Method for extracting and purifying natural active collagen

Abstract

The invention discloses a method for extracting and purifying natural active collagen, particularly relates to a method for purifying type I collagen, and belongs to the technical field of collagen production. By adopting a purification scheme of salting-out and ultrafiltration purification liquid change, high-purity and high-activity natural I-type collagen can be successfully obtained, and the problems of low biological activity and low protein purity caused by long collagen purification period and high non-target protein content in the traditional purification technology are solved. The method for purifying the I-type collagen mainly comprises the following steps: S1, carrying out pretreatment on raw materials, carrying out reaction by adopting an enzymolysis technology, and then obtaining I-type collagen enzymatic hydrolysate by adopting a centrifugation method; s2, preliminarily removing soluble impure protein, pepsin and other impurities by using a salting-out process; and S3, redissolving the crude extract, filtering through a polypropylene folded filter element to obtain a raw material solution, purifying the raw material solution through a tangential flow ultrafiltration method, and concentrating to obtain the I-type collagen purified solution.

Description

Technical Field

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

[0002] As the protein with the highest content and widest distribution in nature, collagen has become a resource-rich industrial raw material, of which type I collagen accounts for up to 90% of the total collagen content. Type I collagen is widely present in the skin, bones, tendons, ligaments, spine, blood vessel walls and other parts as a tissue support in the form of collagen fibers. As the main component of the extracellular matrix, it not only gives each tissue stable mechanical properties, but also gives it the ability to promote cell growth and migration. It also plays an important role in the physiological and pathological processes of cells, tissues and even the entire body. Type I collagen has good biocompatibility, low immunogenicity, biodegradability, and the ability to promote cell proliferation, differentiation and migration, promote platelet aggregation and wound healing. Nowadays, it has been widely used in the clinic and marketed 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, cornea, nerve repair and drug carriers. Type I collagen is the main protein component of human skin, which has the functions of defense, support, protection and nutrition. With the increase of age, its regeneration ability will continue to decline, resulting in the loss of skin collagen, cross-linking and solidification inside the collagen fiber molecules, and the decrease of the content of glycosaminoglycan in the extracellular matrix, which makes the skin lose its elasticity and luster, resulting in the loss of elasticity and luster of skin aging, and the appearance of spots and wrinkles. Therefore, type I collagen is extremely popular in many fields such as medical beauty, medicine, and cosmetics.

[0003] Ultrafiltration is a pressurized membrane separation technology that utilizes the microporous structure of a semipermeable membrane. That is, by using external / internal pressure as a driving force, only soluble substances or solutions such as proteins that are larger than a specific pore size are allowed to pass through the membrane, so that large molecular soluble substances are retained and cannot pass through, thereby achieving a membrane separation method for selective recovery, separation, classification, purification and concentration of substances. 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 increases, the trapped impurities will continue to accumulate on the membrane wall inside 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 the ultrafiltrate permeability of the membrane to decrease and the membrane life to reach the end, which will increase the investment cost and limit 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: 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; 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 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology is used to purify the replacement solution at a pressure of 0.1-5bar and a pump speed of 5-30Hz. During the purification process, replacement fluid is continuously added and the change in conductivity of the waste liquid at the outlet of the replacement fluid is detected until the conductivity no longer changes, indicating that the purification is complete. Ultrapure water is used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer changes, and then it is concentrated to obtain concentrated type I collagen purified solution. The type I collagen purified solution is freeze-dried to obtain collagen freeze-dried sponge.

[0008] Preferably, the protease is one or more combinations of pepsin, ficin, papain, trypsin, pancreatin, and collagenase.

[0009] Preferably, the acidic solution is any one of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, and lactic acid.

[0010] Preferably, the mild enzymatic hydrolysis conditions are 4°C to 35°C, stirring for 96 hours.

[0011] Preferably, the high-concentration salt solution is a 4.5M NaCl aqueous solution; the salting-out system consists of a high-concentration salt solution and an enzymatic hydrolysis supernatant; and the final salt concentration of the salting-out system is 0 mol / L to 2.5 mol / L.

[0012] Preferably, the concentration of the type I collagen ultrafiltration stock solution is between 1.5 mg / mL and 2.5 mg / mL, and the concentration of the type I collagen purified solution is between 3 mg / mL and 4 mg / mL.

