Method for extracting collagen from animal pericardium and application of collagen

Abstract

The invention provides a method for extracting collagen from animal pericardium and application thereof, the method comprises the following steps: (1) physical field crushing: treating an animal pericardium raw material by adopting a high-voltage pulsed electric field system under the conditions that the field intensity is 10-50 kV / cm, the pulse frequency is 50-500 Hz, and the time is 5-10 minutes; (2) compound enzyme controlled enzymolysis: suspending the crushed animal pericardium raw material by adopting a buffer solution, and adding a compound enzyme for enzymolysis; the compound enzyme comprises pepsin, neutral protease and alpha-galactosidase; and (3) protective extraction: mixing the mixture after enzymolysis with a natural cross-linking protective agent to extract collagen. The extraction method disclosed by the invention is mild and efficient, and the high-yield extraction of the collagen with high biological activity is successfully realized through optimized pretreatment, physical field assisted crushing, compound enzyme controlled enzymolysis and application of a natural cross-linking protective agent.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for extracting collagen from animal pericardium and its application. Background Technology

[0002] Collagen is the most abundant structural protein in animals, widely distributed in tissues such as skin, bones, blood vessels, and the heart. Collagen has wide applications in biomedicine, food, and cosmetics, for example, in the preparation of artificial blood vessels, tissue engineering scaffolds, and drug carriers. Collagen membranes made from collagen have extensive uses in the medical field, primarily due to their excellent biocompatibility, biodegradability, and ability to promote tissue regeneration. The pericardium, as a special type of connective tissue, is mainly composed of type I collagen, has a dense fibrous network structure, and contains very little fat, making it an excellent raw material for collagen extraction.

[0003] Traditional collagen extraction methods primarily utilize animal skin or tendons, but research on extraction processes specifically targeting the pericardium is limited. Existing collagen extraction methods mainly include acid extraction, alkaline extraction, and enzymatic extraction. Acid extraction offers mild conditions but has low yields; alkaline extraction is highly efficient but easily damages the triple helix structure of collagen, leading to loss of bioactivity; enzymatic extraction offers controllable conditions but is costly, and the degree of enzymatic hydrolysis is difficult to precisely control. For example, patent CN103966294A uses a combination of acid and enzymatic methods to extract collagen, but this method targets common tissues such as bovine Achilles tendons and pig skin, not the specific material of the pericardium, and uses fig protease in the extraction process, the efficiency of which and its compatibility with cardiac tissue are unknown. CN101230088B develops a method for extracting undenatured natural collagen from animal skin or tendons, using pepsin hydrolysis combined with ultrasonic irradiation, which improves yield, but the enzymatic hydrolysis time is still relatively long, and its applicability to pericardial tissue has not been verified. The CN114907470B patent uses a combination of enzymatic hydrolysis and ultrasonic treatment to improve the extraction rate, but it mainly targets animal skin and does not involve pericardial tissue.

[0004] Existing technologies generally suffer from the following problems: poor adaptability to different types of raw materials (such as pericardium and skin); difficulty in simultaneously maintaining the integrity of the triple helix structure of collagen and achieving a high extraction rate; high impurity content in the product, especially non-collagenous proteins and adipose tissue; and long production cycles, ranging from several days to several weeks, resulting in low efficiency. Therefore, developing a method for efficiently extracting and preserving the bioactivity of collagen from animal pericardium is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for extracting collagen from animal pericardium and its applications. The method achieves a high extraction rate, and the extracted collagen exhibits high bioactivity. The present invention also prepares a collagen membrane based on the extracted collagen, which possesses excellent mechanical properties and biocompatibility.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for extracting collagen from animal pericardium, the method comprising:

[0008] (1) Physical field fragmentation: Animal pericardial raw materials are processed using a high-voltage pulsed electric field (PEF) system. The conditions of the high-voltage pulsed electric field (PEF) system are: field strength 10-50 kV / cm (e.g., 10 kV / cm, 15 kV / cm, 20 kV / cm, 25 kV / cm, 30 kV / cm, 35 kV / cm, 40 kV / cm, 45 kV / cm or 50 kV / cm, etc.), pulse frequency 50-500 Hz (e.g., 50 Hz, 100 Hz, 150 Hz, 200 Hz, 250 Hz, 300 Hz, 350 Hz, 400 Hz, 450 Hz or 500 Hz, etc.), and time 5-10 minutes (e.g., 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes, etc.).

[0009] (2) Controlled enzymatic hydrolysis with compound enzyme: The broken animal pericardium raw material is suspended in buffer solution and enzymatic hydrolysis is carried out by adding compound enzyme; the compound enzyme includes: pepsin, neutral protease and α-galactosidase;

[0010] (3) Protective extraction: The enzymatically hydrolyzed mixture is mixed with a natural cross-linking protectant to extract collagen.

[0011] In this invention, the physical field disruption step can destroy the cell membrane structure and kill microorganisms present in the tissue, increasing the efficiency of subsequent enzymatic hydrolysis and maintaining a sterile environment during the hydrolysis process. High-voltage pulsed electric field (PEF) treatment directly interacts with the cell membrane in the tissue through an external electric field, thereby destroying the cell membrane structure, forming "electroporation," leading to microbial inactivation and cell membrane exposure. When microorganisms are placed in a high-voltage pulsed electric field, the cell membrane is damaged, causing leakage of cell contents and resulting in cell death. Due to the attraction between opposite charges accumulated on the two surfaces of the cell membrane, the membrane is compressed; when the electric field strength increases to a critical value, the permeability of the cell membrane increases dramatically, many small pores appear on the membrane, reducing its strength; further action causes irreparable large perforations in the cell membrane, leading to tissue cell rupture and collapse.

