A preparation kit, preparation method and application of medical grade type I collagen

Abstract

The application provides a preparation kit and a preparation method and application of medical grade type I collagen, and belongs to the technical field of medical and aesthetic polymer materials. The application provides a preparation kit of medical grade type I collagen, which comprises independently packaged chemical degreasing agents, degreasing enzyme solution, swelling liquid, digestive enzyme and purification enzyme. The application realizes physical degreasing, chemical degreasing by using an acetone mixed solution and cooperative degreasing and depilation by using composite enzymes, combines with acid immersion swelling treatment, and maximally retains the triple helix structure of collagen under low-temperature and weak-acid conditions; subsequently, pepsin is used for enzymolysis in a buffer acid system, collagen is released, the extract is subjected to salting-out precipitation and dialysis purification treatment; and terminal peptidase is added to perform enzymolysis on the telopeptide regions at both ends of the collagen molecules, so as to prepare telopeptide-removed collagen, thereby reducing the immunogenicity of the collagen, and improving the biocompatibility and injection safety of the collagen.

Description

Technical Field

[0001] This invention belongs to the field of medical aesthetic polymer materials technology, specifically relating to a preparation kit, preparation method and application of medical-grade type I collagen. Background Technology

[0002] Collagen is a biological macromolecule and a major component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for 25%–30% of total protein. It is closely related to tissue formation and maturation, intercellular communication, joint lubrication, wound healing, calcification, blood clotting, and aging. Collagen is also one of the most crucial raw materials in the biotechnology industry, with wide applications in medical materials, cosmetics, and the food industry.

[0003] Type I collagen has been widely used in food, medicine, tissue engineering, cosmetics and other fields due to its good biocompatibility, biodegradability and bioactivity, such as low antigenicity, easy absorption by the human body, ability to promote cell survival and growth, and ability to promote platelet aggregation.

[0004] Currently, the main methods for extracting type I collagen include enzymatic, acidic, and alkaline methods. One method involves adding solid sodium chloride to a crude collagen solution, allowing it to stand until the collagen solid precipitates, collecting the solid, dissolving it, and dialyzing to obtain type I collagen. However, using current collagen salting-out methods results in collagen with high impurity content and low purity. Furthermore, the preparation process is time-consuming, cumbersome, requires large amounts of reagents, and has a low product yield. In particular, defatting, enzymatic hydrolysis, and purification processes often result in protein structure damage, numerous impurity residues, low extraction efficiency, and high immunogenicity. Summary of the Invention

[0005] This invention provides a reagent kit, preparation method, and application for preparing medical-grade type I collagen, which significantly improves the purity, triple helix structure integrity, and biocompatibility of type I collagen extracted from bovine hide. At the same time, it effectively reduces immunogenicity and batch fluctuations. The overall process is safe, stable, and scalable, and is suitable for the development of high-end medical aesthetics and tissue engineering products.

[0006] This invention provides a kit for preparing medical-grade type I collagen, comprising individually packaged chemical degreasing agent, delipase solution, swelling solution, digestive enzyme and purification enzyme;

[0007] The chemical degreasing agent is a mixture of acetone and water;

[0008] The delipase solution includes elastase, lipase, phospholipase and alkaline protease;

[0009] The swelling solution includes citric acid or acetic acid;

[0010] The digestive enzymes include pepsin;

[0011] The purified enzyme includes trypsin.

[0012] In a preferred embodiment of the present invention, the delipase solution uses water as a solvent and includes elastase, lipase, phospholipase, alkaline protease, salicylic acid, sodium sulfate, PEG, and Ca. 2+ And glutathione or ascorbic acid.

[0013] In a preferred embodiment of the present invention, the delipase solution comprises the following components at the following concentrations: 2500–3000 U / L elastase, 1000–1200 U / L lipase, 600–800 U / L phospholipase, 200–500 U / L alkaline protease, 0.02–0.05 w% salicylic acid, 0.5–1 w% sodium sulfate, 0.5–1 w% PEG, and 50–100 mg / L Ca. 2+ And 100-200 mg / L glutathione or ascorbic acid.