[0013] Preferably, the pore size of the pleated filter element is 5 μm or 10 μm, the molecular weight cutoff 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 ultrafiltration membrane or an external pressure ultrafiltration membrane.

[0014] Preferably, the replacement fluid is hydrochloric acid or acetic acid.

[0015] The present invention provides a method for extracting and purifying natural active collagen, which has the following beneficial effects: 1. The present invention provides a method for purifying type I collagen by crudely extracting an enzymatic hydrolysate, re-dissolving it, and then ultrafiltering it again. Compared with the current large-scale production technology on the market, the method can effectively shorten the purification time of type I collagen and improve the purity of collagen.

[0016] 2. The step-by-step purification method adopted in the present invention allows ultrafiltration purification to achieve the required purity and concentration in just 5 to 8 hours. Compared with the existing purification technology cycle, it can greatly improve its purification efficiency, shorten its purification time, retain protein activity, improve production efficiency, and meet the needs of industrial expansion of production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of a natural active collagen purification method of the present invention; Figure 2 It is a test of the viscosity of the enzymatic hydrolysate at different temperatures over time; Figure 3 The light band changes of SDS-PAGE gel electrophoresis with time of enzyme hydrolysate at different temperatures; Figure 4 This is the circular dichroism (CD) spectrum analysis of collagen in enzymatic hydrolysis systems at different temperatures; Figure 5 The effect of salt precipitation conditions on the changes of light bands in SDS-PAGE gel electrophoresis. Figure 6 This is an ultrafiltration purification test, where (a) is the number of liquid changes and (b) is the purification time; Figure 7 is the collagen extraction rate; Figure 8 SDS-PAGE gel electrophoresis diagram for purity detection and process verification. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Figure 1 , the present invention is further described in conjunction with specific embodiments to provide a better understanding of the present invention.

[0019] 1. Process design: Embodiment 1: 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 disrupt them. The homogenized and disrupted 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 acid 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 completely homogenized and disrupted, and then the supernatant after enzymatic hydrolysis is obtained by high-speed centrifugation.

[0020] S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1, so that the salting-out system reaches a specified salt concentration and then salts out at low temperature for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis solution, stir and re-dissolve, and obtain type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 is filtered through a folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology is used to purify the replacement solution at a pressure of 0.1-5bar and a pump speed of 5-30Hz. During the purification process, the replacement solution is continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution is detected until the conductivity no longer changes, and the purification is completed. Ultrapure water is used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change, and then concentrated to obtain the concentrated type I collagen purified solution. The type I collagen purified solution is freeze-dried to obtain a collagen freeze-dried sponge.