[0012] Preferably, in step (1), the animal pericardium raw material is a pretreated raw material. The pretreatment steps include: removing connective tissue from the animal pericardium and preparing it into small pieces; soaking it in an alkaline solution; soaking it in a salt solution; and rinsing it with a phosphate buffer solution until it is neutral.

[0013] Preferably, the size of the small raw material pieces is 2-5 mm. 3 For example, it could be 2 mm 3 3 mm 3 4 mm 3 or 5 mm 3 wait.

[0014] Preferably, the alkaline solution is any one of sodium bicarbonate solution, sodium lactate solution, sodium hydroxide solution, or sodium carbonate solution.

[0015] Preferably, the concentration of the alkaline solution is 0.05-1 mol / L, for example, it can be 0.05 mol / L, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, 0.7 mol / L, 0.9 mol / L or 1 mol / L, etc., and the pH is 8.5-9.0, for example, it can be 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0, etc.

[0016] Preferably, the alkaline solution soaking treatment is performed under the following conditions: soaking at 0-4℃ (e.g., 0℃, 1℃, 2℃, 3℃ or 4℃, etc.) for 6-12 hours, such as 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours, etc.

[0017] Preferably, the salt solution is any one of a phosphate solution, a sodium chloride solution, a disodium ethylenediaminetetraacetate solution, or a tris(hydroxymethyl)aminomethane hydrochloride solution.

[0018] In this invention, the salt solutions used are conventional buffer solutions or isotonic solutions. For example, the concentration of the phosphate solution used is 0.1 mol / L, the sodium chloride solution is 0.9%, the concentration of the disodium ethylenediaminetetraacetate solution is 0.01 mol / L, and the concentration of the tris(hydroxymethyl)aminomethane hydrochloride solution is 0.05 mol / L.

[0019] Preferably, the conditions for soaking in the salt solution are as follows: temperature 2-8℃ (e.g., 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, or 8℃), time 0.5-24 h (e.g., 0.5 h, 1 h, 4 h, 8 h, 12 h, 16 h, 20 h, or 24 h), rotation speed 50-120 rpm (e.g., 50 rpm, 60 rpm, 80 rpm, 100 rpm, or 120 rpm), and number of cycles 2-10 (e.g., 2, 4, 6, 8, or 10 times).

[0020] In this invention, the connective tissue of fresh or frozen beef or pork pericardium is removed, and it is washed with cooled pure water to remove surface impurities and blood. After washing, it is chopped into 2-5 mm pieces. 3 Small pieces are soaked in a low-concentration alkaline solution (pH 8.5-9.0) at 0-4℃ for 6-12 hours to remove impurities such as fat, proteins, and soluble impurities, and then treated with a low-concentration salt solution to further remove soluble impurities, followed by rinsing with phosphate buffer until neutral. This pretreatment process can significantly reduce impurities in the tissue, including fat, proteins, and soluble impurities, thereby improving the efficiency of subsequent enzymatic hydrolysis and purification.

[0021] Preferably, in step (2), the pH of the buffer solution is 7.0-7.5, for example, it can be 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

[0022] Preferably, in step (2), the mass ratio of pepsin, neutral protease and α-galactosidase is (5-20):(5-20):(0.01-0.4), where "5-20" can be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and "0.01-0.4" can be, for example, 0.01, 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 or 0.4.

[0023] Preferably, in step (2), the concentration of pepsin in the enzymatic hydrolysis system is 0.5-5%, for example, it can be 0.5%, 1%, 2%, 3%, 4% or 5%, etc.

[0024] Preferably, in step (2), the concentration of neutral protease in the enzymatic hydrolysis system is 0.5-5%, for example, it can be 0.5%, 1%, 2%, 3%, 4% or 5%, etc.

[0025] Preferably, in step (2), the concentration of α-galactosidase in the enzymatic hydrolysis system is 0.001-0.1%, for example, it can be 0.001%, 0.0075%, 0.01%, 0.025%, 0.05%, 0.075% or 0.1%, etc.

[0026] Preferably, in step (2), the enzymatic hydrolysis conditions are: gentle stirring at 25-30℃ for 12-24 hours, and the stirring speed is 50-150 rpm, for example, 50 rpm, 60 rpm, 80 rpm, 100 rpm, 120 rpm or 150 rpm, etc.

[0027] In this invention, pepsin can break collagen peptide chains at specific sites in the non-helical regions of collagen chains, selectively cleaving the non-helical telopeptides at both ends, thereby reducing the immunogenicity of collagen. Neutral proteases can degrade various proteins in the extracellular matrix (such as fibronectin), but have a low degradation capacity for fibrous collagen such as type I and II. They are used to disperse tissues and release cells, minimizing the impact of enzymatic degradation on collagen structure. α-Gal antigen is a carbohydrate antigen present on the surface of most mammalian cells, but it is naturally absent in humans. When substances containing α-Gal antigen enter the human body, they bind to pre-existing anti-α-Gal antibodies, activating the complement system and leading to a severe immune response. Therefore, a major obstacle to xenogeneic collagen transplantation is the Gal immune barrier. To mitigate α-Gal-mediated rejection of collagen implants, it is necessary to eliminate the α-Gal antigen. The active site of α-galactosidase contains specific amino acid residues that can specifically recognize and bind to α-galactosidic bonds, eliminating α-Gal antigens in pericardial tissue and obtaining a non-immunogenic collagen solution.