[0014] In a preferred embodiment of the present invention, the citric acid in the swelling solution has a mass-volume percentage of 0.5-1%; and the acetic acid in the swelling solution has a volume percentage of 0.2-0.5%.

[0015] The present invention also provides a method for preparing medical-grade type I collagen using the above-mentioned kit, comprising the following steps: (1) using cowhide as raw material, performing physical degreasing, chemical degreasing agent degreasing and degreasing with degreasing enzyme solution in sequence to obtain degreased cowhide;

[0016] (2) After crushing the defatted cowhide described in step (1), soak it in a swelling solution for 4 to 8 hours to obtain pretreated cowhide;

[0017] (3) The pretreated cowhide in step (2) was digested with the digestive enzyme in an environment of pH 2.5 ± 0.2 for 6 to 12 hours to obtain a digestive solution;

[0018] (4) After centrifuging the digestion solution described in step (3), the supernatant is salted out using a NaCl aqueous solution with a final concentration of 0.8 mol / L. The precipitate is collected and dissolved in acetic acid solution to obtain crude collagen solution.

[0019] (5) Dialyze the crude collagen solution obtained in step (4) using a 30kDa dialysis bag, and then digest the dialyzed collagen solution with a purifying enzyme to obtain a collagen solution with telopeptides removed.

[0020] In a preferred embodiment of the present invention, the defatting stability of the defatting enzyme solution in step (1) is 35-40°C for 120-150 min.

[0021] In a preferred embodiment of the present invention, the centrifugation speed in step (4) is 8000-10000 rpm and the time is 15 min.

[0022] In a preferred embodiment of the present invention, after digestion in step (5), the process further includes freeze drying.

[0023] The present invention also provides medical-grade type I collagen prepared using the above method.

[0024] This invention also provides the application of the above-mentioned medical-grade type I collagen in the preparation of medical aesthetic preparations.

[0025] Beneficial Effects: This invention provides a kit for preparing medical-grade type I collagen, comprising individually packaged chemical degreasing agents, delipase solutions, swelling solutions, digestive enzymes, and purification enzymes. The invention also provides a specific method for using the kit, which involves physical degreasing, chemical degreasing with an acetone mixture, and synergistic degreasing and hair removal using a compound enzyme, combined with acid soaking and swelling treatment, to maximize the preservation of the triple helix structure of collagen under low temperature and weak acid conditions. Subsequently, pepsin is used for enzymatic hydrolysis in a buffered acidic system to release collagen. The extract is then purified by salting out and dialysis. Terminal peptidase is added to enzymatically hydrolyze the telopeptide regions at both ends of the collagen molecule to prepare telopeptide-free collagen, thereby reducing its immunogenicity and improving its biocompatibility and injection safety. The obtained collagen is then freeze-dried at low temperature to obtain high-purity, structurally intact type I collagen powder. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the preparation process of the medical-grade type I collagen of this invention. Detailed Implementation

[0027] This invention provides a kit for preparing medical-grade type I collagen, comprising individually packaged chemical degreasing agent, delipase solution, swelling solution, digestive enzyme and purification enzyme;

[0028] The chemical degreasing agent is a mixture of acetone and water;

[0029] The delipase solution includes elastase, lipase, phospholipase and alkaline protease;

[0030] The swelling solution includes citric acid or acetic acid;

[0031] The digestive enzymes include pepsin;

[0032] The purified enzyme includes trypsin.

[0033] The chemical degreasing agent of the present invention is a mixture of acetone and water, wherein the volume ratio of acetone to water in the mixture can be (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1.