[0021] 2. Effect of extraction temperature on collagen extraction efficiency and activity: Embodiment 2: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 5°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 2.0 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 1.5bar and a pump speed of 25Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0022] Embodiment 3: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 2.0 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2: was filtered through a 5μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 1.5bar and a pump speed of 20Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the pH value of the solution was maintained at 3.5 and no longer changed. The concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0023] Embodiment 4: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 35°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 2.0 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 5μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 2.5bar and a pump speed of 17Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0024] 3. Salting out crude purification exploration: Embodiment 5: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: adding a high-concentration salt solution to the enzymatic supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 0 mol / L, thereby obtaining a type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10 μm folded filter element at a peristaltic pump speed of 160 r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 2.5 bar and a pump speed of 10 Hz. During the purification process, 0.05 mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0025] Embodiment 6: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 0.25 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 5μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 1.0bar and a pump speed of 5Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the pH value of the solution was maintained at 3.5 and no longer changed after purification. The concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0026] Embodiment 7: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 0.5 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 5μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 1.5bar and a pump speed of 10Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0027] Embodiment 8: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 0.75 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 2.3bar and a pump speed of 20Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0028] Embodiment 9: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 1.0 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 2.5bar and a pump speed of 25Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0029] Embodiment 10: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 1.25 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 2.5bar and a pump speed of 25Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0030] Embodiment 11: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 1.5 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 2.5bar and a pump speed of 25Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0031] Embodiment 12: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 1.75 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10μm folded filter element at a peristaltic pump speed of 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the replacement solution at a pressure of 2.5bar and a pump speed of 25Hz. During the purification process, 0.05mol / L acetic acid solution was continuously added and the conductivity change of the waste liquid at the outlet of the replacement solution was detected until the conductivity no longer changed. The purification was completed, and ultrapure water was used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer change. Then, the concentrated type I collagen purified solution was obtained. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0032] Embodiment 13: S1: Select fresh bovine Achilles tendon tissue with animal quarantine certificate and rich in type I collagen, freeze and slice it, wash, sterilize and defatted, then homogenize and break it, defatted and desugared the homogenized and broken tissue again, and then obtain Achilles tendon fibers by high-speed centrifugation, then use 0.5 mol / L acetic acid solution and pepsin to stir at 20°C for 96 hours to extract collagen, and use sodium hydroxide solution to adjust the pH of the enzymatic solution to 5 after extraction. The precipitate is the Achilles tendon fiber that has not been enzymatically hydrolyzed after incomplete homogenization and disruption, and then obtain the supernatant after enzymatic hydrolysis by high-speed centrifugation; S2: Add a high-concentration salt solution to the enzymatic hydrolysis supernatant obtained in S1, so that the NaCl concentration of the salting-out system is 1.0 mol / L, perform low-temperature salting-out for 12 hours, centrifuge to obtain the precipitated protein, then rinse the precipitate with pure water for multiple times and centrifuge again, add the flocculent protein after centrifugation to the same acidic solution as the enzymatic hydrolysis, stir and re-dissolve, and obtain the type I collagen ultrafiltration stock solution; S3: The type I collagen ultrafiltration stock solution obtained in S2 was filtered through a 10 μm pore size folded filter element at a peristaltic pump speed of 160 r / min and then ultrafiltered. The tangential flow ultrafiltration technology was used to purify the liquid at a pressure of 2.5 bar and a pump speed of 10 Hz. During the purification process, 0.05 mol / L acetic acid solution was continuously added until the number of purification cycles was the same as in Example 9, and the purification was completed. Ultrapure water was used to ultrafilter again so that the pH value of the solution was maintained at 3.5 and no longer changed, and then concentrated to obtain the concentrated type I collagen purified solution. The type I collagen purified solution was freeze-dried to obtain a collagen freeze-dried sponge.

[0033] 4. Test on viscosity of enzymatic hydrolysate at different temperatures over time: The viscosity of the enzymatic hydrolyzate at each time point during the enzymatic hydrolysis process in test examples 2 to 4 was changed. The samples were all enzymatic hydrolyzates of the enzymatic hydrolysis system, and the enzymatic hydrolysis sampling points were 12, 24, 36, 48, 60, 72, 84 and 96 hours, respectively. The rheological test was performed by rheology (Discover DHR~2). The plate diameter was 20 mm, the spacing was 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 attached Figure 2 On the basis of the same ratio of acid to enzyme in pre-enzyme treatment, different enzymolysis temperature systems were explored, and by adjusting the enzymolysis temperature, three systems of Examples 2, 3, and 4 were obtained respectively. The viscosity of the enzymolysis solution at each time point under three different temperature conditions was measured, and the viscosity was measured once every 12 hours, and the viscosity at a shear rate of 0.01 was used as the standard for comparison and verification. Figure 2The results show that as the temperature rises, the enzymatic hydrolysis system of Example 4, compared with the enzymatic hydrolysis systems of Examples 1 and 2, rapidly absorbs acid and expands the Achilles tendon fibers within 12 hours at 35°C, and the viscosity reaches 47211.8 Pa.s. As time goes by, the enzymatic hydrolysis reaction proceeds rapidly, and the collagen terminal peptide is rapidly removed. At the same time, the solubility of the expanded Achilles tendon collagen in acetic acid continues to increase, causing the viscosity to decrease continuously, until the viscosity decreases to 48.374 Pa.s after 72 hours, and basically no longer changes with time. Example 2 and Example 3 reach the maximum acid absorption and expansion values ​​of 101752 Pa.s and 89692.9 Pa.s at 36 hours and 48 hours, respectively. Compared with the enzymatic hydrolysis system of Example 2, the 20°C enzymatic hydrolysis system of Example 3 rapidly undergoes enzymatic hydrolysis while absorbing acid and swelling, thereby delaying the swelling time, while the 5°C enzymatic hydrolysis system of Example 2 has low enzyme activity and therefore quickly reaches the acid absorption and swelling value, and the soluble collagen of both is continuously dissolved as the enzymatic hydrolysis time is prolonged, resulting in a slow decrease in viscosity, and the viscosity decrease trend is more obvious compared to the 5°C enzymatic hydrolysis system, while the decrease trend relative to the 35°C enzymatic hydrolysis system is more moderate and controllable. In summary, among the three enzymatic hydrolysis systems, the enzymatic hydrolysis conditions of the 35°C enzymatic hydrolysis system are the most intense, and the enzymatic hydrolysis can be completed in a short time, but the enzymatic hydrolysis reaction is too intense and difficult to control in the actual production process, the enzymatic hydrolysis of the 5°C enzymatic hydrolysis system is the slowest, but the reaction cycle is long and it is difficult to achieve mass production in the actual production process, and the enzymatic hydrolysis conditions of the 20°C enzymatic hydrolysis system are mild, and the enzymatic hydrolysis cycle is suitable. After comparison, the 20°C enzymatic hydrolysis reaction conditions of Example 3 were selected for subsequent process exploration.

