Preparation method for improving integrity of collagen, product and application

Abstract

The invention relates to the field of collagen preparation, in particular to a preparation method for improving the integrity 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 obtained by the extraction process disclosed by the invention contains more type I collagen, the purity exceeds 99%, the thermal stability is excellent, the structural integrity is good, and the collagen can effectively stop bleeding and prevent adhesion, effectively fill an operation cavity, play a stable supporting role and accelerate tissue healing.

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 integrity of collagen. Background Art

[0002] As a natural biological resource, collagen has the functions of biocompatibility, low immunogenicity, biodegradability and absorbability, promotion of new cell formation and promotion of epithelial cell formation that cannot be matched by synthetic polymer materials, and it can make the bones, skin, tendons, etc. of animals have good mechanical properties. Therefore, collagen has a wide range of application prospects in the field of biomedicine, including wound healing, burn repair, nerve damage regeneration, dura mater substitutes, artificial cornea, hemostatic agents and many other aspects. As early as the early 1970s, there were clinical reports of collagen membranes used for burns and trauma, which played a protective, hemostatic and repairing role. Collagen can accelerate hemostasis by attaching and aggregating with platelets. In clinical practice, collagen sponges also have good hemostatic effects and can accelerate the coagulation of wounds. Collagen can also be used as a drug carrier in the clinic to treat various eye diseases, which can accelerate the repair of corneal epithelial damage, the growth of epithelial cells and the healing of corneal wounds.

[0003] At present, a major source of industrial collagen is animal collagen. There are many animal collagen extraction and preparation methods in the prior art. For example, Chinese patent application CN 113462736A discloses a preparation method and collagen for obtaining atelocollagen from pig skin. The preparation method uses three-month-old SPF (specific pathogen-free pig) pig skin as raw material, degreases with a degreasing agent, extracts pig skin collagen by enzymatic hydrolysis and removes the telopeptide, purifies the collagen by salt washing and ultrafiltration, and obtains high-purity sponge-like atelocollagen after freeze drying. At present, type I medical collagen materials have very high requirements for collagen raw materials and are relatively expensive. For example, there are strict regulations for purity, integrity, digestibility, sterility, biocompatibility, etc. Only collagen raw materials that meet the above requirements can use their unique triple helical structure to induce cell adhesion, chemotaxis, and proliferation, provide nutrition for the wound surface, and accelerate the healing of the wound surface, so that they can be promoted and applied in clinical practice. Since the collagen isolated and purified from different tissues has significant differences in purity, physical and chemical properties and collagen configuration, obtaining type I medical collagen materials with higher purity that maintain the unique triple helix structure of collagen has always been the goal of scientific researchers. Summary of the invention

[0004] In view of this, the main purpose of the present invention is to provide a preparation method, product and application for improving the integrity of collagen, in order to at least partially solve the above technical problems.

[0005] In order to achieve the above object, as one aspect of the present invention, a preparation method for improving the integrity of collagen is proposed, comprising the following steps:

[0006] Pre-treatment of the raw materials: freezing the raw materials at below -20°C (for example, -25°C, -30°C, -35°C, -40°C, etc.) and slicing them 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) to remove grease and dirt;

[0007] The pre-treated product is subjected to acid hydrolysis and enzymolysis; wherein the acid hydrolysis and enzymolysis process specifically comprises: using an acetic acid solution having a mass fraction of 0.1%-10% (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, which will not be described in detail due to space limitations) and an appropriate amount of pepsin solution for enzymolysis, at 4-30°C (for example, Filter after enzymatic hydrolysis for 24-96h (for example, it can be 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, 72h, 76h, 80h, 84h, 88h, 92h, 96h or all values ​​in the range, which are not described in detail due to space limitations);

[0008] Salting out the enzymatic hydrolysis product, re-dissolving the salting out product and filtering it;

[0009] The filtered product is ultrafiltered and concentrated to obtain collagen.

