Preparation method for improving biocompatibility of collagen, product and application

Abstract

The invention relates to the field of collagen preparation, in particular to a preparation method for improving the biocompatibility of collagen, a product and application. The preparation method comprises the following steps: pretreating raw materials; carrying out acidolysis and enzymolysis on the pre-treated product; carrying out salting-out on the enzymolysis product, redissolving the salting-out product, and filtering; and carrying out ultrafiltration and concentration on the filtered product to obtain the collagen. The collagen biological terminal peptide obtained through the extraction process is thoroughly removed, immunogenicity can be effectively reduced, biocompatibility is improved, immunological rejection is lower, alpha-Gal antigen residues and proteolytic enzyme residues are both lower than the detection limit, and the collagen can be used for hemostasis and wound healing of major operations.

Description

Technical Field

[0001] The present invention relates to the field of collagen preparation, and in particular to a preparation method, product and application for improving the biocompatibility of collagen. Background Art

[0002] In recent years, with the continuous deepening of research on the structure and function of collagen, the demand for collagen products has grown rapidly worldwide. The application of collagen in the medical field, such as dressings, implant materials and tissue engineering medical products, is growing day by day. Collagen is the main protein component of connective tissue and is a functional protein closely related to the functions of cells, tissues and organs of the body. It has good biocompatibility and biodegradability, and also has properties such as high tensile strength, low immunity, hemostasis and promotion of cell growth. Therefore, the application of collagen in the field of regenerative medicine has attracted much attention, and various large, medium and small enterprises have been involved in the research and development of medical products, showing huge market potential. However, compared with the rapidly developing market, the establishment of collagen quality control technology, standards and specifications is relatively lagging, which leaves hidden dangers for product quality control and safe use, and also restricts the standardized and healthy development of the corresponding industry. The applications of collagen polymers and self-assembled collagen-based products (collectively referred to as collagen products) in the medical field mainly include: as starting materials for surgical implants, scaffolds for tissue engineered medical products (TEMPs), dressings, therapeutic cell scaffolds or drug carriers, and three-dimensional (3D) in vitro tissue systems or models for basic research, drug development and toxicity testing.

[0003] The immunogenicity (i.e., biocompatibility) of collagen varies according to the tissue source of the raw materials. Although it is affected by factors such as purity, impurities, processes, and residual reagents, immune reactions caused solely based on species differences are not common. Common causes of immune rejection reactions also include: collagen fiber quality (including collagen purity, impurity components, and removal of terminal peptides), hydroxyproline percentage, and differences in the structure and chemical properties of collagen. Therefore, biomimetic methods such as chemical cross-linking should be used to determine to what extent changes in the structure of collagen may affect the immunogenicity of collagen after implantation in the human body. General tests include skin sensitization tests, in vivo immunotoxicology tests in animals, etc. If it is a degradable material, the immunogenicity risk during the degradation cycle should also be fully evaluated when conducting immunotoxicology tests, and at least the worst case scenario should be included (a period when the implant material is heavily degraded and potential immunogens are fully exposed) to evaluate whether the remaining immunogenicity risk is acceptable.

[0004] There are many methods for extracting and preparing collagen from animal sources in the prior art. Although they all claim to have achieved good separation effects and ensure the biocompatibility of the obtained collagen, the high price of medical-grade collagen on the market shows that it is not so easy to produce highly biocompatible collagen. How to develop a process that can pass a relatively simple process and ensure product quality is currently an urgent problem to be solved. Summary of the invention

[0005] In view of this, the main purpose of the present invention is to provide a preparation method, product and application for improving the biocompatibility of collagen, in order to at least partially solve the above technical problems.

[0006] In order to achieve the above object, as one aspect of the present invention, a preparation method for improving the biocompatibility of collagen is proposed, comprising the following steps:

[0007] Pre-treatment of raw materials, slicing the raw materials, removing grease and dirt;

[0008] Acid hydrolysis and enzymatic hydrolysis are performed on the pre-treated product to obtain an enzymatic hydrolysis product;

[0009] The enzymatic hydrolysate is mixed with a deep eutectic solvent and then filtered through a sterilizing filter;

[0010] The filtered product is subjected to ultrafiltration, liquid replacement and concentration treatment to obtain the collagen sponge product;

[0011] The deep eutectic solvent is formed by mixing a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is methanesulfonic acid and the hydrogen bond acceptor is choline chloride.

