeze-dried sample (1 mg / mL) obtained under the same number of purification cycles as in Example 13 and Example 9. Figure 8 The results showed that under the same number of ultrafiltration purification cycles, the collagen freeze-dried product obtained in Example 9 had a higher purity. Type I collagen maintained its intact natural triple-helix structure, with the number of bands and molecular weight being the same as those of the standard Type I collagen, and the gel background being the same as that of the standard protein without any other contaminating bands. This indicated that the Type I collagen obtained by this extraction and purification method had a low content of contaminating proteins and a high purity. Calculated according to "YY / T 1453-2016: Characterization Methods for Type I Collagen in Tissue Engineering Medical Device Products", the collagen purity could reach 99.81% of the total protein content.

[0110] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for extracting and purifying natural active collagen, characterized in that: The steps include: S1: Select fresh animal tissues with animal quarantine certificates and rich in type I collagen, freeze and slice them, wash, sterilize and defatted, then homogenize and break them. The homogenized and broken tissues are defatted and desugared again, and then centrifuged at high speed to obtain Achilles tendon fibers. Then, collagen is extracted under mild enzymatic conditions using a combination of acidic solution and protease. After the extraction, the pH of the enzymatic solution is adjusted to 5 using sodium hydroxide solution. The precipitate is the Achilles tendon fibers that have not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then the enzymatic supernatant is obtained by high-speed centrifugation. S2: adding a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in step S1, so that the salting-out system reaches a specified salt concentration, and then salting out at low temperature for 12 hours, centrifuging to obtain the precipitated protein, and then washing the precipitate with pure water for multiple times and centrifuging again, adding the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis solution, stirring and re-dissolving, and obtaining type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in step S2 is filtered through a folded filter element at a peristaltic pump speed of 160 r / min and then ultrafiltered. The tangential flow ultrafiltration technology is used to purify the replacement solution at a pressure of 0.1 to 5 bar and a pump speed of 5 to 30 Hz. During the purification process, replacement fluid is continuously added and the conductivity change of the waste liquid at the outlet of the replacement fluid is detected until the conductivity no longer changes, indicating that the purification is completed. Ultrapure water is used for ultrafiltration again so that the pH value of the solution is maintained at 3.5 and no longer changes, and then concentrated to obtain a concentrated type I collagen purified solution. The type I collagen purified solution is freeze-dried to obtain a collagen freeze-dried sponge; In step S1, the protease is pepsin, the acidic solution is acetic acid solution, the mild enzymatic hydrolysis conditions are 4°C to 35°C, and the stirring is for 96 hours; The high concentration salt solution in step S2 is a 4.5M NaCl aqueous solution; the salting-out system consists of a high concentration salt solution and an enzymatic hydrolysis supernatant; when the final salt concentration of the salting-out system is 1.0 mol / L, the protein reaches the maximum precipitation amount; In step S2, the concentration of the type I collagen ultrafiltration stock solution is 1.5 mg / mL to 2.5 mg / mL, and the concentration of the type I collagen purified solution is 3 mg / mL to 4 mg / mL.

2. The method for extracting and purifying natural active collagen according to claim 1, characterized in that: The pore size of the folded filter element is 5 μm or 10 μm, the molecular weight cutoff of the ultrafiltration membrane in the folded filter element is 10 kDa, the ultrafiltration membrane material is hydrophilic polyethersulfone or regenerated cellulose, and the ultrafiltration membrane is an internal pressure ultrafiltration membrane or an external pressure ultrafiltration membrane.

3. The method for extracting and purifying natural active collagen according to claim 1, characterized in that: The replacement fluid is hydrochloric acid or acetic acid.

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