[0010] Preferably, the conditions for enzymatic hydrolysis are: the temperature for the first 24-48 hours is 23-30°C (for example, it can be 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, 30°C or all values ​​within the range, which are not described in detail due to space limitations), and then the temperature is lowered to 4-8°C to continue the reaction (for example, it can be 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C or all values ​​within the range, which are not described in detail due to space limitations); this setting can remove the propeptide and the terminal peptide in the form of enzymatic hydrolysis during the dissociation of collagen, and then obtain collagen that does not contain any immune components, and can be accompanied by virus inactivation during the enzymatic hydrolysis process.

[0011] Preferably, during the enzymatic hydrolysis reaction, ultrasound is used to assist enzymatic hydrolysis; the ultrasound time is 10-30 min (for example, it can be 10 min, 15 min, 20 min, 25 min, 30 min or all values ​​within the range, which are not described in detail due to space limitations), and the ultrasound power is 280-320 W (for example, it can be 280 W, 285 W, 290 W, 295 W, 300 W, 305 W, 310 W, 315 W, 320 W or all values ​​within the range, which are not described in detail due to space limitations).

[0012] Preferably, the raw material is any one of pig tendon, beef tendon or pig skin, or a combination of at least two of them.

[0013] 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.

[0014] Preferably, the pre-treatment step specifically includes:

[0015] Select and simply clean the raw materials;

[0016] Cryosection at -20℃ into 1-5mm slices;

[0017] Automatic bubble cleaning by machine is used to remove grease and dirt.

[0018] Preferably, the bubble cleaning uses a sodium carbonate solution, a sodium bicarbonate solution, a hydrogen peroxide solution or an ethanol solution or a combination thereof of a set concentration as a cleaning agent.

[0019] Preferably, in the enzymatic hydrolysis step, the purity of pepsin is >90%.

[0020] Preferably, in the pepsin solution, the mass fraction of pepsin is 0.1%-2% (for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or all values ​​within the said range, which are not described again due to space limitations).

[0021] Preferably, in the enzymatic hydrolysis step, the mass ratio of the raw material to the pepsin solution is 1:(50-300), and the mass ratio can be, for example, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, 1:250, 1:260, 1:270, 1:280, 1:290, 1:300 or all values ​​within the range, which will not be repeated due to space limitations.

[0022] Preferably, in the salting-out step, sodium chloride solution is used to salt-out the enzymatic hydrolyzate.

[0023] Preferably, the filtering step is performed using a sterilizing filter.

[0024] Preferably, the sterilizing filter is a single-stage or double-stage filter.

[0025] 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) for ultrafiltration, liquid exchange and concentration treatment.

[0026] Preferably, after the concentration treatment, a freeze-drying step is also included, and the concentrated sample is placed in a freeze dryer for freeze drying, and the sample is dried once or twice, wherein the primary drying time is 12-48 h (for example, it can be 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 42 h, 48 h or all values ​​within the range, which will not be repeated due to space limitations), and the secondary drying time is 4-24 h (for example, it can be 4 h, 8 h, 12 h, 16 h, 20 h, 24 h or all values ​​within the range, which will not be repeated due to space limitations).

[0027] As a second aspect of the present invention, a collagen prepared by the above-mentioned preparation method for improving the integrity of collagen is also proposed.

[0028] As the third aspect of the present invention, a use 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 is also proposed.

[0029] Based on the above technical solutions, it can be seen that the preparation method, product and application of improving the integrity of collagen of the present invention have at least one of the following beneficial effects compared with the prior art:

[0030] 1. Compared with conventional extraction methods, the collagen obtained by the extraction process of the present invention contains more type I collagen, with a purity of more than 99%, excellent thermal stability and good structural integrity. The results of circular dichroism spectrum analysis show that it has basically the same triple helix structure as the standard product; when it is made into a collagen sponge product, the collagen obtained by the extraction process of the present invention can effectively stop bleeding and prevent adhesion, effectively fill the surgical cavity, play a stabilizing and supporting role and accelerate tissue healing.

[0031] 2. The method of the present invention has simple procedures, convenient operation, and saves time and effort.