[0028] Preferably, in step (3), the pH of the enzymatically hydrolyzed mixture is adjusted to 2.5-3.5, for example, it can be 2.5, 3 or 3.5, etc.

[0029] Preferably, in step (3), the natural cross-linking protectant is selected from any one or a combination of at least two of the following: proanthocyanidins, catechins, genipin, tannic acid, chlorogenic acid, gallic acid, or tea polyphenols.

[0030] Preferably, in step (3), the natural cross-linking protectant is selected from any one or a combination of at least two of proanthocyanidins, catechins, or genipin.

[0031] In this invention, the natural cross-linking protectant can also be used in combination.

[0032] Preferably, in step (3), the concentration of the natural crosslinking protectant in the extraction system is 0.001-0.05%, for example, it can be 0.001%, 0.003%, 0.005%, 0.008%, 0.01%, 0.02%, 0.03%, 0.04% or 0.05%, etc.

[0033] In this invention, the cross-linking protectant reacts with the amino and carboxyl groups in collagen, maintaining the collagen's natural triple helix structure and preventing damage to the collagen structure from acids and salts during subsequent separation and purification processes. Simultaneously, these cross-linking protectants possess antioxidant and anti-inflammatory functions, endowing the extracted collagen solution with corresponding biological functions and expanding its clinical application scope.

[0034] Preferably, in step (3), the extraction conditions are: slow stirring extraction for 12-48 hours (e.g., 12 hours, 24 hours, 36 hours or 48 hours) at 0-4℃ (e.g., 0℃, 1℃, 2℃, 3℃ or 4℃, etc.), and the stirring speed is 50-150 rpm, for example, 50 rpm, 60 rpm, 80 rpm, 100 rpm, 120 rpm or 150 rpm, etc.

[0035] Preferably, step (3) further includes a separation and purification step after collagen extraction.

[0036] Preferably, the separation and purification steps are as follows: centrifuge the extract to collect the supernatant, desalt and concentrate it through a tangential flow ultrafiltration system, add sodium chloride to the concentrated supernatant, let it stand to precipitate collagen, centrifuge to collect the precipitate, redissolve it with acetic acid solution, dialyze it with acetic acid solution and pure water in sequence, and prepare the collagen product by freeze drying or spray drying.

[0037] Preferably, the centrifugation conditions are: 0-4℃ (e.g., 0℃, 1℃, 2℃, 3℃ or 4℃, etc.), 10000-15000 rpm (e.g., 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm or 15000 rpm, etc.) for 20-30 minutes (e.g., 20 minutes, 25 minutes or 30 minutes, etc.).

[0038] Preferably, the final concentration of sodium chloride in the supernatant is 0.5-2.0 mol / L, for example, it can be 0.5 mol / L, 1 mol / L, 1.5 mol / L or 2.0 mol / L.

[0039] Preferably, the settling time is 12-24 hours at 0-4℃ (e.g., 0℃, 1℃, 2℃, 3℃ or 4℃, etc.), such as 12 hours, 16 hours, 20 hours or 24 hours.

[0040] Preferably, the concentration of the acetic acid solution is 0.1-0.5 mol / L, for example, it can be 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L or 0.5 mol / L, etc.

[0041] Preferably, the dialysis bag used for dialysis has a molecular weight cutoff of 10-20 kDa, such as 10 kDa, 12 kDa, 15 kDa, 17 kDa, or 20 kDa.

[0042] In a preferred embodiment of the present invention, the method for extracting collagen from animal pericardium includes the following steps:

[0043] (1) Physical field breakdown:

[0044] The connective tissue of the animal pericardium was removed, and it was prepared into 2-5 mm pieces. 3 Small pieces of raw material are soaked in an alkaline solution (sodium bicarbonate, sodium lactate, sodium hydroxide, or sodium carbonate, pH 8.5-9.0) at 0-4℃ for 6-12 hours. Then, they are soaked in a salt solution (phosphate, sodium chloride, disodium ethylenediaminetetraacetate, or tris(hydroxymethyl)aminomethane hydrochloride) at 2-8℃ for 0.5-24 hours, 50-120 rpm for 2-10 times. Finally, they are rinsed with phosphate buffer until neutral.

[0045] Animal pericardial material was treated using a high-voltage pulsed electric field (PEF) system. The conditions of the high-voltage pulsed electric field (PEF) system were: field strength 10-50 kV / cm, pulse frequency 50-500 Hz, and duration 5-10 minutes.