[0012] Deep Eutectic Solvents (DESs) are a type of solvent system composed of salts (hydrogen bond acceptors) and hydrogen bond donors. They have the characteristics of low cost, low toxicity, renewability and good biocompatibility, and are considered to be an environmentally friendly green solvent for effective protein extraction. In addition, DESs can form hydrogen bonds with the amino or carboxyl groups of proteins. Different DESs have different affinities for different amino acids, so they have a certain selectivity, which improves the solubility of collagen during the extraction process, thereby increasing the extraction rate of collagen. The present invention achieves highly selective extraction of collagen from pig or cattle tendons by adjusting the composition of DESs and reaction conditions, thereby improving the recovery rate of collagen.

[0013] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the deep eutectic solvent is 1:(1-2), and the molar ratio can be, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.

[0014] Preferably, the preparation method of the deep eutectic solvent comprises the following steps:

[0015] Methanesulfonic acid and choline chloride are mixed in proportion to obtain a mixed solution A, and deionized water of an equal volume to the mixed solution A is added, heated to 60-68°C, and continuously stirred until it becomes uniform and transparent to obtain a deep eutectic solvent. The temperature may be, for example, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C, 63°C, 63.5°C, 64°C, 64.5°C, 65°C, 65.5°C, 66°C, 66.5°C, 67°C, 67.5°C, 68°C

[0016] Preferably, the liquid-to-solid ratio of the enzymatic hydrolysate to the deep eutectic solvent is 1 g: (20-30) mL (the liquid-to-solid ratio can be, for example, 1 g: 20 mL, 1 g: 21 mL, 1 g: 22 mL, 1 g: 23 mL, 1 g: 24 mL, 1 g: 25 mL, 1 g: 26 mL, 1 g: 27 mL, 1 g: 28 mL, 1 g: 29 mL, 1 g: 30 mL), and is placed at 1-5 ° C (for example, it can be 1 ° C, 1.5 ° C, 2 ° C, 2.5 ° C, 3 ° C, 3.5 ° C, 4 ° C, 4.5 ° C, 5 ° C) and stirred for at least 24 h.

[0017] The acid hydrolysis and enzymolysis processes specifically include: using 0.1%-10% acetic acid solution (the mass fraction of the acetic acid solution can be, for example, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% or all values ​​within the range, due to space limitations The invention relates to a method for preparing an enzymatic hydrolysis product, wherein the enzymatic hydrolysis product is prepared by adding an appropriate amount of a proteolytic enzyme, and the enzymatic hydrolysis product is subjected to enzymatic hydrolysis for 24-96 hours (for example, it can be 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 76 hours, 80 hours, 84 hours, 88 hours, 92 hours, 96 hours or all values ​​within the range, which will not be described in detail due to space limitations), and then filtered to obtain an enzymatic hydrolysis product;

[0018] Preferably, the raw material is tendon, such as pig or cattle tendon, preferably pig tendon.

[0019] Preferably, the pig is a three-month-old pig. Since the ratio of type I to type III collagen is different in pigs of different ages, the tendon of a three-month-old pig has a higher content of type I collagen and a relatively lower fat content.

[0020] Preferably, the method for removing grease and dirt is to use a machine to automatically bubble wash the sliced ​​raw material.

[0021] The pre-treatment steps include:

[0022] Select and simply clean the raw materials;

[0023] Among them, it is preferably frozen at -20°C (for example, -25°C, -30°C, -35°C, -40°C, etc.) and sliced ​​into 1-5 mm (for example, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm or all values ​​within the range, which will not be described in detail due to space limitations);

[0024] Automatic bubble cleaning by machine is used to remove grease and dirt.

[0025] Preferably, simple cleaning only removes general surface dirt such as mud, while bubble cleaning uses a set concentration of sodium carbonate solution, sodium bicarbonate solution, hydrogen peroxide solution or ethanol solution or a combination thereof to generate bubbles through the reaction, or air forced into the outside world, to produce a stirring effect, making it easier to rinse out the dirt in the raw materials cut into small pieces.