[0032] 3. In the method provided by the present invention, the acid hydrolysis and enzymatic hydrolysis steps use different acid hydrolysis and enzymatic hydrolysis processes from the prior art, which can make the end chain enzymatic hydrolysis more thorough, reduce immune rejection, improve the biocompatibility and affinity of the product, and at the same time will not destroy the triple helix structure of collagen. Collagen terminal peptides contain multiple tryptophans. After testing, no tryptophan residues were found in the extracted samples, indicating that the terminal peptides in the samples have been completely removed. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The method and device of the present invention are further described below in conjunction with the accompanying drawings and embodiments:

[0034] Figure 1 It is a flow chart of the preparation method for improving the biological safety of collagen according to the present invention;

[0035] Figure 2 This is the purity detection spectrum of Example 1 based on the national standard GB / T 38482-2021 method. DETAILED DESCRIPTION

[0036] 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.

[0037] 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 with simple process, significant sterilization effect and the ability to ensure the integrity of the prepared product, comprising the following steps:

[0038] Pre-treatment of the raw materials: freezing the raw materials at below -20°C (for example, -25°C, -30°C, -35°C, -40°C, etc.) and slicing them 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) to remove grease and dirt;

[0039] The pre-treated product is subjected to acid hydrolysis and enzymolysis; wherein the acid hydrolysis and enzymolysis process specifically comprises: 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, which will not be described in detail due to space limitations) and an appropriate amount of pepsin solution for enzymolysis, at 4-30°C (for example, Filter after enzymatic hydrolysis for 24-96h (for example, it can be 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, 72h, 76h, 80h, 84h, 88h, 92h, 96h or all values ​​in the range, which are not described in detail due to space limitations);

[0040] Salting out the enzymatic hydrolysis product, re-dissolving the salting out product and filtering it;

[0041] The filtered product is ultrafiltered and concentrated to obtain collagen.

[0042] Wherein, the conditions for enzymatic hydrolysis are: the temperature for the first 24-48 hours is 23-30°C (for example, it can be 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, 30°C or all values ​​within the range, which will not be repeated due to space limitations), and then the temperature is lowered to 4-8°C to continue the reaction (for example, it can be 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C or all values ​​within the range, which will not be repeated due to space limitations); this setting can remove the propeptide and the terminal peptide in the form of enzymatic hydrolysis during the dissociation of collagen, and then obtain collagen that does not contain any immune components, and can be accompanied by virus inactivation during the enzymatic hydrolysis process.

[0043] Wherein, during the enzymatic hydrolysis reaction, ultrasound is used to assist enzymatic hydrolysis; the ultrasound time is 10-30 min (for example, it can be 10 min, 15 min, 20 min, 25 min, 30 min or all values ​​within the range, which will not be repeated due to space limitations), and the ultrasound power is 280-320 W (for example, it can be 280 W, 285 W, 290 W, 295 W, 300 W, 305 W, 310 W, 315 W, 320 W or all values ​​within the range, which will not be repeated due to space limitations).

[0044] Wherein, the raw material is any one of pig tendon, beef tendon or pig skin, or a combination of at least two of them.

[0045] 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.

[0046] Wherein, the pre-processing step includes:

[0047] Select and simply clean the raw materials;

[0048] Cryosection at -20℃ into 1-5mm slices;

[0049] Automatic bubble cleaning by machine is used to remove grease and dirt.

[0050] 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.

[0051] Wherein, in the enzymatic hydrolysis step, the purity of pepsin is greater than 90%.

[0052] Wherein, in the pepsin solution, the mass fraction of pepsin is 0.1%-2% (for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% or all values ​​within the said range, which will not be repeated due to space limitations).

[0053] Wherein, in the enzymatic hydrolysis step, the mass ratio of the raw material to the pepsin solution is 1:(50-300) (for example, it can be 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, 1:250, 1:260, 1:270, 1:280, 1:290, 1:300 or all values ​​within the range, which will not be repeated due to space limitations).

[0054] The acid hydrolysis and enzymolysis steps use different acid hydrolysis and enzymolysis processes from those in the prior art, which can make the end chain enzymolysis more thorough, reduce immune rejection reactions, improve the biocompatibility and affinity of the product, and at the same time will not destroy the triple helix structure of collagen.

