Low-fat collagen as well as preparation method and application thereof

Abstract

The invention relates to low-fat collagen as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out acidolysis and enzymolysis on a pre-treated product; preheating, centrifuging and salting out the enzymolysis product; after salting out, carrying out secondary centrifugation, taking precipitate, re-dissolving the precipitate in an acetic acid solution, carrying out first pasteurization, and filtering; and carrying out ultrafiltration concentration on the filtered product, and carrying out secondary pasteurization on the concentrated solution to obtain the recombinant collagen. The collagen obtained by the extraction process disclosed by the invention is lower in grease content and lower in bacterial endotoxin content, and the purity, the safety, the thermal stability, the structural integrity, the applicability and the like of the collagen are guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of collagen preparation, and in particular to low-fat collagen and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant protein in the human body, accounting for about a quarter of the total protein content. It is the main component of the connective tissue matrix. It is fibrous in the dermis, tendons, and cartilage of animals, and forms collagen fibers after aggregation. As a natural biological resource, collagen has functions that cannot be matched by synthetic polymer materials, such as biocompatibility, low immunogenicity, biodegradability and absorbability, and the promotion of new cell formation and epithelial cell formation. It can also make the bones, skin, tendons, etc. of animals have good mechanical properties. Therefore, collagen has broad application prospects in the field of biomedicine, including wound healing, burn repair, nerve damage regeneration, dura mater substitutes, artificial corneas, hemostatic agents, and many other aspects.

[0003] The fat content in medical recombinant collagen has an important impact on the purity, safety, effectiveness and applicability of collagen. Therefore, when preparing and using medical recombinant collagen, its fat content should be strictly controlled to ensure the quality and safety of the product. For example, the purity of recombinant collagen is one of the important indicators of its quality. Excessive fat content may reduce the purity of recombinant collagen, thereby affecting its overall quality. High-purity recombinant collagen is easier to be accepted and utilized by the human body, reducing the risk of allergies and adverse reactions. In addition, the fat content may also affect the safety of recombinant collagen. If the purification process is not good, harmful substances may remain in the recombinant collagen, such as impurities or contaminants in the fat that are not completely removed, which may pose a potential threat to human health. In addition, allergic reactions to recombinant collagen are also an issue that needs to be considered. Although in theory, collagen from recombinant sources is safer than that from animal sources, it may also cause allergic reactions if the purification process is not done well.

[0004] Excessive fat content may affect the effectiveness of recombinant collagen. Fat may interfere with the active areas of collagen and reduce its ability to interact with cells or other bioactive molecules, thereby affecting its repair and regeneration functions. Recombinant collagen has a variety of biomedical applications, such as skin repair and tissue engineering. In these applications, high-purity recombinant collagen can more effectively promote cell growth and differentiation and accelerate the tissue repair process. The functionality of recombinant collagen is also affected by fat content. For example, recombinant collagen has a stronger ability to promote fibroblast recruitment, adhesion, proliferation and migration, which are crucial in the field of skin repair and skin tissue engineering. Excessive fat content may weaken these functions, thereby affecting its effectiveness in practical applications.

[0005] After the inventor purchased the main commercial products on the market for testing and analysis, it was found that although the commercial products all claimed to have achieved good separation effects and controlled the oil content of the obtained collagen at a low level, the experimental results and the high price of medical-grade collagen on the market showed that it is not so easy to produce collagen with low oil content. How to develop a production process that can pass relatively simple procedures and ensure product quality is an urgent problem that needs to be solved. Summary of the invention

[0006] In view of this, the main purpose of the present invention is to provide a low-fat collagen and a preparation method and application thereof, in order to at least partially solve the above technical problems.