[0026] Preferably, in the salting-out step, a 1%-20% sodium chloride solution (the mass fraction may be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or all values ​​within the range, which will not be described in detail due to space limitations) is used to salt out the enzymatic hydrolysate for 0.5-4h to remove substances such as small molecule proteins and amino acids. After salting out, the precipitate is centrifuged and redissolved in a 0.1%-10% acetic acid solution.

[0027] Preferably, the sterilizing filter is a single-stage or double-stage filter.

[0028] Preferably, the enzymatic hydrolysis time is 24-96 hours.

[0029] Preferably, the ultrafiltration and concentration step uses a hollow fiber column and / or membrane package with a molecular weight cutoff of 10KD-100KD (e.g., 10KD, 30KD, 50KD, 100KD, etc., all values ​​within the range, which will not be repeated due to space limitations) to perform ultrafiltration, liquid exchange and concentration treatment to remove impurities such as inorganic salts.

[0030] Preferably, after the concentration process, a freeze-drying step is also included, for example, the concentrated sample can be placed in a freeze dryer for freeze drying, and the sample can be dried once or twice, wherein the primary drying time is 12-48 hours, and the secondary drying time after the primary drying is 4-24 hours.

[0031] The present invention also provides collagen prepared by the preparation method for improving the biocompatibility of collagen.

[0032] The present invention also proposes an application of the above collagen product in the preparation of wound healing products, burn repair products, nerve damage regeneration products, dura mater substitutes, artificial corneas, hemostatic agents, and drug carriers.

[0033] Based on the above technical solutions, it can be seen that the preparation method, product and application of the present invention for improving the biocompatibility of collagen have at least one of the following beneficial effects compared with the prior art:

[0034] 1. Compared with conventional extraction methods, the present invention selects deep eutectic solvent DESs for protein extraction. DESs can form hydrogen bonds with the amino or carboxyl groups of proteins. Different DESs have different affinities for different amino acids, so they have certain selectivity, which improves the solubility of collagen during the extraction process, thereby increasing the extraction rate of collagen. The present invention achieves highly selective extraction of collagen from pig or beef tendons by adjusting the composition of DESs and reaction conditions, thereby improving the recovery rate of collagen.

[0035] 2. The extraction process of the present invention removes the biological terminal peptide of collagen thoroughly, which can effectively reduce immunogenicity, improve biocompatibility, reduce immune rejection, and the residual α-Gal antigen and residual proteolytic enzyme are both below the detection limit, which can be used for hemostasis and wound healing in major surgery; the collagen has high purity, good thermal stability, and can maintain a complete triple helix structure. The method of the present invention has simple procedures, convenient operation, and saves time and effort. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The method and device of the present invention are further described below in conjunction with the accompanying drawings and embodiments:

[0037] Figure 1 It is a fitting curve diagram of the standard concentration of Example 1 and the residual detection limit of α-gal antigen. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0039] In view of the various collagen preparation methods in the prior art, either the integrity and purity of the prepared product cannot be guaranteed, or the toxicity of the product cannot be reduced. After intensive research, the inventors have proposed a preparation method for improving the biocompatibility of collagen with simple process, significant sterilization effect and the ability to ensure the integrity of the prepared product, comprising the following steps:

[0040] Pre-treatment of raw materials, slicing the raw materials, removing grease and dirt;

[0041] Acid hydrolysis and enzymatic hydrolysis are performed on the pre-treated product to obtain an enzymatic hydrolysis product;

[0042] The enzymatic hydrolysate is mixed with a deep eutectic solvent and then filtered through a sterilizing filter;

[0043] The filtered product is subjected to ultrafiltration, liquid replacement and concentration treatment to obtain the collagen sponge product;

[0044] The deep eutectic solvent is formed by mixing a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is methanesulfonic acid and the hydrogen bond acceptor is choline chloride.

[0045] Deep Eutectic Solvents (DESs) are a type of solvent system composed of salts (hydrogen bond acceptors) and hydrogen bond donors. They have the characteristics of low cost, low toxicity, renewability and good biocompatibility, and are considered to be an environmentally friendly green solvent for effective protein extraction. In addition, DESs can form hydrogen bonds with the amino or carboxyl groups of proteins. Different DESs have different affinities for different amino acids, so they have a certain selectivity, which improves the solubility of collagen during the extraction process, thereby increasing the extraction rate of collagen. The present invention achieves highly selective extraction of collagen from pig or cattle tendons by adjusting the composition of DESs and reaction conditions, thereby improving the recovery rate of collagen.