[0046] (2) Controlled enzymatic hydrolysis with compound enzyme: The ruptured animal pericardium raw material was suspended in a buffer solution with a pH of 7.0-7.5, and a compound enzyme was added for enzymatic hydrolysis. The hydrolysis conditions were: gentle stirring at 25-30℃ for 12-24 hours at a stirring speed of 50-150 rpm. The compound enzyme included pepsin, neutral protease and α-galactosidase. The mass ratio of pepsin, neutral protease and α-galactosidase was (5-20):(5-20):(0.01-0.4). In the hydrolysis system, the concentration of pepsin was 0.5-5%, the concentration of neutral protease was 0.5-5%, and the concentration of α-galactosidase was 0.001-0.1%.

[0047] (3) Protective extraction: The pH of the enzymatically hydrolyzed mixture is adjusted to 2.5-3.5, and collagen is extracted by mixing with a natural cross-linking protectant. The extraction conditions are: slow stirring at 0-4℃ for 12-48 hours, with a stirring speed of 50-150 rpm; the natural cross-linking protectant is selected from any one or a combination of at least two of the following: proanthocyanidins, catechins, genipin, tannic acid, chlorogenic acid, gallic acid, or tea polyphenols; the concentration of the natural cross-linking protectant in the extraction system is 0.001-0.05%;

[0048] The extract was centrifuged at 0-4℃ and 10000-15000 rpm for 20-30 minutes. The supernatant was collected and desalted and concentrated using a tangential flow ultrafiltration system. Sodium chloride was added to the concentrated supernatant, with a final concentration of 0.5-2.0 mol / L. The mixture was allowed to stand at 0-4℃ for 12-24 hours to precipitate collagen. The precipitate was collected by centrifugation and redissolved in 0.1-0.5 mol / L acetic acid solution. The mixture was then dialyzed sequentially with acetic acid solution and pure water. The molecular weight cutoff of the dialysis bag was 10-20 kDa. The collagen product was prepared by freeze drying or spray drying.

[0049] Secondly, the present invention provides a method for preparing a collagen membrane, the method comprising:

[0050] (S1) Prepare a 1-3% (e.g., 1%, 1.5%, 2%, 2.5% or 3%) collagen acetic acid solution, wherein the collagen in the collagen acetic acid solution is prepared by the method for extracting collagen from animal pericardium as described in the first aspect;

[0051] (S2) Place the solution from step (S1) on a flat mold and dry it to form a film to obtain a collagen film.

[0052] Preferably, in step (S1), the solvent of the collagen acetic acid solution is an acetic acid solution, and the concentration of the acetic acid solution is 0.05-0.2 mol / L, for example, it can be 0.05 mol / L, 0.1 mol / L, 0.15 mol / L or 0.2 mol / L, etc.

[0053] Preferably, in step (S2), the drying film formation conditions are: drying at 35-45°C, for example, 35°C, 40°C, or 45°C.

[0054] Preferably, step (S1) further includes adding any one of silver ion solution, magnesium ion solution, bone morphogenetic protein or vascular endothelial growth factor to the collagen acetic acid solution to obtain a mixed solution.

[0055] Preferably, the final concentration of silver ions in the mixed solution is 1-1000 μg / mL, for example, it can be 1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 800 μg / mL or 1000 μg / mL, etc.

[0056] Preferably, the final concentration of magnesium ions in the mixed solution is 120-2400 μg / mL, for example, it can be 120 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL, 1200 μg / mL, 1400 μg / mL, 1600 μg / mL, 1800 μg / mL, 2000 μg / mL, 2200 μg / mL or 2400 μg / mL, etc.

[0057] Preferably, the final concentration of bone morphogenetic protein in the mixed solution is 0.1-500 μg / mL, for example, it can be 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL or 500 μg / mL, etc.

[0058] Preferably, the final concentration of vascular endothelial growth factor in the mixed solution is 0.1-1000 ng / mL, for example, it can be 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 800 ng / mL or 1000 ng / mL, etc.

[0059] The collagen solution prepared in this invention has extremely low immune response and provides antioxidant and anti-inflammatory biological functions through natural cross-linking protection. On this basis, metal ion networks (such as silver / magnesium), bone morphogenetic proteins, vascular endothelial growth factor and other components are introduced into the collagen solution to prepare a film, thereby endowing it with multiple activities such as antibacterial, osteoproliferative and angiogenesis-promoting effects.

[0060] Thirdly, the present invention provides the application of the method for extracting collagen from animal pericardium as described in the first aspect or the method for preparing collagen membrane as described in the second aspect in the preparation of pharmaceuticals, skin repair dressings, biomaterials or medical devices.

[0061] The collagen extraction method of this invention is a high-value resource utilization technology that can turn waste into treasure, increase industrial added value, solve environmental problems, and transform "waste" into high-value biomaterials, such as medical dressings (sponges, membranes), hemostatic materials, drug sustained-release carriers, artificial skin or bone repair scaffolds, etc.; functional skin care ingredients, medical dressings (masks, patches) or medical aesthetic fillers; collagen peptide beverages, powders or dietary supplements, etc.

[0062] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) The method for extracting collagen in this invention preserves the natural triple helix structure of collagen to the greatest extent. The cross-linking protectant used in this invention can react with the amino and carboxyl groups in collagen, so that collagen maintains its natural triple helix structure and avoids the damage to the collagen structure caused by acids and salts in the subsequent separation and purification process.