[0034] The lipase solution of this invention uses water as a solvent and includes elastase, lipase, phospholipase, alkaline protease, salicylic acid, sodium sulfate, PEG (polyethylene glycol), and Ca. 2+ And glutathione or ascorbic acid. In the delipase solution, the concentration of the elastase can be 2500–3000 U / L, such as 2500 U / L, 2600 U / L, 2700 U / L, 2800 U / L, 2900 U / L, or 3000 U / L. In the delipase solution, the concentration of the lipase can be 1000–1200 U / L, such as 1000 U / L, 1100 U / L, or 1200 U / L. In the delipase solution, the concentration of the phospholipase can be 600–800 U / L, such as 600 U / L, 700 U / L, or 800 U / L. In the delipase solution, the concentration of the alkaline protease can be 200–500 U / L, such as 200 U / L, 300 U / L, 400 U / L, or 500 U / L. The delipase solution of the present invention further includes salicylic acid, the concentration of which is 0.02-0.05 wt%, such as 0.02 wt%, 0.03 wt%, 0.04 wt%, or 0.05 wt%. The delipase solution of the present invention also includes sodium sulfate, the concentration of which is 0.5-1 wt%, such as 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, or 1 wt%. The delipase solution of the present invention also includes PEG, the concentration of which is 0.5-1 wt%, such as 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, or 1 wt%. The delipase solution of the present invention also includes Ca. 2+ The Ca 2+ The concentration is 50–100 mg / L, such as 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, or 100 mg / L. The Ca described in this invention... 2+ The lipase solution can be selected from calcium chloride, calcium acetate, calcium lactate, calcium citrate, calcium gluconate, calcium nitrate, or calcium formate, with calcium chloride or calcium acetate being preferred. The lipase solution of this invention also includes glutathione or ascorbic acid, wherein the concentration of glutathione or ascorbic acid is 100–200 mg / L, such as 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, or 200 mg / L.

[0035] The lipase solution described in this invention is a water-based composite enzyme / chemical aid system designed to efficiently remove impurities such as fats and phospholipids from raw materials under mild conditions, while preserving the triple helix structure and bioactivity of type I collagen. The components work synergistically, and its design rationale and function are as follows:

[0036] Elastase, lipase, phospholipase, and alkaline protease are the core enzyme components in this invention. Their concentrations are set to ensure the decomposition of various impurity components in the tissue during the defatting process: Elastase can degrade elastin and a small amount of non-collagenous proteins, assisting in the release of collagen fibers; lipase is used to hydrolyze triglycerides and remove neutral lipids from animal tissues; phospholipase can effectively decompose phospholipids in cell membranes, which helps to clear cell debris; alkaline protease has a broad-spectrum proteolytic ability and is used to assist in the removal of impurity proteins, but its concentration is controlled in a low range (200-500 U / L) to avoid damaging the target collagen.

[0037] Salicylic acid is a mild organic acid with anti-inflammatory, degreasing, keratolytic, and antibacterial properties. In an enzymatic environment, it can further disrupt the structure between the fat layer and protein, improving enzyme permeability and degreasing efficiency.

[0038] Sodium sulfate is a neutral salt that can regulate the ionic strength and osmotic pressure of the system. To a certain extent, it can inhibit the adsorption of non-specific proteins, maintain the collagen swelling state, and help improve enzyme treatment efficiency.

[0039] PEG is a nonionic surfactant with good wetting, emulsifying and diffusion-promoting functions. It can enhance the contact between enzymes and the matrix and reduce protein aggregation.

[0040] Ca 2+ Ions act as important activators of metalloenzymes, especially promoting the activity of certain proteases (such as phospholipase A2). Furthermore, Ca... 2+ It can also stabilize the cross-linked structure between collagen molecules to a certain extent, preventing structural collapse. 2+ It can be provided by a variety of soluble calcium salts, including calcium chloride, calcium acetate, calcium lactate, calcium citrate, calcium gluconate, calcium nitrate or calcium formate, with calcium chloride or calcium acetate being preferred because of their high solubility, good stability and no toxic residue.

[0041] Glutathione or ascorbic acid, as reducing antioxidants, have a dual function: first, to prevent collagen from oxidizing and denaturing during processing and to maintain its activity; second, to participate in the removal of free radicals induced by lipid oxidation byproducts and metal ions, thereby improving the quality of the extract.

[0042] In summary, the components of the delipase solution of this invention are functionally complementary and chemically compatible, synergistically improving delipidation efficiency while effectively protecting the integrity and activity of type I collagen, making it particularly suitable for the pretreatment of medical-grade collagen.