[0046] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the deep eutectic solvent is 1:(1-2), and the molar ratio can be, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.

[0047] Preferably, the preparation method of the deep eutectic solvent comprises the following steps:

[0048] Methanesulfonic acid and choline chloride are mixed in proportion to obtain a mixed solution A, and deionized water of an equal volume to the mixed solution A is added, heated to 60-68°C, and continuously stirred until it becomes uniform and transparent to obtain a deep eutectic solvent. The temperature may be, for example, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C, 63°C, 63.5°C, 64°C, 64.5°C, 65°C, 65.5°C, 66°C, 66.5°C, 67°C, 67.5°C, 68°C

[0049] Preferably, the liquid-to-solid ratio of the enzymatic hydrolysate to the deep eutectic solvent is 1 g: (20-30) mL (the liquid-to-solid ratio can be, for example, 1 g: 20 mL, 1 g: 21 mL, 1 g: 22 mL, 1 g: 23 mL, 1 g: 24 mL, 1 g: 25 mL, 1 g: 26 mL, 1 g: 27 mL, 1 g: 28 mL, 1 g: 29 mL, 1 g: 30 mL), and is placed at 1-5 ° C (for example, it can be 1 ° C, 1.5 ° C, 2 ° C, 2.5 ° C, 3 ° C, 3.5 ° C, 4 ° C, 4.5 ° C, 5 ° C) and stirred for at least 24 h.

[0050] The acid hydrolysis and enzymatic hydrolysis process specifically includes: using 0.1%-10% acetic acid solution (the mass fraction of the acetic acid solution can be, for example, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% or all values ​​within the range), by The invention relates to a method for preparing a filtration apparatus comprising: ...

[0051] The filtered product is subjected to ultrafiltration, liquid exchange and concentration treatment to obtain the collagen sponge product.

[0052] Wherein, the raw material is tendon, such as pig or cattle tendon, preferably pig tendon.

[0053] Among them, the pig is preferably a three-month-old pig. Since the ratio of type I to type III collagen is different in pigs of different ages, the tendon of a three-month-old pig has a higher content of type I collagen and a relatively lower fat content.

[0054] Among them, the method of removing grease and dirt is to use a machine to automatically bubble clean the sliced ​​raw materials.

[0055] The pre-treatment steps include:

[0056] Select and simply clean the raw materials;

[0057] Wherein, the frozen slices are cut into 1-5 mm (for example, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm or all values ​​within the range, which will not be described in detail due to space limitations) at a temperature below -20°C (for example, -25°C, -30°C, -35°C, -40°C, etc.);

[0058] Automatic bubble cleaning by machine is used to remove grease and dirt.

[0059] Among them, simple cleaning only removes general surface dirt such as mud, while bubble cleaning uses a set concentration of sodium carbonate solution, sodium bicarbonate solution, hydrogen peroxide solution or ethanol solution or a combination thereof to produce bubbles through the reaction, or air forced into the outside world, to create a stirring effect, making it easier to rinse out the dirt in the raw materials cut into small pieces.

[0060] Wherein, the sterilizing filter is a single-stage or double-stage filter.

[0061] Among them, the ultrafiltration and concentration step, for example, uses a hollow fiber column and / or membrane package with a molecular weight cutoff of 10KD-100KD (for example, 10KD, 30KD, 50KD, 100KD, etc., all values ​​within the range, which will not be repeated due to space limitations) to perform ultrafiltration, liquid exchange and concentration treatment to remove impurities such as inorganic salts.

[0062] Among them, after the concentration process, for example, a freeze-drying step is also included. For example, the concentrated sample can be placed in a freeze dryer for freeze drying. The sample can be dried once or twice, wherein the primary drying time is 12-48 hours, and the secondary drying time after the primary drying is 4-24 hours.

[0063] In a preferred embodiment, the method for preparing collagen of the present invention comprises the following steps:

[0064] 1. Pretreatment: Collect frozen tendons and slow them down, wash and remove impurities such as fascia, freeze and slice them at -20℃ to 1-5mm. After slicing, use cleaning agents of different concentrations (sodium carbonate solution, sodium bicarbonate solution, hydrogen peroxide solution, ethanol solution) to wash again to remove grease and other substances.