[0065] (2) The method for extracting collagen in this invention significantly reduces the immunogenicity of the product. This invention uses a complex enzyme for enzymatic hydrolysis. Among them, pepsin can break collagen peptide chains at specific sites in the non-helical region of the collagen chain, selectively removing the non-helical telopeptides at both ends, thereby reducing the immunogenicity of collagen. Neutral protease can degrade a variety of proteins in the extracellular matrix, but its ability to degrade fibrous collagen such as type I and II is low. It is used to disperse tissues and release cells, minimizing the impact of the enzymatic hydrolysis process on the collagen structure. α-galactose (α-Gal) antigen is a carbohydrate antigen present on the surface of most mammalian cells, but it is naturally absent in the human body. When a substance containing α-Gal antigen enters the human body, it will bind to the pre-existing anti-α-Gal antibody in the human body, activating the complement system and leading to a severe immune response. Therefore, a major obstacle to xenogeneic collagen transplantation is the Gal immune barrier. In order to reduce α-Gal-mediated rejection of collagen implants, it is necessary to eliminate α-Gal antigen. The active site of α-galactosidase contains specific amino acid residues that can specifically recognize and bind to α-galactosidic bonds, eliminating α-Gal antigens in pericardial tissue and obtaining a non-immunogenic collagen solution.

[0066] (3) The method for extracting collagen in this invention improves the extraction efficiency (over 85%) and yield (18-25%) of collagen.

[0067] (4) The method for extracting collagen in this invention reduces the use of chemical reagents and reduces environmental pollution.

[0068] (5) The method for extracting collagen in this invention is suitable for large-scale industrial production. Attached Figure Description

[0069] Figure 1 These are the results of SDS-PAGE analysis.

[0070] Figure 2 This is the result of circular dichroism chromatographic analysis.

[0071] Figure 3 This is the result of quantitative detection of α-Gal antigen. Detailed Implementation

[0072] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0073] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0074] Example 1

[0075] Extraction of collagen from bovine pericardium.

[0076] 1. Raw material pretreatment.

[0077] Take 200 g of fresh beef heart and rinse it thoroughly with sterile saline to remove surface fat and blood vessels. Cut it into small pieces of approximately 3 mm × 3 mm and place them in a 0.2 mol / L NaHCO3 solution (pH 8.7). Stir at 80 rpm for 8 hours at 4°C. Then soak them in a 0.2 mol / L NaCl solution at 4°C for 12 hours. After soaking, rinse with phosphate buffer until neutral and drain.

[0078] 2. Physical field breakdown.

[0079] The pretreated bovine pericardium was placed in a high-voltage pulsed electric field treatment chamber, and 4 times the volume of pre-cooled deionized water was added. The field strength was adjusted to 25 kV / cm, the pulse frequency was 150 Hz, and the treatment time was 8 minutes.

[0080] 3. Controlled enzymatic hydrolysis using compound enzymes.

[0081] The crushed raw material was suspended in phosphate buffer at pH 7.2 with a solid-liquid ratio of 1:10 (w / v). A complex enzyme system (0.5% pepsin, 0.5% neutral protease, and 0.01% α-galactosidase) was added, and the mixture was gently stirred at 28°C for 16 hours. During the process, the absorbance change at 220 nm was monitored using an online UV monitoring system to control the enzymatic hydrolysis endpoint.

[0082] 3. Protective extraction.

[0083] The enzymatically hydrolyzed mixture was adjusted to pH 3.0 with 0.5 mol / L hydrochloric acid, and 0.01% proanthocyanidins were added as a cross-linking protectant. The mixture was then slowly extracted at 4°C with stirring for 24 hours.

[0084] 4. Separation and purification.

[0085] The acid-soluble extract (enzymatic hydrolysis extract containing a cross-linking protectant, adjusted to acidity with hydrochloric acid) was centrifuged at 4°C and 12,000 rpm for 25 minutes, and the supernatant was collected. Desalting and concentration were performed using a tangential flow ultrafiltration system (molecular weight cutoff 30 kDa), and finally, the product was freeze-dried to obtain a white fibrous collagen product. The collagen was prepared into a 2% acetic acid solution, cast into a film, and dried at 45°C to obtain a transparent collagen membrane.

[0086] Results: The yield was 21.5%. SDS-PAGE analysis showed complete α1 and α2 chain structures, such as... Figure 1As shown; circular dichroism chromatographic analysis confirmed that the triple helix structure retention rate reached 96.2%, as... Figure 2 As shown; quantitative detection of α-Gal antigen showed that α-Gal antigen was not detected, as... Figure 3 As shown, the immunogenicity of the extracted collagen is significantly reduced.

[0087] Example 2

[0088] Extraction of collagen from porcine pericardium.

[0089] 1. Raw material pretreatment.

[0090] Take 300 g of fresh pig heart and rinse it thoroughly with running water. Cut it into small pieces of about 2 mm × 2 mm. First, soak it in 0.05 mol / L NaOH solution (pH 9.0) at 4℃ for 5 hours, and then soak it in 0.3 mol / L NaCl solution for 4 hours to remove non-collagenous components.

[0091] 2. Physical field breakdown.

[0092] The pretreated pig pericardium was placed in a high-voltage pulsed electric field treatment chamber, and 4 times the volume of pre-cooled deionized water was added. The field strength was adjusted to 10 kV / cm, the pulse frequency was 60 Hz, and the treatment time was 5 minutes.