[0043] The swelling solution of the present invention is an aqueous solution of citric acid or acetic acid, wherein the citric acid aqueous solution contains 0.5% to 1% by mass volume, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%; and the acetic acid in the swelling solution contains 0.2% to 0.5% by volume, such as 0.2%, 0.3%, 0.4%, or 0.5%.

[0044] This invention also provides a method for preparing medical-grade type I collagen using the above-mentioned kit, such as... Figure 1 As shown, the process includes the following steps: (1) using cowhide as raw material, physical degreasing, chemical degreasing agent degreasing, and degreasing enzyme solution degreasing are carried out in sequence to obtain degreased cowhide;

[0045] (2) After crushing the defatted cowhide described in step (1), soak it in a swelling solution for 4 to 8 hours to obtain pretreated cowhide;

[0046] (3) The pretreated cowhide in step (2) was digested with the digestive enzyme in an environment of pH 2.5 ± 0.2 for 6 to 12 hours to obtain a digestive solution;

[0047] (4) After centrifuging the digestion solution described in step (3), the supernatant is salted out using a NaCl aqueous solution with a final concentration of 0.8 mol / L. The precipitate is collected and dissolved in acetic acid solution to obtain crude collagen solution.

[0048] (5) Dialyze the crude collagen solution obtained in step (4) using a 30kDa dialysis bag, and then digest the dialyzed collagen solution with a purifying enzyme to obtain a collagen solution with telopeptides removed.

[0049] This invention uses cowhide as raw material to prepare collagen. Healthy cowhide is selected, and after cleaning and disinfection, it undergoes the aforementioned degreasing treatment. The invention first degreases the cowhide, including physical degreasing, chemical degreasing agent degreasing, and degreasing enzyme solution degreasing.

[0050] The physical degreasing method described in this invention includes high-pressure water jet degreasing. For example, by using a 3-5 MPa high-pressure water jet at 4-10°C for 0.5-1 hour, 40-60% of the fat on the surface of cowhide can be removed.

[0051] In this invention, after physical degreasing, the cowhide is placed in a chemical degreasing solution for chemical degreasing, including shaking and soaking at 4°C for 4 hours. The shaking speed in this invention is 80-120 rpm.

[0052] This invention involves placing chemically degreased cowhide in a degreasing enzyme solution, wherein the mass of the degreasing enzyme solution is 2 to 3 times the mass of the cowhide. The invention utilizes a stirring and soaking method to complete the degreasing with the degreasing enzyme solution. The degreasing temperature is 35–40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. The pH value of the degreasing enzyme solution system is required to be 7.5–8.5. The stirring speed during soaking is 100–200 rpm, such as 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, or 200 rpm, and the soaking time is 120–150 min, such as 120 min, 125 min, 130 min, 135 min, 140 min, 145 min, or 150 min.

[0053] After defatting, the cowhide is washed and then pulverized. In the embodiment, the cowhide is pulverized to a particle size of 0.5-2 mm using a tissue shredder. It is then soaked in a swelling solution at a temperature of 4-10°C for 4-8 hours to remove non-collagenous impurities and stabilize the collagen structure.

[0054] This invention utilizes digestive enzymes to treat swollen bovine hide granules. Specifically, the pretreated bovine hide granules are placed in a reaction vessel, and the pH of the system is adjusted to 2.5 ± 0.2 using 0.5M acetate buffer. Then, digestive enzymes (pepsin) are added. The amount of pepsin added is 1 / 100 of the mass of the raw bovine hide, and the pepsin activity should be ≥3000 U / mg to ensure batch-to-batch consistency. During the enzymatic digestion process, a pH meter is used to monitor and adjust the pH in real time to ensure that the buffer solution volume is sufficient to maintain pH stability during the 6–12 h reaction period. If the pH deviates from ± 0.2, a small amount of 1 mol / L hydrochloric acid or NaOH solution is added. The enzymatic digestion described in this invention is carried out at a temperature of 25–30°C with slow stirring under constant temperature conditions. The stirring speed can be 50–100 rpm, such as 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, or 100 rpm, and is maintained for 6–12 hours. Samples are taken every 2 hours during this period, and the concentration of soluble protein is determined by the BCA method to judge the reaction progress. This allows collagen in bovine hide to be released under mild conditions, preserving the triple helix structure to the greatest extent.