[0065] 2. Enzymatic hydrolysis: Use 0.1%-10% acetic acid solution and an appropriate amount of proteolytic enzyme for enzymatic hydrolysis. After 24-96 hours of enzymatic hydrolysis, filter to obtain the enzymatic hydrolysis product of collagen.

[0066] 3. Sterile filtration: Mix the enzymatic hydrolysate with the deep eutectic solvent, and filter the mixed solution using a sterilizing filter; the sterilizing filter is a single-stage or double-stage filter.

[0067] 4. Ultrafiltration and concentration: Use hollow fiber columns or membrane packages with a molecular weight cutoff of 10KD-100KD for ultrafiltration and liquid exchange and concentration to remove impurities such as inorganic salts.

[0068] 5. Freeze drying: Put the concentrated sample into the freeze dryer for freeze drying. The sample is pre-frozen for 5-12 hours and then dried once. The first drying time is 12-48 hours and the second drying time is 4-24 hours.

[0069] The present invention also provides collagen prepared by the preparation method for improving the biocompatibility of collagen.

[0070] The present invention also proposes an application of the above collagen product in the preparation of wound healing products, burn repair products, nerve damage regeneration products, dura mater substitutes, artificial corneas, hemostatic agents, and drug carriers.

[0071] The present invention will be further described below through specific examples. It should be noted that the following examples are only for illustration and are not intended to limit the present invention.

[0072] Experimental preparation

[0073] Centrifuge Brand: Xiangyi Centrifuge Instrument Co., Ltd. Model: H2050R

[0074] Reactor Brand: Zhengzhou Great Wall Science & Technology Co., Ltd. Model: GR-10

[0075] Slicer Brand: Beijing Century Mingyang Food Machinery Co., Ltd. Model: QY-30

[0076] Electrophoresis instrument brand: Beijing Liuyi Biotechnology Co., Ltd. Model: DYY-60

[0077] Gel imaging system Brand: American BIO-RAD Model: GelDocGo

[0078] UV-Vis Spectrophotometer Brand: Shimadzu Instruments Co., Ltd. Model: UV-2600

[0079] Micro-volume differential scanner Brand: Malvern Panalytical Model: PEAQ-DSC

[0080] Circular Dichroism Spectrometer Brand: JASCO Model: J-1500

[0081] Pigs aged 3-6 months: Large tendons, more type I collagen, and better purity

[0082] Proteolytic enzyme for quantitative detection Brand: US Pharmacopeia, Product Number: 1510051 (United States Pharmacopeia (USP) Reference Standard), CAS Number: 9001-75-6

[0083] Example 1

[0084] Specific experimental steps:

[0085] 1. Pretreatment: The collected frozen tendons are slowed down, washed to remove impurities such as fascia, and then frozen and sliced ​​to 5 mm at below -20°C. After slicing, 10 wt% sodium carbonate aqueous solution is used as a cleaning agent to wash again to remove grease and other substances.

[0086] 2. Enzymatic hydrolysis: Use 8% by mass acetic acid solution and an appropriate amount of proteolytic enzyme for enzymatic hydrolysis. After 24-96 hours of enzymatic hydrolysis, filter and obtain the enzymatic hydrolysis product of collagen.

[0087] 3. Sterile filtration: Mix the enzymatic hydrolysate with the deep eutectic solvent, and filter the mixed solution using a sterilizing filter; the sterilizing filter is a single-stage or double-stage filter;

[0088] The preparation steps of the deep eutectic solvent are as follows:

[0089] Methanesulfonic acid and choline chloride are mixed in proportion to obtain a mixed solution A, deionized water having an equal volume to the mixed solution A is added, the mixture is heated to 68° C., and the mixture is continuously stirred until it becomes uniform and transparent, thereby obtaining a deep eutectic solvent.

[0090] 4. Ultrafiltration and concentration: Use a hollow fiber column or membrane package with a molecular weight cutoff of 50KD for ultrafiltration and liquid exchange and concentration to remove impurities such as inorganic salts.

[0091] 5. Freeze drying: Put the concentrated sample into the freeze dryer for freeze drying. The sample is pre-frozen for 5 hours and then dried once. The drying time is 24 hours.