[0007] In order to achieve the above object, as a first aspect of the present invention, a method for preparing a low-fat collagen product is proposed, comprising the following steps:

[0008] Freeze the raw materials into slices to remove grease and dirt;

[0009] Acid hydrolysis and enzymatic hydrolysis are performed on the pre-treated product;

[0010] The enzymatic hydrolysate is preheated, centrifuged and salted out, wherein the preheating is preheating to 55-65° C. (for example, it can be 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C. or all values ​​within the range, which will not be described in detail due to space limitations);

[0011] After salting out, centrifuge twice to obtain the precipitate, redissolve it in acetic acid solution, perform the first pasteurization, and then filter it with a sterilizing filter; the temperature of the first pasteurization is 75-80°C (for example, it can be 75°C, 75.5°C, 76°C, 76.5°C, 77°C, 77.5°C, 78°C, 78.5°C, 79°C, 79.5°C, 80°C, 80.5°C, 81°C, 81.5°C, 82°C, 82.5°C, 83°C, 83.5°C, 84°C, 84.5°C, 85°C or all values ​​within the range, which are not described in detail due to space limitations), and the sterilization time is 15-20s (for example, it can be 15s, 15.5s, 16s, 16.5s, 17s, 17.5s, 18s, 18.5s, 19s, 19.5s, 20s or all values ​​within the range, which are not described in detail due to space limitations);

[0012] The filtered product is subjected to ultrafiltration and concentration treatment, and the concentrate is pasteurized for a second time to obtain recombinant collagen; the temperature of the second pasteurization is 70-72°C (for example, it can be 70°C, 70.2°C, 70.4°C, 70.6°C, 70.8°C, 71°C, 71.2°C, 71.4°C, 71.6°C, 71.8°C, 72°C or all values ​​within the range, which are not described again due to space limitations), and the sterilization time is 15-20s (for example, it can be 15s, 15.5s, 16s, 16.5s, 17s, 17.5s, 18s, 18.5s, 19s, 19.5s, 20s or all values ​​within the range, which are not described again due to space limitations).

[0013] Preferably, the pig is preferably a pig of three to six months old. Since the ratio of type I to type III collagen is different in pigs of different ages, the tendons of three to six months old pigs have a higher content of type I collagen and a relatively lower fat content.

[0014] Preferably, the pre-treatment step comprises:

[0015] Select and simply clean the raw materials;

[0016] Cryosectioning at below -20°C (e.g., -25°C, -30°C, -35°C, -40°C, etc.) into 1-5 mm (e.g., 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, or any value within the range, which will not be described in detail due to space limitations);

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

[0018] Preferably, simple cleaning is used to remove general surface dirt such as mud and impurities such as fascia on the surface of tendons; while bubble cleaning uses a set concentration of sodium carbonate solution, sodium bicarbonate solution, hydrogen peroxide solution or ethanol solution or a combination thereof, and produces bubbles through the reaction, or air forced into the outside world, to produce a stirring effect, so that the dirt in the raw materials cut into small pieces can be more easily rinsed out.

[0019] Preferably, a proteolytic enzyme is used in the enzymatic hydrolysis step, preferably an acetic acid solution with 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 a proteolytic enzyme for enzymatic hydrolysis; 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 without any immune components, and can be accompanied by virus inactivation during the enzymatic hydrolysis process.

[0020] Preferably, the speed of the centrifugation after preheating is 6000-6500rpm (for example, it can be 6050rpm, 6100rpm, 6150rpm, 6200rpm, 6250rpm, 6300rpm, 6350rpm, 6400rpm, 6450rpm, 6500rpm or all values ​​within the range, which are not described in detail due to space limitations), and the centrifugation time is 60-90s (for example, it can be 60s, 62s, 64s, 66s, 68s, 70s, 72s, 74s, 76s, 78s, 80s, 82s, 84s, 86s, 88s, 90s or all values ​​within the range, which are not described in detail due to space limitations). This centrifugation step can, on the basis of removing grease in the first step, again settle the easily condensed fat to the bottom of the centrifuge to form a fat layer, thereby playing a role of secondary degreasing, and can also preliminarily remove impurities, and then take the supernatant for salting out. The purpose of preheating is to fully melt the residual fat in the liquid, so that the degreasing effect is better in the subsequent centrifugation step.

[0021] Preferably, in the salting-out step, a sodium chloride solution having a mass fraction of 1%-20% (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 are not described in detail due to space limitations) is used to salt out the enzymatic solution for 0.5-4h (for example, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h or all values ​​within the range, which are not described in detail due to space limitations) to remove small molecule proteins and amino acid impurities. .

[0022] Preferably, the precipitate is centrifuged after salting out and redissolved in an acetic acid solution with 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 repeated due to space limitations).