[0055] In this invention, the above-mentioned enzymatic hydrolysate is coarsely filtered through double-layer sterile gauze to remove incompletely degraded macromolecular impurities and tissue fragments. Then, simple purification is performed by centrifugation at 4°C at a speed of 8000–10000 rpm (e.g., 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, or 10000 rpm) for a limited time of 15 minutes. The supernatant is collected for further purification. The further purification method of this invention includes salting out, such as slowly adding sodium chloride to the supernatant to achieve a final concentration of 0.8 M, and allowing it to stand at 4°C for 12 hours to precipitate collagen. After salting out, the precipitate is collected by centrifugation at 5000 rpm for 10 minutes and resuspended in a 0.5 mol / L acetic acid solution for later use.

[0056] This invention provides a method for deep purification of the acetic acid resuspension, including dialysis. The dialysis process involves placing the acetic acid resuspension into a dialysis bag with a molecular weight cutoff of 30 kDa and dialyzing it in 0.1 M acetic acid for at least 48 hours, during which the dialysis solution is changed at least three times to remove small molecule impurities and inorganic salts. After dialysis, the solution is further dialyzed in distilled water for 24 hours to remove residual acidic components and improve the purity of collagen.

[0057] After obtaining the dialysis-processed collagen, this invention further includes a telopeptide removal treatment. This telopeptide removal treatment involves using the purification enzymes provided in the kit. The treatment is performed by mild enzymatic hydrolysis with trypsin. The amount of trypsin used is 0.1–1 wt% of the collagen protein mass, or the enzyme activity is 10–100 U / mg collagen. The hydrolysis temperature is 30–37°C, and the reaction time is 30–60 min. This selectively removes the antigenic peptides at both ends of the collagen molecules, thereby improving its biocompatibility and injection safety. After the enzymatic hydrolysis reaction is completed, ultrafiltration / dialysis (molecular weight cutoff of 10–30 kDa) is performed to remove residual trypsin.

[0058] This invention involves freeze-drying the collagen solution containing the determinated peptides to obtain high-purity type I collagen powder. The drying process is maintained at a low temperature (<-40°C) to avoid any heat damage.

[0059] This invention involves dissolving the collagen obtained after telopeptide removal in sterile water to prepare a solution of a certain concentration, and then using a 0.22μm microporous membrane for sterilization filtration. The filtrate is then placed in a freeze-drying bottle and placed in a freeze-drying system for vacuum freeze-drying to preserve the natural triple helix structure of the collagen. After drying, the finished collagen is packaged in sterile bottles and sealed under vacuum to prevent moisture and secondary contamination.

[0060] The present invention also provides medical-grade type I collagen prepared using the above method.

[0061] The collagen prepared using the method described in this invention has a higher hydroxyproline content than commercially available medical-grade type I collagen standards, and exhibits typical negative peaks at 197 nm and positive peaks at 220 nm, indicating its intact triple helix structure. The medical-grade type I collagen of this invention did not induce a significant specific IgG antibody response after repeated subcutaneous injections, comparable to the PBS control group, indicating that the method described in this invention can effectively reduce immunogenicity and improve biosafety.

[0062] This invention also provides the application of the above-mentioned medical-grade type I collagen in the preparation of medical aesthetic preparations.

[0063] This invention significantly improves the purity, triple helix structure integrity, and biocompatibility of type I collagen extracted from bovine hide, while effectively reducing immunogenicity and batch variation. The overall process is safe, stable, and scalable, making it suitable for the development of high-end medical aesthetics and tissue engineering products.

[0064] This invention does not specifically limit the dosage form of the medical aesthetic preparation. It can be formulated into different physical forms according to subsequent application needs, including liquid states (such as diluted solutions and colloidal suspensions) or lyophilized solid powder states. Collagen solutions can be directly used for clinical or cosmetic applications such as injection, application, and instillation, while lyophilized powder states offer better stability and shelf life. Before use, it can be reconstituted to the required concentration with sterile water, PBS, or physiological saline, making it suitable for various scenarios such as medical aesthetics, tissue repair, and drug delivery.