[0093] 3. Controlled enzymatic hydrolysis using compound enzymes.

[0094] The ruptured porcine pericardium was suspended in Tris-HCl buffer at pH 7.4 at a solid-liquid ratio of 1:8 (w / v). A complex enzyme system (0.5% pepsin, 0.5% neutral protease, and 0.01% α-galactosidase) was added, and the mixture was gently stirred at 30°C for 14 hours.

[0095] 4. Protective extraction.

[0096] The pH was adjusted to 2.8 with 1.0 mol / L acetic acid, and 0.05% catechin was added as a cross-linking protectant. The mixture was then slowly stirred and extracted at 4°C for 12 hours.

[0097] 5. Separation and purification.

[0098] The supernatant was collected by centrifugation (under the same conditions as in Example 1), desalted and concentrated by tangential flow ultrafiltration system, and finally spray-dried to obtain a white powdered collagen product.

[0099] Results: The yield was 19.8%. SDS-PAGE analysis showed intact collagen bands, such as... Figure 1 As shown; circular dichroism chromatographic analysis confirmed that the triple helix structure retention rate reached 96.2%, as... Figure 2 As shown; quantitative detection of α-Gal antigen showed that α-Gal antigen was not detected, as... Figure 3 As shown, the immunogenicity of the extracted collagen is significantly reduced.

[0100] Example 3

[0101] Extraction of collagen from donkey pericardium.

[0102] The main steps in this embodiment are the same as in embodiment 1, except that:

[0103] The main ingredient is 250 g of fresh donkey pericardium.

[0104] Physical crushing uses a high-voltage pulsed electric field with a field strength of 28 kV / cm, a pulse frequency of 120 Hz, and a processing time of 10 minutes.

[0105] The compound enzymatic hydrolysis temperature was 26℃ and the time was 18 hours.

[0106] The acid extraction was performed at pH 3.2 for 30 hours.

[0107] Results: The yield was 22.3%. SDS-PAGE analysis showed intact collagen bands, such as... Figure 1 As shown; circular dichroism spectroscopy confirmed that the triple helix structure retention rate reached 96.8%, as... Figure 2 As shown; quantitative detection of α-Gal antigen showed that α-Gal antigen was not detected, as... Figure 3 As shown.

[0108] Table 1 shows a comparison of the effects of the present invention and traditional extraction methods.

[0109] Table 1

[0110]

[0111] Example 4

[0112] Fresh porcine pericardium, after cleaning and removal of excess fat and connective tissue, was cut into slices approximately 2 cm × 2 cm and randomly grouped. Control group A (traditional mechanical disruption group): Pericardium tissue was placed directly in 4℃ physiological saline without PEF treatment and disrupted using a conventional homogenizer for 5 minutes. Experimental groups were treated with a high-voltage pulsed electric field; specific parameters are shown in Table 2 below. During treatment, the samples were suspended in a 4℃, 0.9% NaCl solution, and the temperature was maintained below 10℃ throughout.

[0113] Table 2

[0114]

[0115] Subsequent standardized processing: After the above pretreatment, all samples were processed using the enzymatic hydrolysis and purification method described in Example 2 to extract collagen. Results: Collagen yield (%, dry basis): The experimental groups were generally significantly higher than control group A (22.3% ± 0.5% vs 12.1% ± 1.2%). Collagen integrity (SDS-PAGE electrophoresis analysis): The collagen extracted from all experimental groups showed intact α1 and α2 chain bands characteristic of type I collagen, with fewer degradation products, indicating that PEF treatment can effectively protect the triple helix structure of collagen while efficiently disrupting tissue. Control group A had fainter bands due to incomplete extraction. Energy consumption and efficiency: The experimental groups had shorter processing time, lower overall energy consumption, and higher extraction rate, showing better process efficiency. This example confirms that using a high-voltage pulsed electric field for physical field disruption can significantly improve the extraction efficiency and integrity of collagen from animal pericardium.

[0116] Comparison of the results of experimental group 1 and control experimental groups 1 and 2 shows that the collagen yield of control experimental group 1 was relatively low (10.8% ± 0.7%), indicating that collagen could not be completely extracted under the low high-voltage pulse electric field parameter. Control experimental group 2, under the high high-voltage pulse electric field parameter, showed poor collagen integrity (SDS-PAGE electrophoresis analysis) and more degradation products, indicating that the high-voltage pulse electric field parameter should not be too high and needs to be controlled within a certain range.

[0117] Example 5

[0118] Bovine pericardial fragments treated under the preferred physical field disruption conditions (25 kV / cm, 150 Hz, 8 min) in Example 1 were grouped as shown in Table 3. All groups were prepared as a 10% (w / v) suspension using phosphate buffer (pH 7.2) and enzymatically hydrolyzed at 25°C with constant shaking (100 rpm) for 12 hours. After enzymatic hydrolysis, all groups were treated using the collagen purification method described in Example 1.

[0119] Table 3

[0120]

[0121] Collagen yield (%, dry basis): Control group B (single pepsin) yield was 13.2% ± 0.8%. Control group C (single neutral protease) yield was 9.4% ± 0.7%. Control group D (binary complex, without α-galactosidase) yield was 16.7% ± 1.3%. The yields of all ternary complex experimental groups (4, 5, 6) were significantly higher than those of the control groups (p < 0.05). Among them, the yield of experimental group 4 was 18.3% ± 0.9%, the yield of experimental group 6 was 23.6% ± 1.7%, and the extraction yield of experimental group 5 (pepsin, neutral protease, and α-galactosidase concentrations were 2%, 1%, and 0.02%, respectively) reached the peak at 25.6% ± 1.2%.

