A medical collagen extraction method

Abstract

The application discloses a medical collagen extraction method and belongs to the technical field of biomedical materials. The extraction steps are as follows: after removing surface foreign matters from a bovine Achilles tendon, the bovine Achilles tendon is cut into particles, washed by a NaCl solution and soaked by a Tris-HCl buffer solution containing EDTA-Na2; the pretreated bovine Achilles tendon tissue is added into a swelling treatment liquid prepared by taking lactic acid and betaine as raw materials to perform soaking, so that swelled bovine Achilles tendon tissue is obtained; the swelled bovine Achilles tendon tissue is subjected to enzymolysis by pepsin, so that a crude collagen extraction liquid is obtained; poly(N-isopropyl acrylamide) is added into the crude collagen extraction liquid to precipitate a collagen-containing precipitate; after resuspension of the precipitate, constant-volume ultrafiltration diafiltration purification is performed, so that a collagen concentrate liquid is obtained, and the medical collagen extraction is completed. The product has the characteristics of high purity, structural integrity and low endotoxin, meets medical-grade safety standards and is suitable for large-scale popularization and use.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a method for extracting medical collagen. Background Technology

[0002] Existing collagen extraction methods mainly include acid extraction, alkaline extraction, enzymatic extraction, and combined acid-enzyme methods. However, they still have the following shortcomings: incomplete removal of lipids and impurities affects purity and transparency; microbial load and endotoxins are difficult to control effectively, making it difficult to meet injection-grade requirements; strong acid, strong alkali, or ultrasonic treatment can easily damage the triple helix structure, leading to collagen chain breakage; membrane filtration and dialysis have low efficiency, making it difficult to achieve large-scale production. Therefore, there is an urgent need for a medical-grade collagen extraction method that can balance high purity, structural integrity, low endotoxins, and scalability.

[0003] Content of this invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for extracting medical collagen.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for extracting medical collagen includes the following steps:

[0007] (a) After removing foreign matter from the surface of the bovine Achilles tendon, it was granulated, washed with NaCl solution, and soaked in Tris-HCl buffer containing EDTA-Na2 to obtain pretreated bovine Achilles tendon tissue.

[0008] (b) The pretreated bovine Achilles tendon tissue was immersed in a swelling treatment solution prepared with lactic acid and betaine to obtain swollen bovine Achilles tendon tissue.

[0009] (c) The swollen bovine Achilles tendon tissue was enzymatically hydrolyzed with pepsin to obtain a crude collagen extract;

[0010] (d) Add poly(N-isopropylacrylamide) to the crude collagen extract to precipitate collagen-containing precipitate;

[0011] (e) After resuspending the precipitate, tangential flow ultrafiltration and constant volume perfiltration were performed to purify it, and collagen concentrate was obtained, thus completing the extraction of medical collagen.

[0012] The present invention is further configured such that, in step (a), the soaking temperature is 3-6°C.

[0013] The present invention is further configured such that, in step (b), a swelling treatment solution is prepared by using lactic acid and betaine in a molar ratio of 1:1, and the concentration of lactic acid in the swelling treatment solution is 0.2 mol / L.

[0014] The present invention is further configured such that, in step (b), the swelling temperature is 3-8°C.

[0015] The present invention is further configured such that step (c) specifically involves: adding the swollen bovine Achilles tendon tissue to an acetic acid solution, adding pepsin, enzymatically hydrolyzing at 35°C, and then cooling to 25°C to continue enzymatic hydrolysis; then adjusting the pH of the system to 7.5, quenching the reaction in an ice bath, centrifuging, and collecting the supernatant to obtain the crude collagen extract.

[0016] The present invention is further configured such that, in step (d), poly(N-isopropylacrylamide) is added to a final concentration of 0.2-0.6 wt%.

[0017] The present invention is further configured such that, in step (e), the precipitate is resuspended at a ratio of 1g precipitate to 20mL buffer solution, wherein the buffer solution is an acetate-sodium acetate buffer solution.