[0034] 5. Changes in SDS-PAGE gel electrophoresis bands of enzyme hydrolysates at different temperatures over time: The changes in the light bands of collagen in the solution system at different time points during the enzymatic hydrolysis process in test examples 2 to 4. The samples were all enzymatic hydrolyzates of the enzymatic hydrolysis system, and the enzymatic hydrolysis sampling points were 24, 48, 72, 84 and 96 hours, respectively. The light band change test was carried out by SDS-PAGE gel electrophoresis. The thickness of the gel plate is 1.5 mm, the mass fraction of the concentrated gel is 5%, the mass fraction of the separation gel is 8%, and the collagen loading concentration is 1 mg / mL. The initial constant voltage electrophoresis is 80V for about 40 minutes, and the later constant voltage electrophoresis is about 80 minutes, until the bromophenol blue dye stops electrophoresis at the bottom of the colloid. The concentrated gel was cut, and the separation gel was stained in Coomassie Brilliant Blue Rapid Staining Solution P0017 for 10 minutes, and distilled water was decolorized until the bands were clear. The decolorized film was analyzed using the Bio-Rad Gel Imager Image Lab image analysis system to record the band optical density. The test results are attached. Figure 3 .

[0035] On the basis of the same ratio of acid and enzyme before enzymatic hydrolysis, different enzymatic hydrolysis temperature systems were explored, and by adjusting the enzymatic hydrolysis temperature, three systems of Examples 2, 3, and 4 were obtained. The enzymatic hydrolyzate at each time point under three different temperature conditions was weighed and diluted with acid solution in the same proportion to analyze the changes in the light bands of collagen, and the same time point was selected for comparison and verification of the changes in the bands and band optical density. Normally, 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 enzyme cleavage forms a third ɑ chain ɑ3. The dimer peptide chain formed by two ɑ peptide chains in the three ɑ peptide chains of type I collagen molecules is called a β peptide chain, indicating that the triple helix structure of type I collagen is complete. Figure 3 The results show that as time goes on, the light stripes of 100KDa to 130KDa in Example 2 and Example 3 change from three to two, and the brightness of the stripes is also increasing, indicating that the enzymatic process is constantly going on, and by comparison, compared with Example 2, Example 3 has become two stripes within 72 hours, and the increase in optical density is more obvious, indicating that the enzymatic speed is faster. In Example 4, the stripes have become two in 48 hours, and the density of the light stripes hardly changes as time goes on, and the light stripe density is lower than that of Example 2 and Example 3 after 96 hours, indicating that Example 3 is faster in enzymatic hydrolysis, but the entire enzymatic hydrolysis process is too fast to control, and there is a risk of over-enzymatic hydrolysis. In summary, the changes in light stripes and density in the three enzymatic hydrolysis systems are consistent with the viscosity test results. After comparison, the 20°C enzymatic hydrolysis reaction conditions of Example 3 were selected for subsequent process exploration.