[0065] The present invention specification provides some exemplary data. It is worth noting that the listed data only represents a portion of the data that can be used to complete the experiment. It can also be any number between two numbers in integer form, or a non-integer number in decimal or fraction form.

[0066] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a kit for preparing medical-grade type I collagen, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] 1. Hide pretreatment: Select healthy cowhide, and after cleaning and disinfection, proceed with the following treatment steps:

[0069] ① Physical degreasing: The cowhide was treated with a 4MPa high-pressure water jet at 4℃ for 0.75 hours to remove about 50% of the fat from the surface.

[0070] ② Chemical degreasing: The cowhide is placed in a mixture of acetone and water (volume ratio 3:1) and shaken at 100 rpm for 4 hours at 4°C for further degreasing;

[0071] ③ Enzymatic degreasing and hair removal: Place the cowhide in a degreasing enzyme solution three times its weight (containing 2500 U / L elastase, 1100 U / L lipase, 700 U / L phospholipase, 400 U / L alkaline protease, 0.04 w% salicylic acid, 0.8 w% sodium sulfate, 0.5 w% PEG, and 80 mg / L Ca). 2+ 150 mg / L glutathione or ascorbic acid, with the remainder being water), was stirred and soaked for 120 minutes at 37°C and pH 8.0, followed by thorough rinsing;

[0072] ④ Acid soaking and swelling treatment: Chop the cowhide into particles of 0.5-2 mm using a tissue mincer, place it in a 0.5% citric acid solution, and soak it at 6°C for 6 hours to remove non-collagen impurities and stabilize the collagen structure;

[0073] 2. Pepsin Enzymatic Extraction: Pre-treated bovine hide granules were placed in a reaction vessel, and the pH of the system was adjusted to 2.5 with 0.5 mol / L acetate buffer. Under these acidic conditions, pepsin was added to the system at a mass ratio of 1:100 (w / w) to the bovine hide substrate. The pepsin activity should be ≥3000 U / mg to ensure batch-to-batch consistency. The reaction was carried out under constant temperature of 28℃ with slow stirring at 80 rpm to avoid particle breakage or enzyme denaturation caused by excessive stirring. The reaction was carried out for 10 hours, with samples taken every 2 hours. The concentration of soluble protein was determined using the BCA method to assess the reaction progress, ensuring the release of collagen from the bovine hide under mild conditions and maximizing the preservation of the triple helix structure.

[0074] 3. Preliminary purification: The above enzymatic hydrolysate was coarsely filtered through double-layer sterile gauze to remove incompletely degraded macromolecular impurities and tissue fragments. The filtrate was centrifuged at 9000 rpm for 15 min at 4℃, and the supernatant was collected for further purification. Sodium chloride was slowly added to the supernatant to bring the final concentration of the system to 0.8 mol / L, and the mixture was allowed to stand at 4℃ for 12 h to precipitate collagen. The precipitate was collected by centrifugation at 5000 rpm for 10 min and resuspended in 0.5 mol / L acetic acid solution for later use.

[0075] 4. Deep purification and concentration: The above acetic acid resuspension was placed into a dialysis bag with a molecular weight cutoff of 30 kDa and dialyzed in 0.1 mol / L acetic acid for no less than 48 hours, during which the dialysate was changed no less than 3 times to remove small molecule impurities and inorganic salts; after dialysis, it was dialyzed in distilled water for 24 hours to remove residual acidic components and improve the purity of collagen.

[0076] The obtained collagen solution was then subjected to terminal peptide removal treatment by adding trypsin for mild enzymatic hydrolysis. The enzyme dosage was 0.5 wt% of the collagen protein, with an enzyme activity of 50 U / mg. The reaction temperature was 36°C, and the reaction time was 45 min. This selectively removed the antigenic peptide segments at both ends of the collagen molecules, thereby improving its biocompatibility and injection safety. After enzymatic hydrolysis, the collagen was dialyzed in distilled water for 48 h using a 30 kDa dialysis bag to remove residual enzymes and small molecule impurities.