[0023] Preferably, the temperature of the ultrafiltration concentration is 5-15°C, for example, it can be 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C or all values ​​within the range, which will not be described again due to space limitations.

[0024] 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.) to perform ultrafiltration and concentration treatment to remove impurities such as inorganic salts and small molecular proteins.

[0025] Preferably, after the secondary pasteurization, 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, 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 are not described in detail due to space limitations), and the secondary drying time after the primary drying 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 are not described in detail due to space limitations).

[0026] As a second aspect of the present invention, a collagen product prepared by the above-mentioned method for preparing a low-fat collagen product is also proposed.

[0027] 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.

[0028] Based on the above technical solutions, it can be seen that the preparation method, product and application of the present invention for improving the biosafety of collagen have at least one of the following beneficial effects compared with the prior art:

[0029] Compared with conventional extraction methods, the extraction process of the present invention adopts a combination of filtration and ultrafiltration, as well as reasonable pasteurization process parameters, which not only has a bactericidal effect, but also can reduce the lipid content of collagen from a macroscopic perspective, so that the obtained collagen has a lower oil content and a lower bacterial endotoxin content; at the same time, due to repeated verification and testing, the appropriate low-temperature sterilization process is adopted, and the original structure and properties of the collagen are maintained, and the purity, safety, thermal stability, structural integrity, biocompatibility, degradability and applicability of the collagen are guaranteed, so that it can be used in more scenarios of close contact with the human body; the method of the present invention has simple procedures, convenient operation, and saves time and effort.

[0030] In addition, although pasteurization does not completely destroy all structures of bacteria (e.g., bacterial spores may survive), it is sufficient to kill most harmful microorganisms and destroy their cell structures, especially cell membranes and cell walls, thereby rendering them inactive or dead. The destruction of cell structures can cause the substances in the cells (including lipid substances) to leak out and mix with the lipids in the collagen, and be removed by the centrifugation, filtration, ultrafiltration and other steps in the present invention. DETAILED DESCRIPTION

[0031] 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 references.

[0032] In view of the various collagen preparation methods in the prior art, either the fat content of the prepared product cannot be kept at a low level, or the toxicity of the product cannot be reduced. After intensive research, the inventors have proposed a preparation method for a collagen product with a simple process, significant sterilization effect and the ability to ensure a low fat content in the prepared product, comprising the following steps:

[0033] Freeze the raw materials into slices to remove grease and dirt;

[0034] Acid hydrolysis and enzymatic hydrolysis are performed on the pre-treated product;

[0035] The enzymatic hydrolysate is preheated, centrifuged and salted out, wherein the preheating is preheating to 55-65° C. (for example, it can be 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C. or all values ​​within the range, which will not be described in detail due to space limitations);

[0036] After salting out, centrifuge twice to obtain the precipitate, redissolve it in acetic acid solution, perform the first pasteurization, and then filter it with a sterilizing filter; the temperature of the first pasteurization is 75-80°C (for example, it can be 75°C, 75.5°C, 76°C, 76.5°C, 77°C, 77.5°C, 78°C, 78.5°C, 79°C, 79.5°C, 80°C, 80.5°C, 81°C, 81.5°C, 82°C, 82.5°C, 83°C, 83.5°C, 84°C, 84.5°C, 85°C or all values ​​within the range, which are not described in detail due to space limitations), and the sterilization time is 15-20s (for example, it can be 15s, 15.5s, 16s, 16.5s, 17s, 17.5s, 18s, 18.5s, 19s, 19.5s, 20s or all values ​​within the range, which are not described in detail due to space limitations);

[0037] The filtered product is subjected to ultrafiltration and concentration treatment, and the concentrate is pasteurized for a second time to obtain recombinant collagen; the temperature of the second pasteurization is 70-72°C (for example, it can be 70°C, 70.2°C, 70.4°C, 70.6°C, 70.8°C, 71°C, 71.2°C, 71.4°C, 71.6°C, 71.8°C, 72°C or all values ​​within the range, which are not described again due to space limitations), and the sterilization time is 15-20s (for example, it can be 15s, 15.5s, 16s, 16.5s, 17s, 17.5s, 18s, 18.5s, 19s, 19.5s, 20s or all values ​​within the range, which are not described again due to space limitations).