[0036] 6. Analysis of collagen circular dichroism (CD) spectra of enzymatic hydrolysis systems at different temperatures: The CD spectrum of the collagen freeze-dried sponge in the enzymatic hydrolysis system in test examples 2 to 4 was changed. The samples were all freeze-dried products after 96 hours of enzymatic hydrolysis in each group of enzymatic hydrolysis systems. The secondary structure of type I collagen was compared and analyzed by circular dichroism spectrometer (JASCO-1700). Type I collagen was prepared with a concentration of 0.1 mg / mL and scanned in the wavelength range of 190-250 nm. Setting parameters: cuvette thickness 1 mm, scanning speed 50 nm / min, room temperature scanning, slit width 1 nm. See attached for test results. Figure 4 .

[0037] On the basis of the same ratio of pre-enzymatic treatment and acid enzyme, different enzymatic hydrolysis temperature systems were explored, and by adjusting the enzymatic hydrolysis temperature, three systems of Examples 2, 3, and 4 were obtained respectively. The collagen freeze-dried sponges of each group of Example I were re-dissolved with acetic acid solution and then subjected to CD analysis, and the collagen concentration of each example group was the same. The special repeating sequence structure of collagen causes it to be optically active, and a CD characteristic graph similar to the polyproline II type helical conformation of protein will appear, that is, there is a weak positive Colton effect between 225nm and 250nm, and a negative Colton effect between 180nm and 225nm. When the collagen is completely denatured, the ellipticity value at 225nm to 250nm decreases, and the CD curve becomes smooth. Secondly, the CD spectrum was analyzed by DichroWeb~Online Circular Dichroism Analysis to obtain the proportion composition of α helix, β fold, β turn and disordered fold in the secondary structure of collagen and standard type I collagen in Example 2 and Example 3 (Table 1, Table 2, Table 3 and Table 4 are Example 2, Example 3, Example 4 and standard type I collagen respectively). Figure 4 The results show that as the temperature continues to rise, Examples 2, 3 and 4 all show obvious positive and negative Colton effects at 225nm-250nm and 180nm-225nm, respectively, indicating that the natural helical structure of the freeze-dried collagen sponges obtained in each example is not destroyed, and the enzymatic hydrolysis process well maintains the natural conformation of collagen. Figure 3 We can see that as the temperature continues to rise, the CD curve obviously shifts upward and becomes smoother, indicating that as the temperature rises, although collagen maintains its natural helical conformation, the stability of the helical conformation is also decreasing. The secondary structure ratio was further analyzed by DichroWeb~Online Circular Dichroism Analysis. The results in Tables 1 to 4 show that as the temperature continues to rise, the secondary structure ratio continues to decrease, and compared with the standard type I collagen, both the CD curve and the total content of the secondary structure in Example 3 are closer to the standard type I collagen results. In summary, 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 20°C enzymatic hydrolysis reaction conditions of Example 3 were selected for subsequent process exploration.

[0038] Table 1 CD spectrum secondary result analysis data in Example 2 result Spiral 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 Phase 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 Table 2 CD spectrum secondary result analysis data in Example 3 result Spiral 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 Phase 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 Table 3 CD spectrum secondary result analysis data in Example 4 result Spiral 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 Phase 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 Table 4 Secondary analysis data of standard type I collagen CD spectrum result Spiral 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 Phase 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 7. Analysis of the effect of salting-out conditions on collagen purification: Test Examples 5 to 12 for the effect of salting-out concentration on the extraction rate of collagen. All samples were supernatants after salting-out of equal volumes and different concentrations in each group of salting-out systems. The changes in protein light bands in the supernatant were tested by SDS-PAGE gel electrophoresis. The thickness of the gel plate was 1.5 mm, the mass fraction of the concentrated gel was 5%, and the mass fraction of the separation gel was 10%. The supernatant after centrifugation was diluted three times for sample preparation. The initial constant voltage electrophoresis was 80 V for about 40 min, and the later constant voltage electrophoresis was about 80 min until the bromophenol blue dye reached the bottom of the colloid and stopped electrophoresis. The concentrated gel was cut, and the separation gel was stained in Coomassie Brilliant Blue Rapid Stain P0017 for 10 min, decolorized with distilled water until the bands were clear, and the decolorized film was analyzed using the Bio-Rad Gel Imager Image Lab imaging analysis system to record the band optical density. See Attachment for the test results. Figure 5 .