[0018] In summary, the present invention has the following beneficial effects:

[0019] This invention effectively utilizes lactic acid / betaine swelling and two-stage temperature-controlled enzymatic hydrolysis to precisely remove impurities and lipids under mild conditions, while effectively avoiding the damage to collagen caused by strong acids and alkalis, maintaining the triple helix structure of collagen at over 95%. Furthermore, by combining poly(N-isopropylacrylamide) precipitation technology with tangential flow ultrafiltration purification technology, it achieves highly efficient removal of impurities and endotoxins, resulting in a product purity of over 98% and endotoxin content strictly controlled below 0.05 EU / mL. The product of this invention possesses high activity, high purity, and high safety, meeting medical-grade safety standards and suitable for large-scale promotion and use. Attached Figure Description

[0020] Figure 1 This is a comparison of the SDS-PAGE electrophoresis results of the collagen obtained in Example 1 with other proteins. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1

[0023] (1) Pretreatment: Remove blood, blood vessels, fat, fascia and other foreign matter from the surface of the cow Achilles tendon, clean it with purified water, and then cut the cow Achilles tendon into 0.5-1cm pieces. 3Particles; Bovine Achilles tendon particles were placed in 0.9wt% NaCl solution (solid-to-liquid ratio 1g:15mL), and washed with shaking at 4℃ for 2h. Then, they were transferred to Tris-HCl buffer (pH=7.4) containing 0.05wt% EDTA-Na2 and soaked at 4℃ for 6h (solid-to-liquid ratio 1g:15mL, the soaking temperature can be adjusted to 3-6℃ as needed). The bovine Achilles tendon particles were filtered out, washed with purified water until neutral, and drained to obtain pretreated bovine Achilles tendon tissue.

[0024] (2) Swelling: Mix lactic acid and betaine at a molar ratio of 1:1 and stir at 60°C until a transparent liquid is formed. Dilute with purified water to obtain a swelling treatment solution with a lactic acid concentration of 0.2 mol / L. Add the pretreated bovine Achilles tendon tissue to the swelling treatment solution at a material-to-liquid ratio of 1 g:10 mL and let it stand at 4°C for 4 hours to swell (the soaking temperature can be adjusted to 3-8°C as needed). Then wash with purified water 3 times to obtain the swollen bovine Achilles tendon tissue.

[0025] (3) Enzymatic hydrolysis: The swollen bovine Achilles tendon tissue was added to 0.1 mol / L acetic acid solution at a material-to-liquid ratio of 1 g: 15 mL. Pepsin (purchased from Tianjin Guangfu Technology Development Co., Ltd., product number gfsh304) was added at an enzyme activity of 500 U per g of bovine Achilles tendon tissue. The tissue was hydrolyzed at 35°C for 2 h and then cooled to 25°C for 4 h. After that, 1 mol / L NaOH solution was added to adjust the pH of the system to 7.5. The reaction was quenched in an ice bath. The tissue was centrifuged and the supernatant was collected to obtain the crude collagen extract.

[0026] (4) Add poly(N-isopropylacrylamide) with a number-average molecular weight of 30,000 Da to the crude collagen extract to a final concentration of 0.3 wt%, heat to 35 °C and maintain for 30 min, then centrifuge to collect the precipitate; the concentration of poly(N-isopropylacrylamide) is adjusted to 0.2-0.6 wt% according to different animal tissues;

[0027] (5) Purification: The precipitate obtained in step (4) was resuspended in 20 mmol / L acetate-sodium acetate buffer (pH=3.6) at a ratio of 1 g precipitate to 20 mL buffer. Using 20 mmol / L acetate-sodium acetate buffer (pH=3.6) as the replacement fluid, constant volume perfiltration was performed using a tangential flow ultrafiltration (TFF) system (conditions: 100 kDa hollow fiber column, transmembrane pressure (TMP) ≤0.1 MPa, cross-flow rate 60 mL / min, temperature 4-8℃). After 5 replacement volumes, the retentate in the membrane was collected to obtain collagen concentrate, thus completing the extraction of medical collagen.

[0028] Comparative Example 1

[0029] Medical collagen was extracted according to Example 1, except that the swelling treatment solution in step (2) was replaced with 0.11 mol / L acetic acid solution.

[0030] Comparative Example 2

[0031] Medical collagen was extracted according to Example 1, the difference being that the enzymatic hydrolysis process in step (3) was carried out at 35°C in the dark for 6 hours, instead of two-stage temperature-controlled enzymatic hydrolysis.

[0032] Comparative Example 3

[0033] Medical collagen was extracted according to Example 1, except that in step (4) poly(N-isopropylacrylamide) was replaced with PEG-6000 and PEG-6000 was added to a final concentration of 8% (w / v).

[0034] Comparative Example 4

[0035] Medical collagen was extracted according to Example 1, except that step (4) was changed to: the pH of the crude collagen extract was adjusted to 7.5 with 20wt% NaOH solution, NaCl was added until the final NaCl concentration reached 0.5mol / L, the mixture was allowed to stand at 5℃ for 18h, and then centrifuged and the precipitate was collected; the above salting out was repeated three times and all the precipitates were collected.