[0077] 5. The collagen solution after terminal processing is sterilized by filtration through a 0.22μm microporous membrane. The filtrate is then placed into freeze-drying bottles and placed in a freeze-drying system at a temperature below -40℃ to preserve its natural triple helix structure. After freeze-drying, the finished collagen is packaged in aseptic bottles and vacuum-sealed for storage to prevent moisture and secondary contamination.

[0078] Experiments were conducted on the medical-grade type I collagen prepared in Example 1:

[0079] 1. Determination of hydroxyproline content

[0080] To evaluate the purity of collagen samples prepared using different processes, hydroxyproline content was determined colorimetrically as a characteristic indicator. Approximately 10 mg of each sample was weighed and hydrolyzed in 6 mol / L hydrochloric acid at 105 °C for 16 h. After neutralization, a suitable amount of the hydrolysate was sequentially added to chloramine T and p-dimethylaminobenzaldehyde reagent for oxidation and colorimetric reactions, and the absorbance was measured at 560 nm. The hydroxyproline concentration was calculated using a standard curve, and its mass percentage of the total protein was determined to characterize collagen purity.

[0081] The following sample groups were set up for the experiment: Group A was the collagen sample extracted in Example 1; Group B was the bovine collagen control sample extracted according to the conventional acid-enzyme method (Schmidt, MM, Dornelles, RCP, Mello, RO, et al. Collagen extraction process[J]. International Food Research Journal, 2016, 23(3):913-922.); Group C was the commercially available medical grade type I collagen standard.

[0082] Table 1 Hydroxyproline content of different collagens

[0083] Sample group Hydroxyproline content (%) Purity evaluation Group A 14.5±0.2 high Group B 12.1±0.3 medium Group C 13.7±0.1 high

[0084] The hydroxyproline content of samples A, B, and C was detected by colorimetry. The results showed that the hydroxyproline content of the group of the present invention (group A) was close to that of the standard group (group C) and significantly higher than that of the traditional process group (group B), indicating that the process of the present invention has advantages in removing impurities and improving purity.

[0085] 2. Circular dichroism (CD) scan

[0086] To assess the integrity of the collagen triple helix structure, four groups of samples were selected for testing:

[0087] Group A: Collagen samples extracted in Example 1;

[0088] Group B: Bovine collagen control sample extracted according to the conventional acid-enzyme method (Schmidt, MM, Dornelles, RCP, Mello, RO, et al. Collagen extraction process[J]. International Food Research Journal, 2016, 23(3):913-922.);

[0089] Group C: Commercially available medical-grade type I collagen standard;

[0090] Group D: Thermally denatured collagen obtained from Group A samples after heating at 90℃ for 30 minutes.

[0091] The sample was dissolved in 0.01 mol / L acetic acid solution to prepare a concentration of 0.3 mg / mL. A JASCO J-815 circular dichroism spectrometer was used to scan the sample in a 1 mm optical path quartz cuvette within the wavelength range of 190–250 nm. Scanning parameters were set to a bandwidth of 1 nm, a response time of 4 seconds, and a scan rate of 100 nm / min. Each sample was scanned three times, and the average curve was recorded. The presence of a negative peak at 197 nm and a positive peak at 220 nm was observed, and the ellipticity value (mdeg) was used to characterize the degree of triple helix structure retention.

[0092] Table 2. Degree of Preservation of Triple Helix Structure

[0093]

[0094] CD scanning revealed that samples in groups A and C both exhibited typical negative peaks at 197 nm and positive peaks at 220 nm, indicating that their triple helix structure was intact. Group B showed weaker peak shapes, suggesting partial structural damage. No characteristic double peaks were observed in group D, indicating that its triple helix structure had completely degenerated.