[0038] In a preferred embodiment, the raw material is tendon, such as pig or cattle tendon, preferably pig tendon.

[0039] In a preferred embodiment, the pig is preferably a three to six month old pig. Due to the different ratios of type I and type III collagen in pigs of different ages, the tendons of three to six month old pigs have a higher content of type I collagen and a relatively lower fat content.

[0040] In a preferred embodiment, the pre-treatment step comprises:

[0041] Select and simply clean the raw materials;

[0042] Cryosectioning at below -20°C (e.g., -25°C, -30°C, -35°C, -40°C, etc.) into 1-5 mm (e.g., 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, or any value within the range, which will not be described in detail due to space limitations);

[0043] Automatic bubble cleaning by machine is used to remove grease and dirt.

[0044] Among them, simple cleaning is used to remove general surface dirt such as mud and impurities such as fascia on the surface of tendons; while bubble cleaning uses a set concentration of sodium carbonate solution, sodium bicarbonate solution, hydrogen peroxide solution or ethanol solution or a combination thereof, and produces 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.

[0045] In a preferred specific embodiment, a proteolytic enzyme is used in the enzymolysis step, preferably an acetic acid solution with 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 are not described in detail due to space limitations) and an appropriate amount of a proteolytic enzyme for enzymolysis; this setting can remove the propeptide and the terminal peptide in the form of enzymolysis during the dissociation of collagen, and then obtain collagen without any immune components, and can be accompanied by virus inactivation during the enzymolysis process.

[0046] In a preferred specific embodiment, the speed of the centrifugation after preheating is 6000-6500rpm (for example, it can be 6050rpm, 6100rpm, 6150rpm, 6200rpm, 6250rpm, 6300rpm, 6350rpm, 6400rpm, 6450rpm, 6500rpm or all values ​​within the range, which are not described in detail due to space limitations), and the centrifugation time is 60-90s (for example, it can be 60s, 62s, 64s, 66s, 68s, 70s, 72s, 74s, 76s, 78s, 80s, 82s, 84s, 86s, 88s, 90s or all values ​​within the range, which are not described in detail due to space limitations). This centrifugation step can, on the basis of removing oil in the first step, again settle the easily condensed fat to the bottom of the centrifuge to form a fat layer, thereby playing a role of secondary degreasing, and can also preliminarily remove impurities, and then take the supernatant for salting out. The purpose of preheating is to fully melt the residual fat in the liquid, so that the degreasing effect is better in the subsequent centrifugation step.

[0047] In a preferred specific embodiment, in the salting-out step, a sodium chloride solution having a mass fraction of 1%-20% (the mass fraction can 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 are not described in detail due to space limitations) is used to salt out the enzymatic solution for 0.5-4h (for example, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h or all values ​​within the range, which are not described in detail due to space limitations) to remove impurities such as small molecule proteins and amino acids.

[0048] In a preferred specific embodiment, the precipitate is centrifuged after salting out and redissolved in an acetic acid solution with 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 repeated due to space limitations.

[0049] In a preferred embodiment, the temperature of the ultrafiltration concentration is 5-15°C, for example, it can be 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C or all values ​​within the range, which will not be described again due to space limitations.

[0050] In the ultrafiltration and concentration step, for example, a hollow fiber column and / or membrane package with a molecular weight cutoff of 10KD-100KD (e.g., 10KD, 30KD, 50KD, 100KD, etc.) is used for ultrafiltration and concentration treatment to remove impurities such as inorganic salts and small molecular proteins.

[0051] In a preferred embodiment, after the secondary pasteurization, 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, 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 are not described in detail due to space limitations), and the secondary drying time after the primary drying 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 are not described in detail due to space limitations).

[0052] In a preferred embodiment, the method for preparing collagen of the present invention comprises the following steps:

[0053] 1. Pretreatment: Collect frozen tendons of pig trotters aged three to six months, slow down the thawing, clean and remove impurities such as fascia, freeze and slice at -20℃ to 1-5mm, use cleaning agents of different concentrations (sodium carbonate solution, sodium bicarbonate solution, hydrogen peroxide solution, ethanol solution) and use a machine to automatically bubble clean to remove grease and other substances.