[0036] The collagen obtained in Example 1 and each comparative example was subjected to performance tests (triple helix structure retention rate and purity were determined according to YY / T 1985-2025 "Tissue-engineered Medical Device Collagen", and endotoxin content was determined according to the "Pharmacopoeia of the People's Republic of China" 2020 edition, Part IV, General Chapter 1143, Bacterial Endotoxin Test Method). The results are shown in Table 1. Figure 1 The image shows a comparison of SDS-PAGE electrophoresis results of collagen obtained in Example 1 with other proteins (in the image: lane 1 corresponds to the collagen sample extracted in Example 1; lane 2 corresponds to the collagen standard (bovine type I collagen reference, purchased from the National Institutes for Food and Drug Control, specification: 380008-202001); lane 3 corresponds to the product obtained after enzymatic hydrolysis in Example 1; lane 4 corresponds to purified water; lane 5 corresponds to 0.02 mg / mL bovine serum albumin sample solution; lane 6 corresponds to 0.01 mg / mL bovine serum albumin sample solution; lane 7 corresponds to 0.005 mg / mL bovine serum albumin sample solution; lane 8 corresponds to 0.0025 mg / mL bovine serum albumin sample solution).

[0037] Table 1

[0038]

[0039] As shown in Table 1, the collagen obtained in Example 1 of this invention has a complete triple helix structure, high purity, and low endotoxin level, and all its properties are superior to those of the comparative examples. This indicates that the lactic acid and betaine compound swelling system used in this invention can effectively protect the natural structure of collagen; the two-stage gradient cooling enzymatic hydrolysis can reduce excessive collagen degradation; and the poly(N-isopropylacrylamide) precipitation method is superior to traditional PEG salting-out and sodium chloride salting-out in removing impurities and reducing endotoxins.

[0040] The collagen extraction method provided by this invention can achieve high purity, high concentration, low endotoxin, and large-scale production while maintaining the triple helix structure. It is suitable for various application scenarios such as injection, implantation, and medical aesthetics, and has significant industrialization value.

[0041] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting medical collagen, characterized in that, Includes the following steps: (a) After removing foreign matter from the surface of the bovine Achilles tendon, it was granulated, washed with NaCl solution, and soaked in Tris-HCl buffer containing EDTA-Na2 to obtain pretreated bovine Achilles tendon tissue. (b) The pretreated bovine Achilles tendon tissue was immersed in a swelling treatment solution prepared with lactic acid and betaine to obtain swollen bovine Achilles tendon tissue. (c) The swollen bovine Achilles tendon tissue was enzymatically hydrolyzed with pepsin to obtain a crude collagen extract; (d) Add poly(N-isopropylacrylamide) to the crude collagen extract to precipitate collagen-containing precipitate; (e) After resuspending the precipitate, tangential flow ultrafiltration and constant volume perfiltration were performed to purify it, and collagen concentrate was obtained, thus completing the extraction of medical collagen.

2. The method for extracting medical collagen according to claim 1, characterized in that, In step (a), the soaking temperature is 3-6℃.

3. The method for extracting medical collagen according to claim 1, characterized in that, In step (b), a swelling treatment solution is prepared by using lactic acid and betaine in a molar ratio of 1:1, and the concentration of lactic acid in the swelling treatment solution is 0.2 mol / L.

4. The method for extracting medical collagen according to claim 1, characterized in that, In step (b), the swelling temperature is 3-8℃.

5. The method for extracting medical collagen according to claim 1, characterized in that, Step (c) is as follows: the swollen bovine Achilles tendon tissue is added to acetic acid solution, pepsin is added, and enzymatic hydrolysis is carried out at 35°C and then cooled to 25°C for further enzymatic hydrolysis; then the pH of the system is adjusted to 7.5, the reaction is quenched in an ice bath, centrifuged, and the supernatant is collected to obtain the crude collagen extract.

6. The method for extracting medical collagen according to claim 1, characterized in that, In step (d), poly(N-isopropylacrylamide) is added to a final concentration of 0.2-0.6 wt%.

7. The method for extracting medical collagen according to claim 1, characterized in that, In step (e), the precipitate is resuspended at a ratio of 1g precipitate to 20mL buffer solution, which is an acetate-sodium acetate buffer solution.

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