[0095] 3. Immunogenicity test by subcutaneous injection in mice

[0096] To verify whether the determinated peptide collagen prepared in this invention has low immunogenicity, a mouse subcutaneous injection model was used to detect its induced IgG antibody level. Six- to eight-week-old SPF-grade BALB / c mice were randomly divided into three groups of six each. Each group was injected with the determinated peptide collagen prepared in Example 1 (Group A), bovine skin collagen extracted using a conventional acid-enzyme method (Schmidt, MM, Dornelles, RCP, Mello, RO, et al. Collagen extraction process[J]. International Food Research Journal, 2016, 23(3):913-922.) (Group B), and a PBS blank control (Group C).

[0097] Each group of samples was prepared with sterile PBS to a concentration of 10 mg / mL. 0.2 mL was subcutaneously injected into the back of each mouse, three times consecutively (once a week). Mice were sacrificed on day 21, and serum was collected from the heart. The level of anti-collagen IgG antibodies in the serum was detected using enzyme-linked immunosorbent assay (ELISA), expressed as OD0.05. 450 Absorbance is expressed.

[0098] Table 3 Serum IgG levels in each group

[0099]

[0100]

[0101] The results showed that the collagen with telopeptides removed in this invention did not induce a significant specific IgG antibody response after repeated subcutaneous injections, which was comparable to the PBS control group and significantly better than the control group with telopeptides retained by traditional extraction. This suggests that the structural modification strategy can effectively reduce immunogenicity and improve biosafety.

[0102] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kit for preparing medical-grade type I collagen, characterized in that, This includes individually packaged chemical degreasing agents, delipidase solutions, swelling solutions, digestive enzymes, and purified enzymes; The chemical degreasing agent is a mixture of acetone and water; The delipase solution includes elastase, lipase, phospholipase and alkaline protease; The swelling solution includes citric acid or acetic acid; The digestive enzymes include pepsin; The purified enzyme includes trypsin.

2. The reagent kit according to claim 1, characterized in that, The delipase solution uses water as a solvent and includes elastase, lipase, phospholipase, alkaline protease, salicylic acid, sodium sulfate, PEG, and Ca. 2+ And glutathione or ascorbic acid.

3. The reagent kit according to claim 2, characterized in that, The delipase solution comprises the following components at the following concentrations: 2500–3000 U / L elastase, 1000–1200 U / L lipase, 600–800 U / L phospholipase, 200–500 U / L alkaline protease, 0.02–0.05 w% salicylic acid, 0.5–1 w% sodium sulfate, 0.5–1 w% PEG, and 50–100 mg / L Ca. 2+ And 100-200 mg / L glutathione or ascorbic acid.

4. The kit according to claim 1, characterized in that, The citric acid in the swelling solution has a mass-volume percentage of 0.5-1%; the acetic acid in the swelling solution has a volume percentage of 0.2-0.5%.

5. A method for preparing medical-grade type I collagen using the kit described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Using cowhide as raw material, physical degreasing, chemical degreasing agent degreasing and degreasing enzyme solution degreasing are carried out in sequence to obtain degreased cowhide; (2) After crushing the defatted cowhide described in step (1), soak it in a swelling solution for 4 to 8 hours to obtain pretreated cowhide; (3) The pretreated cowhide in step (2) was digested with the digestive enzyme in an environment of pH 2.5 ± 0.2 for 6 to 12 hours to obtain a digestive solution; (4) After centrifuging the digestion solution described in step (3), the supernatant is salted out using a NaCl aqueous solution with a final concentration of 0.8 mol / L. The precipitate is collected and dissolved in acetic acid solution to obtain crude collagen solution. (5) Dialyze the crude collagen solution obtained in step (4) using a 30kDa dialysis bag, and then digest the dialyzed collagen solution with a purifying enzyme to obtain a collagen solution with telopeptides removed.

6. The method according to claim 5, characterized in that, The defatting stability of the defatting enzyme solution in step (1) is 35-40℃ for 120-150 min.

7. The method according to claim 5, characterized in that, In step (4), the centrifugation speed is 8000-10000 rpm and the time is 15 min.

8. The method according to claim 5, characterized in that, After digestion as described in step (5), the process also includes freeze-drying.

9. Medical-grade type I collagen prepared by the method according to any one of claims 5 to 8.

10. The application of the medical-grade type I collagen according to claim 9 in the preparation of medical aesthetic preparations.

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