[0054] 2. Acid hydrolysis and enzymatic hydrolysis: Use 0.1%-10% acetic acid solution and an appropriate amount of proteolytic enzyme for enzymatic hydrolysis, and then filter to obtain collagenase hydrolysate.

[0055] 3. Salting out: preheating, centrifuging and salting out the enzymatic hydrolysate, wherein the preheating is preheating to 55-65°C; the centrifugal speed is 6000-6500rpm, and the centrifugal time is 60-90s; using a sodium chloride solution with a mass fraction of 1%-20% to salt out the enzymatic hydrolysate for 0.5-4h to remove small molecular proteins, amino acids and other substances.

[0056] 4. Redissolution: After salting out, centrifuge twice to obtain the precipitate and re-dissolve it in 0.1%-10% acetic acid solution for the first pasteurization; the first pasteurization temperature is 75-80℃, and the sterilization time is 15-20s;

[0057] 5. Sterilization filtration: Use a sterilization filter to filter the solution after the first pasteurization. The sterilization filter is a single-stage or double-stage filter.

[0058] 6. Ultrafiltration and concentration: Use hollow fiber columns and / or membrane packages with a molecular retention rate of 10KD-100KD to ultrafilter and concentrate the filtered product to remove impurities such as inorganic salts.

[0059] 7. Secondary sterilization: The concentrated liquid is subjected to a second pasteurization to obtain recombinant collagen; the temperature of the second pasteurization is 70-72°C, and the sterilization time is 15-20s.

[0060] 8. Freeze drying: Place the sample after secondary sterilization into a freeze dryer for freeze drying. Pre-freeze the sample for 5-12 hours before primary or secondary drying. The primary drying time is 12-48 hours, and the secondary drying time is 4-24 hours.

[0061] The present invention also provides a collagen product prepared by the method for preparing the low-fat collagen product.

[0062] 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.

[0063] 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.

[0064] Experimental preparation

[0065] Centrifuge Brand: Xiangyi Centrifuge Instrument Co., Ltd. Model: H2050R

[0066] Reactor Brand: Zhengzhou Great Wall Science & Technology Co., Ltd. Model: GR-10

[0067] Slicer Brand: Beijing Century Mingyang Food Machinery Co., Ltd. Model: QY-30

[0068] Electrophoresis instrument brand: Beijing Liuyi Biotechnology Co., Ltd. Model: DYY-60

[0069] Gel imaging system Brand: American BIO-RAD Model: GelDocGo

[0070] UV-Vis Spectrophotometer Brand: Shimadzu Instruments Co., Ltd. Model: UV-2600

[0071] Micro-volume differential scanner Brand: Malvern Panalytical Model: PEAQ-DSC

[0072] Circular Dichroism Spectrometer Brand: JASCO Model: J-1500

[0073] Pigs aged 3-6 months: Large tendons, more type I collagen, and better purity

[0074] Proteolytic enzyme for quantitative detection Brand: US Pharmacopeia, Product Number: 1510051 (United States Pharmacopeia (USP) Reference Standard), CAS Number: 9001-75-6

[0075] Example 1

[0076] 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.

[0077] 2. Acid hydrolysis and enzymatic hydrolysis: Use 8% by mass acetic acid solution and an appropriate amount of proteolytic enzyme for enzymatic hydrolysis, and filter after enzymatic hydrolysis to obtain collagenase hydrolysate.

[0078] 3. Salting out: preheating, centrifuging and salting out the enzymatic hydrolysate, wherein the preheating is preheating to 55°C; the centrifugal speed is 6000rpm, and the centrifugal time is 90s; the enzymatic hydrolysate is salted out for 1h using a 10% sodium chloride solution to remove small molecular proteins, amino acids and other substances.

[0079] 4. Redissolution: After salting out, centrifuge twice to obtain the precipitate and re-dissolve it in 8% acetic acid solution for the first pasteurization. The first pasteurization temperature is 75°C and the sterilization time is 15s.

[0080] 5. Sterilization filtration: Use a sterilization filter to filter the solution after the first pasteurization. The sterilization filter is a single-stage filtration.