[0039] Based on the previous temperature extraction exploration, the effect of different salting-out concentrations on the precipitation efficiency of type I collagen in the process system was studied, and nine systems including Example 3, Example 5 to Example 12 were obtained by adjusting the same volume of different salting-out concentrations. The supernatant of the salting-out liquid of 9 different salting-out systems was centrifuged and diluted with an acid solution to analyze the change of the total light band, and the total light band density of each group was compared and verified (3, 5 to 12 in the figure represent Example 3 and Example 5 to 12, respectively). Figure 5 The results show that under the same extraction process conditions, as the salting-out concentration continues to increase, collagen is continuously precipitated. When the salting-out concentration reaches 0M-0.75M, multiple protein strips can be seen on the colloid, and as the salting-out concentration increases, the total light band density in the supernatant of the salting-out liquid continues to weaken. When the concentration reaches 1.0M, the protein light band becomes three, and the number of bands no longer changes with the increase of the salting-out concentration, and the light intensity no longer changes. The results show that as the salt concentration continues to increase, the amount of protein precipitation continues to increase. When the salt concentration of the salting-out system reaches 1.0M, the protein reaches the maximum precipitation amount, and it no longer changes with the continuous increase of the salting-out concentration. In summary, among the 9 salting-out systems, when the salting-out concentration reaches 1.0M in Example 10, the amount of protein precipitation in the salting-out system is the largest. In the actual production process, the input amount of salt raw materials is lower than the 2.0M and 4.0M commonly used in traditional processes, which can effectively increase the time required for later purification and desalting, and shorten the process cycle.

[0040] 8. Ultrafiltration purification test: By recording the fluid replacement times, conductivity of the replaced fluid and purification time during the ultrafiltration process of the type I collagen ultrafiltrate of Example 2 to Example 12, a comparison is made between Examples 2 to Example 12 to obtain the optimal extraction and purification example. The ultrafiltration membrane material is hydrophilic polyethersulfone, the ultrafiltration system is tangential flow ultrafiltration, the membrane package pressure during the ultrafiltration process is 0.1 to 5 bar, and the feed pump speed frequency is 5 to 30 Hz. The ultrafiltration exchange fluid is 0.05 mol / L acetic acid solution and ultrapure water. The conductivity of the outlet end of the replacement fluid is measured every time the volume is replaced, and the actual fluid replacement times and time are recorded according to the material status and purification efficiency. The actual production efficiency of the purified product is calculated based on the total protein content of the Achilles tendon. The test results are attached. Figure 6 , Figure 7 .

[0041] Based on various previous tests and analyses, a comparison of the purification efficiencies of 11 groups of type I collagen raw material solutions from Examples 2 to 12 was performed. Figure 6 , Figure 7 The results show that in the process of exploring the extraction temperature in Examples 2 to 4, the purification efficiency of the type I collagen raw liquid is related to the extraction rate of collagen. The higher the collagen extraction rate, the more times the purification liquid is replaced, the longer the purification time, and the higher the yield. In the process of exploring the crude purification of salting out in Examples 5 to 12, the purification efficiency of the type I collagen raw liquid is related to the amount of collagen precipitated. In the salt concentration range of 0.25 to 0.75 mol / L, the lower the salt concentration, the lower the collagen precipitation rate, the lower the number of purification liquid changes, the shorter the purification time, and the lower the collagen yield. When the salt concentration reaches 0.75 to 2.0 mol / L, the collagen precipitation reaches the maximum, and it no longer changes with the increase of salt concentration. Within this range, the protein yield no longer changes, and 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 embodiments have a great influence on the number of subsequent purification liquid changes and the purification liquid change time, that is, the purification efficiency. Therefore, considering comprehensively, Embodiment 10 is the best embodiment among all extraction embodiment groups.