[0055] In the salting out step, sodium chloride solution is used to salt out the enzymatic hydrolysate to remove small molecule proteins, amino acids and other substances. After salting out, the precipitate is centrifuged and redissolved in 0.1%-10% acetic acid solution.

[0056] The filtering step is performed, for example, using a sterilizing filter.

[0057] The sterilizing filter is single-stage or double-stage filtration.

[0058] 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.

[0059] Among them, after the concentration treatment, for example, a freeze-drying step is also included. For example, the cross-linked sample can be placed in a freeze dryer for freeze drying. The sample can be dried once or twice, and the primary drying time is 12-48h (for example, it can be 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 42h, 48h or all values ​​within the range, which will not be repeated due to space limitations), and the secondary drying time after the primary drying is 4-24h (for example, it can be 4h, 8h, 12h, 16h, 20h, 24h or all values ​​within the range, which will not be repeated due to space limitations).

[0060] In a preferred embodiment, the method for preparing collagen of the present invention comprises the following steps:

[0061] 1. Pretreatment: Take the collected frozen raw materials and slow down, wash and remove impurities such as fascia, and then freeze and slice them into 1-5mm at below -20℃. 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;

[0062] The raw material is any one of pig tendons, beef tendons or pig skin, or a combination of at least two of them;

[0063] The pigs are three-month-old pigs;

[0064] 2. Acid hydrolysis and enzymolysis: Use 0.1%-10% acetic acid solution for acid hydrolysis, use an appropriate amount of pepsin solution for enzymolysis, and filter to obtain collagenase hydrolysate after enzymolysis at 4-30°C for 24-96h. The purity of pepsin is >90%, the mass fraction is 0.1%-2%, and the mass ratio of raw material to pepsin solution is 1:(50-300); wherein, the temperature for the first 24-48h is 23-30°C, and then the temperature is lowered to 4-8°C to continue the reaction; while the enzymolysis reaction is being performed, ultrasound is used to assist the enzymolysis (ultrasound time 10-30min, ultrasonic power 280-320W);

[0065] 3. Salting out and re-dissolving: Use sodium chloride solution to salt out the enzymatic solution;

[0066] 4. Sterilization and filtration: Use a sterilization filter to filter the reconstituted collagen solution;

[0067] 5. 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] 6. Freeze drying: Place the cross-linked sample into a freeze dryer for freeze drying. Pre-freeze the sample for 5-12 hours before drying. 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 biological safety 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] Pepsin for quantitative detection Brand: US Pharmacopeia, Product Number: 1510051 (United States Pharmacopeia (USP) Reference Standard), CAS Number: 9001-75-6

[0083] Example 1

[0084] 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.

[0085] 2. Enzymolysis: Use 8% acetic acid solution and an appropriate amount of pepsin for enzymolysis, react for a total of 48 hours, of which 0-24 hours are reacted at 26°C, and the 25-48 hours are reacted at 6°C, and the collagenase hydrolysate is obtained by filtration. The mass fraction of pepsin is 1%, and the mass ratio of raw material to pepsin solution is 1:50;

[0086] 3. Salting out and re-dissolving: Use sodium chloride solution to salt out the enzymatic hydrolysate to remove small molecule proteins, amino acids and other substances. After salting out, centrifuge and take the precipitate and re-dissolve it in 8% acetic acid solution.

[0087] 4. Sterile filtration: Use a sterilizing filter to filter the reconstituted collagen solution.

[0088] 5. Ultrafiltration and concentration: Use hollow fiber columns or membrane packages with a molecular weight cutoff of 50KD for ultrafiltration and liquid exchange and concentration to remove impurities such as inorganic salts.

[0089] Example 2-11

[0090] The specific operation steps are the same as those in Example 1, except that some parameters are adjusted as described in Table 1 below.

[0091] Table 1 List of specific operating parameters of Examples 1-11

[0092]

[0093]

[0094] Performance test and conclusion:

[0095] I. Collagen purity test

[0096] 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.

[0097] Figure 2 This is the purity detection graph of Example 1, wherein a is the collagen sample band, b is the collagen sample band after collagenase treatment, and c is the collagenase band. The purity detection and calculation were performed according to the GB / T 38482-2021 method. The results showed that there were no impurity 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.