[0081] 6. Ultrafiltration and concentration: Use a hollow fiber column with a molecular retention rate of 10KD to ultrafiltration and concentrate the filtered product to remove impurities such as inorganic salts.

[0082] 7. Secondary sterilization: The concentrated liquid is subjected to a second pasteurization to obtain recombinant collagen; the temperature of the second pasteurization is 72° C., and the sterilization time is 20 seconds.

[0083] 8. Freeze drying: Place the concentrated sample into a freeze dryer for freeze drying. Pre-freeze the sample for 5 hours and then dry it for 48 hours.

[0084] Example 2-11

[0085] The specific operation steps are the same as those in Example 1, except that some parameters are adjusted as described in Table 1 below.

[0086] Table 1-1 List of key operating parameters of Examples 1-11

[0087]

[0088]

[0089] Table 1-2 Key operating parameters of Examples 1-11

[0090]

[0091] Performance test and conclusion:

[0092] I. Oil content detection

[0093] The oil content in the collagen prepared in Example 1-11 was detected according to the method described in Clause 4.11 of "YY / T 1453-2016 Characterization Method of Type I Collagen for Tissue Engineering Medical Device Products", and the total fat content in the sample was determined according to GB / T 22223-2008, and the results were calculated based on the dry weight of the sample.

[0094] The results are shown in Table 2. It can be seen that the fat content in the collagen prepared in Examples 1-11 is less than 0.1%. The above examples remove fat more thoroughly through multiple centrifugation, filtration, ultrafiltration and other steps, which is far lower than the industry standard requirement of no more than 1%.

[0095] II. Bacterial endotoxin content detection

[0096] Collagen plays an important role in the biomedical field, but bacterial endotoxin contamination limits its use. Bacterial endotoxin is a unique structure on the outer membrane of the cell wall of Gram-negative bacteria (GNB). When a trace amount of bacterial endotoxin enters the human body, it will cause symptoms such as high fever and diarrhea. A large dose can cause fainting or even death. Therefore, there are very clear regulations on the minimum standard for the bacterial endotoxin content of medical devices.

[0097] According to the bacterial endotoxin test method of Pharmacopoeia 1143, the limulus amebocyte lysate is used to detect or quantify the bacterial endotoxin produced by Gram-negative bacteria to determine whether the limit of bacterial endotoxin in the test sample meets the regulations; the endotoxin content results in the examples are shown in Table 2, and it can be seen that the bacterial endotoxin content of Examples 1-11 is lower than 0.01EU / mL, which is far lower than the limit of 0.5EU / mL required for collagen implants.

[0098] III. Collagen purity test

[0099] 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.

[0100] 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. The purity detection patterns of the collagen prepared in Examples 2-11 were similar to those in Example 1, and the purity was greater than 99%.

[0101] IV. Circular Dichroism Spectrum Analysis

[0102] The scanning results of the standard product using a 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.

[0103] The scanning results of the collagen prepared in Example 1 show that its positive peak wavelength is 221.3nm, the peak value is 36.4587, the negative peak wavelength is 198.3nm, the peak value is 275.1532, and the absolute value ratio of the positive peak to the negative peak is 0.1325. The above scanning results show that Example 1 has basically the same triple helix structure as the standard, and has good integrity and stability. The scanning results of the collagen prepared in Examples 2-11 are similar to those in Example 1, and all have basically the same triple helix structure as the standard. It is speculated that the reason may be that the method provided by the present invention adopts the form of secondary pasteurization, which avoids the contact of collagen with the high temperature environment on the basis of reducing endotoxins, so that it can maintain its original structure as much as possible, or as little structural change as possible, thereby achieving a better effect of integrity and stability.

[0104] Ⅴ. Detection of terminal peptide residues

[0105] 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.

[0106] 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.

[0107] The above-mentioned various parameter fine-tuning can achieve the purpose of the invention, but it has a slight impact on the filtration time, the amount of consumables and the purity of the final product. Through the combination of various parameters, it can be found that the comprehensive performance of Example 1, Example 8 and Example 10 is better. The specific performance test results are shown in Table 2.

[0108] Table 2 Test results of Examples 1-11

[0109]

[0110]

[0111] Comparison test

[0112] 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 "Product A" and "Product B").