[0042] 9. Purity testing and process verification: The purity of the lyophilized collagen product obtained in Example 10 was tested. The sample was the lyophilized collagen sponge obtained in Example 10. The purity of type I collagen was determined by SDS gel electrophoresis according to the pharmaceutical industry standard of the People's Republic of China "Characterization Method of Type I Collagen for Tissue Engineering Medical Devices YY / T 1453-2016". The thickness of the gel plate was 1.5 mm, the mass fraction of the concentrated gel was 4%, the mass fraction of the separation gel was 7%, and the lyophilized collagen sponge was prepared to obtain a protein concentration of 1 mg / mL (sample 2), a collagenase digestion solution of the same concentration of collagen (sample 4), and an enzymatic solution with the same enzyme concentration as the digestion solution (sample 5). The concentrations of bovine serum albumin (BSA) were 0.025 mg / mL, 0.015 mg / mL, 0.005 mg / mL, and 0.001 mg / mL (samples 6, 7, 8, and 9), respectively. Load 20μm sample in each well, perform constant voltage electrophoresis at 80V for about 40min in the early stage and about 80min in the later stage, until the bromophenol blue dye reaches the bottom of the colloid and the electrophoresis is stopped. Cut the concentrated gel, stain the separation gel in Coomassie Brilliant Blue Fast Staining Solution P0017 for 10min, decolorize with distilled water until the bands are clear, analyze the decolorized film with Bio-Rad Gel Imager Image Lab Image Analysis System, and record the band optical density. See the attached test results Figure 8 .

[0043] The collagen has a triple helix structure that other proteins do not have, and the specific collagenase is used to detect the total amount of impurities contained in the collagen sample by determining the staining limit of bovine serum albumin (BSA) with Coomassie Brilliant Blue. Purity testing and process verification were performed on the collagen freeze-dried sponge obtained by the process of Example 9. Sample preparation was carried out according to the "Characterization Method of Type I Collagen for Tissue Engineering Medical Device Products YY / T 1453-2016", where sample 2 is standard type I collagen (1 mg / mL), and sample 4 is a collagen freeze-dried sample (1 mg / mL) obtained under the same purification cycle conditions as Example 13 and Example 9. Figure 8 The results show that under the same number of ultrafiltration purification cycles, the collagen freeze-dried product obtained in Example 9 has higher purity, the type I collagen maintains a complete natural triple helix structure, the number of bands and molecular weight are the same as those of the standard type I collagen, and the gel background is the same as the standard protein without other miscellaneous bands, indicating that the type I collagen obtained by this extraction and purification method has a low content of impurity protein and high purity. According to the "Characterization Method of Type I Collagen for Tissue Engineering Medical Device Products YY / T 1453-2016", the purity of the collagen can reach 99.81% of the total protein content.

[0044] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of 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, 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; 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 160r / min and then ultrafiltered. The tangential flow ultrafiltration technology is used to purify the replacement solution at a pressure of 0.1-5bar and a pump speed of 5-30Hz. During the purification process, replacement fluid is continuously added and the change in conductivity of the waste liquid at the outlet of the replacement fluid is detected until the conductivity no longer changes, indicating that the purification is complete. Ultrapure water is used for ultrafiltration again to keep the pH value of the solution at 3.5 and no longer changes, and then it is concentrated to obtain concentrated type I collagen purified solution. The type I collagen purified solution is freeze-dried to obtain collagen freeze-dried sponge.

2. The method for extracting and purifying natural active collagen according to claim 1, characterized in that: The protease is one or more combinations of pepsin, ficin, papain, trypsin, pancreatin, and collagenase.

3. The method for extracting and purifying natural active collagen according to claim 1, characterized in that: The acidic solution is any one of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid and lactic acid.

4. The method for extracting and purifying natural active collagen according to claim 1, characterized in that: The mild enzymatic hydrolysis conditions are 4°C to 35°C and stirring for 96 hours.

5. The method for extracting and purifying natural active collagen according to claim 1, characterized in that: The high-concentration salt solution is a 4.5M NaCl aqueous solution; the salting-out system consists of a high-concentration salt solution and an enzymatic hydrolysis supernatant; and the final salt concentration of the salting-out system is 0 mol / L to 2.5 mol / L.

6. The method for extracting and purifying natural active collagen according to claim 1, characterized in that: The concentration of the type I collagen ultrafiltration stock solution is between 1.5 mg / mL and 2.5 mg / mL, and the concentration of the type I collagen purified solution is between 3 mg / mL and 4 mg / mL.

7. 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.

8. 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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