[0098] 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%.

[0099] II. Collagen Tm value detection

[0100] The above-mentioned collagen solution to be tested is freeze-dried and cross-linked by the same method (a 0.5% mass fraction 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 when heated, it has a specific phase transition temperature and a corresponding thermal enthalpy change accompanied by a heat absorption process. When collagen unwinds its triple helix structure, a specific transition temperature can be observed, represented by Tm. It can be seen from the differential scanning calorimetry diagrams of Examples 1-11 that the Tm values ​​of the collagen raw materials prepared in Examples 1-11 are all higher than 65.5°C.

[0101] III. Detection of terminal peptide residues

[0102] The immunogenicity of type I collagen is mainly distributed in the terminal peptide region of the molecular chain. Therefore, it is necessary to inactivate the terminal peptide during the extraction of collagen to prepare collagen without terminal peptide and reduce its immunogenicity. Therefore, the degree of terminal peptide 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.

[0103] 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.

[0104] 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 2.

[0105] Table 2 Performance test results of Examples 1-11

[0106]

[0107]

[0108] Comparison test

[0109] 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 the collagen of the prior art (hereinafter referred to as "Product A", "Product B", "Product C", "Product D").

[0110] Among them, product A uses the method described in CN106367460A "A method for preparing collagen sponge under acidic conditions" to prepare collagen, wherein the enzymatic hydrolysis step is to mix the particles prepared in the previous step with a 0.1-1 mol / L acetic acid solution containing 0.1-1 mg / ml pepsin, stir at 37°C for 30-48 hours, centrifuge, and take the supernatant; wherein the mass volume ratio of the particles and the acetic acid solution is 1-12 mg:1 mL.

[0111] Product B uses the method described in TWI396693B "Preparation of High Purity Collagen" to prepare collagen, wherein the enzymatic hydrolysis step is to immerse any of the above connective tissues in an appropriate amount of acid solution, and after an appropriate period of time, the connective tissue is expanded to the required standard, that is, the thickness is at least 50% thicker than the original thickness (preferably 2-10 times the original thickness). The above acid solution can be prepared by an organic acid, such as formic acid, carboxylic acid, oxalic acid, and acetic acid. The cleaning solution may contain one or more proteolytic enzymes, such as ficin, pepsin, and trypsin.

[0112] Product C uses the method described in CN113462736A "Method for preparing de-telopeptide collagen from pig skin and collagen" to prepare collagen, wherein the enzymatic hydrolysis step is to extract collagen from pig skin with pepsin and hydrochloric acid / acetic acid to obtain an extract; the amount of pepsin added is 1 / 100 to 1 / 20 of the mass of the homogenized defatted pig skin; the hydrochloric acid / acetic acid is used at a concentration of 0.5 mol / L, and the amount added is 10 times the volume of the homogenized defatted pig skin; the extraction temperature of the collagen is 4°C, and the extraction time is 24 to 48 hours.

[0113] Product D is prepared by the method described in CN114540452A "A collagen, its inactivation and extraction method and facial filler containing the collagen", wherein the enzymatic hydrolysis step is to add the tissue precipitate into an acidic solution, stir and dissolve it, then add the enzyme solution to continue the reaction, and obtain the supernatant after centrifugation; the concentration of the acidic solution is 0.1-1 mol / L, and the acidic solution is acetic acid solution; the enzyme solution is pepsin solution, the concentration of the enzyme solution is 0.3-3wt.%, and the reaction time is 7-10d.

[0114] I. Thermal stability study

[0115] When collagen undergoes a phase change when heated, it absorbs heat and has a specific phase transition temperature and corresponding thermal enthalpy change. When collagen unwinds its triple helix structure, a specific transition temperature can be observed, represented by Tm. The results showed that the Tm value of the self-developed product was 65.8°C, which was higher than that of product AD (Tm values ​​were: product A: 61.0°C, product B: 56.3°C, product C: 60.8°C, product D: 53.7°C), which shows that the triple helix structure of the self-developed product has a higher integrity and better stability than commercially available products.