[0113] Among them, product A adopts the method described in Example 1 of CN106367460A "A method for preparing collagen sponge under acidic conditions" to prepare collagen; product B adopts the method described in TWI396693B "Preparation of high-purity collagen" to prepare collagen; product C adopts the method described in Example 1 of CN113462736A "Preparation method of de-terminated collagen and collagen obtained from pig skin" to prepare collagen; product D adopts the method described in Example 1 of CN114540452A "A collagen, its inactivation extraction method and facial filler containing the collagen" to prepare collagen.

[0114] I. Thermal stability test

[0115] 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 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 66.4°C, which is higher than the Tm of other commercially available products (Tm value of product A: 61.0°C; Tm value of product A: 56.3°C; Tm value of product C: 52.7°C; Tm value of product D: 59.8°C), which indicates that the triple helix structure of the self-developed product has a higher integrity and better stability than the above-mentioned prior art products.

[0116] II. Endotoxin content test

[0117] The endotoxin content of product AD was tested according to the above method, and the results are as follows:

[0118] Product A: less than 0.002EU / mL

[0119] Product B: less than 0.184EU / mL

[0120] Product C: less than 0.057EU / mL

[0121] Product D: less than 0.093EU / mL

[0122] Self-developed product: 0.001EU / mL

[0123] It can be seen from the results that the bacterial endotoxin content of the self-developed product is much lower than that of products B, C, and D, and is equivalent to that of product A, meeting the limit of 0.5EU / mL required by the standard, making the recombinant collagen prepared by the process of the present invention suitable for the preparation of medical products.

[0124] III. Oil content and purity test

[0125] The oil content and purity of product AD were tested according to the above method, and the results are as follows:

[0126] Table 3 Oil content and purity test of product AD

[0127]

[0128] from Oil content and purity testing The results show that the fat content of the self-developed product (Example 1) is much lower than that of products A, B, C, and D, and the purity is higher than that of products A, C, and D, which is equivalent to that of product B; this indicates that the collagen obtained by the extraction process of the present invention has a lower fat content and a lower bacterial endotoxin content, and the purity, safety, thermal stability, structural integrity, effectiveness and applicability of the collagen are guaranteed, making it suitable for more scenarios with close contact with the human body.

[0129] 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 method for reducing the fat content of collagen, characterized in that: The steps include: Freeze slice the stock to remove grease and dirt; Acid hydrolysis and enzymatic hydrolysis are performed on the pre-treated product; Preheating, centrifuging and salting out the enzymatic hydrolysis product, wherein the preheating is preheating to 55-65° C.; After salting out, the precipitate is centrifuged twice and redissolved in acetic acid solution for the first pasteurization, and then filtered with a sterilizing filter; the first pasteurization temperature is 75-80°C, and the sterilization time is 15-20s; The filtered product is subjected to ultrafiltration and concentration treatment, and the concentrated liquid is subjected to a second pasteurization to obtain recombinant collagen; the second pasteurization temperature is 70-72° C., and the sterilization time is 15-20 seconds.

2. The preparation method according to claim 1, characterized in that: The raw material is pig or cattle tendon, and the pig is a pig of three to six months old.

3. 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.

4. The preparation method according to claim 3, characterized in that: The bubble cleaning uses a sodium carbonate solution, a sodium bicarbonate solution, a hydrogen peroxide solution or an ethanol solution or a combination thereof with a set concentration as a cleaning agent.

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

6. The preparation method according to claim 1, characterized in that: In the enzymolysis step, the preheated enzymolysis product is salted out using a sodium chloride solution.

7. The preparation method according to claim 1, characterized in that: The centrifugal speed after salting out is 6000-6500 rpm, and the centrifugal time is 60-90 s.

8. The preparation method according to claim 1, characterized in that: After the salting out, the precipitate is centrifuged and redissolved in an acetic acid solution with a mass fraction of 0.1%-10%.

9. The preparation method according to claim 1, characterized in that: The temperature of the ultrafiltration concentration is 5-15°C.

10. 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 for ultrafiltration and concentration.

11. 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.

12. Collagen prepared by the method for improving the biological safety of collagen as claimed in any one of claims 1 to 11.

13. Use of the collagen as claimed in claim 12 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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