[0092] Example 2-11

[0093] The specific operation steps are the same as those in Example 1, except that some parameters are adjusted as described in Table 1 below.

[0094] Table 1 List of specific operating parameters of Examples 1-11

[0095]

[0096]

[0097] Performance test and conclusion:

[0098] I. Detection of terminal peptide residues

[0099] There is no tryptophan in the de-telopeptide collagen, but there are multiple tryptophans in the telopeptide. Therefore, the degree of telopeptide removal can be determined by testing the tryptophan content in the sample. The specific testing method is based on YY 0954-2015 "Type I collagen implant", Appendix E "Tryptophan test". It is mainly through the reaction of the indole group of tryptophan with acetaldehyde to generate a purple-red substance after the sample is subjected to alkaline hydrolysis.

[0100] After testing, there was no purple ring at the interface of the acetic acid and sulfuric acid liquids in Examples 1-11, indicating that there was no tryptophan residue, indicating that the terminal peptide of the collagen product prepared in Examples 1-11 had been completely removed, which can effectively reduce the immunogenicity and thus improve the biocompatibility.

[0101] II. α-Gal Antigen Detection

[0102] According to the method given in the industry standard "Tissue Engineering Medical Device Products - Detection of Residual α-Gal Antigen in Animal-Derived Scaffold Materials", the detection is carried out using the enzyme-linked immunosorbent assay (competitive ELISA or ELISA inhibition method) of specific anti-Gal antibodies, that is: under the premise of ensuring an excess of specific antibodies in the antigen-antibody reactants, firstly, the α-Gal antigen of the standard curve sample and the test sample reacts with the specific antibody (consuming part of the antibody); then, using the artificially synthesized Gal antigen (Gal-BSA) as the solid phase antigen, the remaining antibodies in the supernatant after the first reaction are detected by the ELISA method; and then the standard curve can be used to calculate the α-Gal antigen content in the test object.

[0103] According to the antibody binding inhibition rate (%) and the antigen mass concentration (mg / L) in the standard sample in Table 2, Figure 1 The standard curve fitting equation is shown. After the samples are treated, they are detected by an ELISA instrument and the antibody binding inhibition rate (%) is calculated. Substituting this value into the above standard curve equation, the α-Gal antigen concentration can be calculated. The test results of Examples 1-11 are shown in Table 3. After testing, the α-Gal antigen in the samples prepared in Examples 1-11 is lower than the detection limit of α antigen detection in YY / T 1561-2017, indicating that the optimized preparation process provided by the present invention has a significant effect on the removal of α antigen.

[0104] Table 2

[0105]

[0106] III. Collagen purity test

[0107] The purity of type I collagen prepared in Example 1-11 was determined according to GB / T 38482-2021 Determination of animal-derived type I collagen composition-polyacrylamide gel electrophoresis. The determination method utilizes the triple helical structure of type I collagen that other proteins do not have, cooperates with the specific collagenase, and determines the staining limit of bovine serum albumin (BSA) with Coomassie brilliant blue, and uses the SDS-PAGE method to determine the composition of type I collagen.

[0108] The purity of Example 1 was detected and calculated according to the GB / T 38482-2021 method. The results showed that there were no mixed bands during electrophoresis detection. The purity of the collagen sample prepared in Example 1 was greater than 99% as determined by the BSA staining limit.

[0109] The purity detection graph of the collagen prepared in Example 2-11 is similar to that in Example 1, and the purity is more than 99%.

[0110] The fine adjustment of the above parameters can achieve the purpose of the invention, but it has a slight impact on the final yield. Through the combination of various parameters, it can be found that Examples 1, 10, and 11 have better performance. The specific performance test results are shown in Table 3.

[0111] Table 3 Performance test results of Examples 1-11

[0112]

[0113] Comparison test

[0114] In order to further compare with the prior art, the present invention selected Example 1 as a representative (hereinafter referred to as "self-developed product") and conducted a series of experiments with two collagen raw materials purchased on the market (hereinafter referred to as "commercially available product A" and "commercially available product B").