[0122] α-Gal antigen residual rate (determined by ELISA): The residual rates of α-Gal antigen in control groups B, C, and D were relatively high, ranging from 1.5% to 5.3%. In all experimental groups containing α-galactosidase (4, 5, and 6), the residual rates were significantly reduced, falling below the detection limit. This demonstrates that α-galactosidase effectively removes key antigenic epitopes in pericardial tissue that cause immune rejection.

[0123] Example 6

[0124] The enzymatically hydrolyzed mixture after treatment in Example 1 (physical field disruption and controlled enzymatic hydrolysis with compound enzymes) was used as the raw material for protective extraction. Equal volumes of the raw material were taken, and the pH was adjusted to 3.0 with 1 M HCl. Different types and concentrations of natural cross-linking protectants were added. Extraction was carried out at 4°C and 80 rpm for 18 hours with shaking. The groups are shown in Table 4 below. Subsequent separation and purification were performed according to the steps in Example 1 to obtain the collagen product.

[0125] Table 4

[0126]

[0127] The final dry weight yield showed a significant difference between the groups with added cross-linking protectant and the control group E (19.5%-24.9% vs 14.6%), indicating that the protectant did not significantly affect the dissolution and recovery of collagen. SDS-PAGE analysis showed that the products obtained from all experimental groups and the control group E exhibited typical type I collagen patterns, indicating that the protectant did not introduce impurities. Circular dichroism spectroscopy analysis showed that the triple helix structure retention rate of collagen in all experimental groups reached 94.3-97.7%, while that in the control group E was only 73.8%. The retention rates for experimental groups 7 and 11 were 94.3%, 97.7%, 95.8%, 97.2%, and 96.7%, respectively. The comparison of the results from experimental groups 7-11 indicates that the addition of the cross-linking protectant resulted in a more intact triple helix structure of collagen, which was well preserved even after separation and purification processes involving acetic acid and salt.

[0128] This embodiment demonstrates that introducing a low concentration of natural cross-linking protectant during the extraction stage can achieve "protective extraction" of collagen. This treatment gently enhances the thermal stability and resistance to enzymatic degradation of collagen without significantly altering the extraction process or final yield, while perfectly maintaining the product's biocompatibility. This step effectively compensates for the potential weakening of collagen's ultrastructure that may be caused by preceding physical and enzymatic treatments, providing a crucial guarantee for obtaining collagen products with high structural integrity and high stability. This protective extraction condition is an important step in the method of this invention for improving product performance.

[0129] Example 7

[0130] This embodiment provides a method for preparing a collagen membrane.

[0131] The collagen extracted in Example 2 was dissolved in 0.1 mol / L acetic acid solution to prepare a 2% (w / w) collagen solution. Silver ion solution was then added to obtain a mixed solution with a final silver ion concentration of 50 μg / mL. The mixed solution was poured onto a flat mold and dried at 40°C to form a film. After peeling, an antibacterial collagen film was obtained.

[0132] Example 8

[0133] This embodiment provides a method for preparing a collagen membrane.

[0134] The collagen extracted in Example 2 was dissolved in 0.1 mol / L acetic acid solution to prepare a 2% (w / w) collagen solution. Bone morphogenetic protein was then added to obtain a mixed solution with a final concentration of 500 ng / mL. The mixed solution was poured onto a flat mold and dried at 40°C to form a film. After peeling, a collagen membrane with bone regeneration-promoting function was obtained.

[0135] Example 9

[0136] This embodiment provides a method for preparing a collagen membrane.

[0137] The collagen extracted in Example 2 was dissolved in 0.1 mol / L acetic acid solution to prepare a 2% (w / w) collagen solution. Vascular endothelial growth factor (VEGF) was then added to obtain a mixed solution with a final VEGF concentration of 500 ng / mL. The mixed solution was poured onto a flat mold and dried at 40°C to form a film. After peeling, a collagen membrane with angiogenesis-promoting function was obtained.

[0138] In summary, this invention has developed a method for efficiently extracting and preserving the bioactivity of collagen from animal pericardium, and has prepared collagen membranes with different biological functions based on the collagen extracted by the above method. The extraction method and the obtained products have broad application prospects in the fields of biomedicine, food, and cosmetics.

[0139] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for extracting collagen from animal pericardium, characterized in that, The method includes: (1) Physical field crushing: Animal pericardial raw materials are processed by a high-voltage pulse electric field system. The conditions of the high-voltage pulse electric field system are: field strength 10-50 kV / cm, pulse frequency 50-500 Hz, and time 5-10 minutes. (2) Controlled enzymatic hydrolysis with compound enzyme: The broken animal pericardium raw material is suspended in buffer solution and enzymatically hydrolyzed by adding compound enzyme; the compound enzyme includes: pepsin, neutral protease and α-galactosidase; (3) Protective extraction: The enzymatically hydrolyzed mixture is mixed with a natural cross-linking protectant to extract collagen.