[0116] In addition, the scanning results of the Japanese Spectroscopic Circular Dichroism Spectrometer of the standard product 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;

[0117] 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.

[0118] 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] II. Pepsin Residue Detection

[0120] Pepsin residues were tested according to the pepsin activity test method described in the Chinese Pharmacopoeia 2020. The results are as follows:

[0121] Product A: 43.1EU / g

[0122] Product B: 3.5EU / g

[0123] Product C: 6.7EU / g

[0124] Product D: 5.9EU / g

[0125] Self-developed products: 3.1EU / g

[0126] This indicates that the residual amount of pepsin in the self-developed product is maintained at an extremely low level, and is lower than or equivalent to the products prepared by the prior art. Therefore, the 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 pepsin, thereby playing a stabilizing supporting role and being more suitable for medical use.

[0127] In summary, the collagen obtained by the extraction process of the present invention contains more type I collagen, with a purity of more than 99%, excellent thermal stability and good structural integrity. The results of circular dichroism spectrum analysis show that it has substantially the same triple helix structure as the standard; compared with commercially available products, its triple helix structure has higher integrity and better stability; when it is made into a collagen sponge product, the collagen obtained by the extraction process of the present invention can effectively stop bleeding and prevent adhesion, perform effective surgical cavity filling, play a stable support role and accelerate tissue healing. The method of the present invention has simple procedures, convenient operation, time-saving and labor-saving. In addition, in the method provided by the present invention, the acid hydrolysis and enzymolysis steps can make the end chain enzymolysis more thorough, reduce immune rejection, improve the biocompatibility and affinity of the product, and will not destroy the triple helix structure of collagen. The collagen telopeptide contains multiple tryptophans, and no tryptophan residues are found in the extracted sample after detection, indicating that the telopeptide in the sample has been completely removed.

[0128] 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 scope of protection of the present invention.

Claims

1. A preparation method for improving the integrity of collagen, characterized in that: The steps include: Pre-treatment of raw materials, freezing and slicing the raw materials, removing grease and dirt; The pre-treated product is subjected to acid hydrolysis and enzymolysis; wherein the acid hydrolysis and enzymolysis process specifically includes: using 0.1%-10% by mass acetic acid solution and pepsin solution for enzymolysis, and filtering after enzymolysis for 24-96 hours at 4-30°C; Salting out the enzymatic hydrolysis product, re-dissolving the salting out product and filtering it; Ultrafiltration and concentration are performed on the filtered product to obtain collagen; The purity of pepsin is greater than 90%; the mass fraction of pepsin in the pepsin solution is 0.1%-2%; and the mass ratio of the raw material to the pepsin solution is 1:(50-300).

2. The preparation method according to claim 1, characterized in that: During the enzymatic hydrolysis process, the temperature is 23-30° C. for the first 24-48 hours, and then the temperature is lowered to 4-8° C. to continue the reaction.

3. The preparation method according to claim 1 or 2, characterized in that: During the enzymatic hydrolysis reaction, ultrasound is used to assist the enzymatic hydrolysis; the ultrasound time is 10-30 minutes, and the ultrasound power is 280-320W.

4. The preparation method according to claim 1, characterized in that: The raw material is any one of pig tendons, beef tendons or pig skin, or a combination of at least two of them; the pig is a three-month-old pig.

5. The preparation method according to claim 1, characterized in that: The pre-processing steps specifically include: Select and simply clean the raw materials; Cryosection at -20℃ into 1-5mm slices; Automatic bubble cleaning by machine is used to remove grease and dirt.

6. The preparation method according to claim 5, characterized in that: The bubble cleaning uses 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: In the salting-out step, sodium chloride solution is used to salt out the enzymatic hydrolyzate.

8. The preparation method according to claim 7, characterized in that: The sterilizing filter is single-stage or double-stage filtration.

9. 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.

10. The preparation method according to claim 1, characterized in that: After the concentration process, a freeze-drying step is also included, in which the concentrated 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.

11. Collagen prepared by the method for improving the integrity of collagen according to any one of claims 1 to 10.

12. Use of the collagen as claimed in claim 10 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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