[0115] I. Thermal stability study

[0116] The above-mentioned collagen solution to be tested is freeze-dried and cross-linked by the same method (a 0.5% mass fraction of glutaraldehyde solution is added to the collagen solution, mixed and then placed at 4°C for cross-linking, and then transferred to a mold for freeze-drying) to form a collagen sponge, which is then heated. When the collagen sponge undergoes a phase change during heating, it has a specific phase transition temperature and a corresponding thermal enthalpy change accompanied by a heat absorption process. When collagen unravels the triple helix structure, a specific transition temperature can be observed, represented by Tm. The results show that the Tm value of the self-developed product is 65.8°C, which is higher than 61.0°C and 56.3°C of other commercially available products. This shows that the triple helix structure of the self-developed product has a higher integrity and better stability than commercially available products.

[0117] II. Circular dichroism spectrum analysis

[0118] The scanning results of the standard product using the Japanese Spectroscopic Circular Dichroism Spectrometer showed that its positive peak wavelength was 221.4nm, the peak value was 35.0465, the negative peak wavelength was 198.5nm, the peak value was -273.83, and the absolute value ratio of the positive peak to the negative peak was 0.1280; the scanning results of the self-developed product showed that its positive peak wavelength was 221.1nm, the peak value was 37.0072, the negative peak wavelength was 197.7nm, the peak value was 269.729, and the absolute value ratio of the positive peak to the negative peak was 0.1372. The above scanning results show that the self-developed product and the standard product have basically the same triple helix structure, with good integrity and stability.

[0119] III. α-Gal antigen detection

[0120] According to the method described above, α-Gal antigen testing was performed on the self-developed product, commercially available product A and commercially available product B. The results showed that the residual α-Gal antigen of commercially available product B and the self-developed product were both below the detection limit (0.03125 mg / L), while that of commercially available product A was above the detection limit.

[0121] Table 4

[0122]

[0123] IV. Proteolytic enzyme residue detection

[0124] Proteolytic enzyme residue detection was performed according to the proteolytic enzyme activity detection method described in the Chinese Pharmacopoeia 2020. The results are as follows:

[0125] Commercially available product A: 43.1U / g

[0126] Commercially available product B: 3.5EU / g

[0127] Self-developed products: 3.1EU / g

[0128] This indicates that the residual amount of proteolytic enzymes in the self-developed product is maintained at an extremely low level, and is lower than or equivalent to commercially available products. Therefore, the recombinant collagen in the self-developed product has a lower possibility of being dissolved into fibrous collagen and can maintain the integrity of the collagen structure. The triple helix structure is not easily degraded by proteolytic enzymes, thereby playing a stabilizing supporting role and being more suitable for medical use.

[0129] V. Comparative testing of DESs with different compositions

[0130] The method of the present invention was compared with the traditional method of extracting collagen (Hu Kang, Zhang Wei. The role and significance of collagen as a medical biomaterial in the repair, regeneration and reconstruction of defective tissues [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(2): 317-322. DOI: 10.3969 / j.issn.2095-4344.0650, 1.3 The 8 articles on the source and extraction method of collagen were compared and tested), and the collagen recovery rate and collagen structure identification were performed. It was found that the collagen extraction rate of the process of the present invention is higher. Subsequently, DESs of different compositions (trimethylbenzylammonium chloride-methanesulfonic acid, triethylbenzylammonium chloride-methanesulfonic acid, tripropylbenzylammonium chloride-methanesulfonic acid, tributylbenzylammonium chloride-methanesulfonic acid, trimethylethylbenzylammonium chloride-methanesulfonic acid, methyltrioctylammonium chloride-methanesulfonic acid, methyltriethylammonium chloride-methanesulfonic acid, methyltributylammonium chloride-methanesulfonic acid, tetramethylammonium chloride-methanesulfonic acid, tetraethylammonium chloride-methanesulfonic acid, tetrapropylammonium chloride-methanesulfonic acid, tetrabutylammonium chloride-methanesulfonic acid, urea-propionic acid, urea-oxalic acid, urea-citric ... The collagen in pig's trotters was extracted by using the following DESs: betaine-malic acid, urea-ethylene glycol, urea-methanesulfonic acid, betaine-formic acid, betaine-acetic acid, betaine-propionic acid, betaine-lactic acid, betaine-oxalic acid, betaine-citric acid, betaine-malic acid, betaine-ethylene glycol, betaine-methanesulfonic acid, choline chloride-formic acid, choline chloride-acetic acid, choline chloride-propionic acid, choline chloride-lactic acid, choline chloride-oxalic acid, choline chloride-citric acid, choline chloride-malic acid, choline chloride-ethylene glycol) in the same manner as in Example 1, except that the types of DESs were different.