2. The method for extracting collagen from animal pericardium according to claim 1, characterized in that, In step (1), the animal pericardium raw material is a pretreated raw material. The pretreatment steps include: removing connective tissue from the animal pericardium and preparing it into small pieces; soaking it in an alkaline solution; soaking it in a salt solution; and rinsing it with a phosphate buffer solution until it is neutral.

3. The method for extracting collagen from animal pericardium according to claim 2, characterized in that, The size of the small raw material pieces is 2-5 mm. 3 ; Preferably, the alkaline solution is any one of sodium bicarbonate solution, sodium lactate solution, sodium hydroxide solution, or sodium carbonate solution; Preferably, the concentration of the alkaline solution is 0.05-1 mol / L, and the pH is 8.5-9.0; Preferably, the alkaline solution soaking treatment is performed under the following conditions: soaking at 0-4℃ for 6-12 hours; Preferably, the salt solution is any one of a phosphate solution, a sodium chloride solution, a disodium ethylenediaminetetraacetate solution, or a tris(hydroxymethyl)aminomethane hydrochloride solution; Preferably, the conditions for soaking in the salt solution are: temperature of 2-8℃, time of 0.5-24 h, rotation speed of 50-120 rpm, and number of times of soaking 2-10.

4. The method for extracting collagen from animal pericardium according to any one of claims 1-3, characterized in that, In step (2), the pH of the buffer solution is 7.0-7.5; Preferably, in step (2), the mass ratio of pepsin, neutral protease and α-galactosidase is (5-20):(5-20):(0.01-0.4); Preferably, in step (2), the concentration of pepsin in the enzymatic hydrolysis system is 0.5-5%; Preferably, in step (2), the concentration of neutral protease in the enzymatic hydrolysis system is 0.5-5%; Preferably, in step (2), the concentration of α-galactosidase in the enzymatic hydrolysis system is 0.001-0.1%; Preferably, in step (2), the enzymatic hydrolysis conditions are: gentle stirring at 25-30°C for 12-24 hours, with a stirring speed of 50-150 rpm.

5. The method for extracting collagen from animal pericardium according to any one of claims 1-4, characterized in that, In step (3), the pH of the enzymatically hydrolyzed mixture is adjusted to 2.5-3.5; Preferably, in step (3), the natural cross-linking protectant is selected from any one or a combination of at least two of the following: proanthocyanidins, catechins, genipin, tannic acid, chlorogenic acid, gallic acid, or tea polyphenols; Preferably, in step (3), the concentration of the natural cross-linking protectant in the extraction system is 0.001-0.05%; Preferably, in step (3), the extraction conditions are: slow stirring extraction at 0-4℃ for 12-48 hours, with a stirring speed of 50-150 rpm.

6. The method for extracting collagen from animal pericardium according to any one of claims 1-5, characterized in that, In step (3), after extracting collagen, a separation and purification step is also included; Preferably, the separation and purification steps are as follows: centrifuge the extract to collect the supernatant, desalt and concentrate it through a tangential flow ultrafiltration system, add sodium chloride to the concentrated supernatant, let it stand to precipitate collagen, centrifuge to collect the precipitate, redissolve it with acetic acid solution, dialyze it with acetic acid solution and pure water in sequence, and prepare the collagen product by freeze drying or spray drying.

7. The method for extracting collagen from animal pericardium according to claim 6 and its application, characterized in that, The centrifugation conditions are: 0-4℃, 10000-15000 rpm for 20-30 minutes; Preferably, the final concentration of sodium chloride in the supernatant is 0.5-2.0 mol / L; Preferably, the settling time is 12-24 hours at 0-4℃; Preferably, the concentration of the acetic acid solution is 0.1-0.5 mol / L; Preferably, the dialysis bag used for dialysis has a molecular weight cutoff of 10-20 kDa.

8. A method for preparing a collagen membrane, characterized in that, The preparation method includes: (S1) Prepare a 1-3% collagen acetic acid solution, wherein the collagen in the collagen acetic acid solution is prepared by the method of extracting collagen from animal pericardium as described in any one of claims 1-7; (S2) Place the solution from step (S1) on a flat mold and dry it to form a film to obtain a collagen film.

9. The method for preparing a collagen membrane according to claim 8, characterized in that, In step (S1), the solvent for the collagen acetic acid solution is an acetic acid solution with a concentration of 0.05-0.2 mol / L. Preferably, in step (S2), the drying film formation conditions are: drying at 35-45°C; Preferably, step (S1) further includes adding any one of silver ion solution, magnesium ion solution, bone morphogenetic protein or vascular endothelial growth factor to the collagen acetic acid solution to obtain a mixed solution; Preferably, the final concentration of silver ions in the mixed solution is 1-1000 μg / mL; Preferably, the final concentration of magnesium ions in the mixed solution is 120-2400 μg / mL; Preferably, the final concentration of bone morphogenetic protein in the mixed solution is 0.1-500 μg / mL; Preferably, the final concentration of vascular endothelial growth factor in the mixed solution is 0.1-1000 ng / mL.

10. The method for extracting collagen from animal pericardium according to any one of claims 1-7 or the method for preparing collagen membrane according to claim 8 or 9, and its use in the preparation of pharmaceuticals, skin repair dressings, implants, biomaterials or medical devices.

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