[0131] The test found that the collagen recovery rates of DESs (methyltrioctylammonium chloride-methanesulfonic acid, urea-ethylene glycol, betaine-ethylene glycol, choline chloride-malic acid, choline chloride-ethylene glycol) were all over 80%, while the collagen recovery rates of DESs (methyltriethylammonium chloride-methanesulfonic acid, methyltributylammonium chloride-methanesulfonic acid, tetramethylammonium chloride-methanesulfonic acid, betaine-propionic acid) were all less than 40%; the remaining DESs of different formulas were unable to extract collagen from pig trotters.

[0132] In summary, the extraction process of the present invention removes the biological terminal peptide of collagen thoroughly, can effectively reduce immunogenicity, improve biocompatibility, lower immune rejection reaction, and the residual α-Gal antigen and residual proteolytic enzyme are both below the detection limit, which can be used for hemostasis and wound healing in major surgery; the collagen has high purity, good thermal stability, and can maintain a complete triple helix structure. The method of the present invention has simple procedures, convenient operation, and saves time and effort.

[0133] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method for improving the biocompatibility of collagen, characterized in that: The steps include: Pre-treatment of raw materials, slicing the raw materials, removing grease and dirt; Acid hydrolysis and enzymatic hydrolysis are performed on the pre-treated product to obtain an enzymatic hydrolysis product; The enzymatic hydrolysate is mixed with a deep eutectic solvent and then filtered through a sterilizing filter; The filtered product is subjected to ultrafiltration, liquid replacement and concentration treatment to obtain the collagen sponge product; The deep eutectic solvent is formed by mixing a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is methanesulfonic acid and the hydrogen bond acceptor is choline chloride.

2. The preparation method according to claim 1, characterized in that: The molar ratio of hydrogen bond acceptors to hydrogen bond donors of deep eutectic solvents is 1:(1-2).

3. The preparation method according to claim 1 or 2, characterized in that: The preparation method of the deep eutectic solvent comprises the following steps: Methanesulfonic acid and choline chloride are mixed in proportion to obtain a mixed solution A, deionized water having an equal volume to the mixed solution A is added, the mixture is heated to 60-68° C., and the mixture is continuously stirred until it becomes uniform and transparent to obtain a deep eutectic solvent.

4. The preparation method according to claim 1, characterized in that: The liquid ratio of the enzymatic hydrolysate to the deep eutectic solvent is 1 g: (20-30) mL, and the mixture is stirred and mixed at 1-5° C. for at least 24 hours.

5. The preparation method according to claim 1, characterized in that: The raw material is pig or cattle tendon, and the pig is a three-month-old pig.

6. The preparation method according to claim 5, characterized in that: The method for removing grease and dirt is to use a machine to automatically bubble clean the sliced ​​raw material, and the bubble cleaning uses a set concentration of sodium carbonate solution, sodium bicarbonate solution, hydrogen peroxide solution or ethanol solution or a combination thereof as a cleaning agent.

7. The preparation method according to claim 1, characterized in that: The acid hydrolysis and enzymatic hydrolysis process specifically includes: using 0.1%-10% acetic acid solution for acid hydrolysis; using proteolytic enzyme for enzymatic hydrolysis.

8. The preparation method according to claim 1, characterized in that: The ultrafiltration and concentration step uses a hollow fiber column and / or a membrane package with a molecular weight cutoff of 10KD-100KD to perform ultrafiltration, liquid exchange and concentration treatment.

9. The preparation method according to claim 1, characterized in that: After the concentration treatment, a freeze-drying step is also included, in which the cross-linked sample is placed in a freeze dryer for freeze drying. The sample is dried once or twice, wherein the first drying time is 12-48 hours and the second drying time is 4-24 hours.

10. Collagen prepared by the preparation method for improving the biocompatibility of collagen according to any one of claims 1 to 9.

11. Use of the collagen as claimed in claim 11 in the preparation of wound healing products, burn repair products, nerve damage regeneration products, dura mater substitutes, artificial corneas, hemostatic agents, and drug carriers.

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