Preparation method for telopeptide-retained collagen and telopeptide-retained collagen

Abstract

The present invention relates to the technical field of collagen preparation. Disclosed are a preparation method for a telopeptide-retained collagen and the telopeptide-retained collagen. The preparation method for the telopeptide-retained collagen comprises: preparing a collagen suspension; using a protease to perform enzymatic treatment on the collagen suspension to remove non-collagenous proteins, so as to obtain a collagen extract, wherein the enzymolysis efficiency of the protease for the non-collagenous proteins is higher than that for telopeptides of collagen; and dissolving and homogenizing the collagen extract to obtain the telopeptide-retained collagen. By means of the described method, the prepared locked telopeptide-retained collagen exhibits an intact three-helix structure, and has less non-collagenous impurities, high purity, and a high telopeptide retention rate that can reach 80% or higher.

Description

Preparation method of septum collagen and septum collagen

[0001] This application claims priority to Chinese Patent Application No. 2024106888640, filed on May 29, 2024, entitled "Method for preparing saliva collagen and saliva collagen". [Technical Field]

[0002] This application relates to the field of collagen preparation technology, and in particular to a method for preparing stubby collagen and stubby collagen. [Background Technology]

[0003] Animal-derived collagen has been widely used in the medical and health fields. Initially, due to its high conservation, collagen was thought to have almost no immunogenicity. However, with further research and application, collagen has shown some immunogenicity. The telopeptide region of collagen is more immunogenic than its helical domain. Therefore, most commercially available collagen currently removes telopeptides to reduce its immunogenicity, especially soluble collagen. Furthermore, most collagen reported in domestic patents are also prepared using the telopeptide removal method. However, from a materials science perspective, telopeptide retention is significant, as it preserves a more complete material structure and more cross-linking sites, and provides more possibilities for downstream applications of collagen. Studies have shown that telopeptides play important roles in fiber formation, thermal stability, triple helix structure stability, mechanical properties, and cell recognition and adhesion. Therefore, how to prepare telopeptide-retained collagen (retaining both N-terminal and C-terminal peptides) has become an urgent problem to be solved.

[0004] [Summary of the Invention]

[0005] The main technical problem addressed by this application is to provide a method for preparing collagen and a collagen product that can produce collagen with a high telopeptide retention rate.

[0006] To solve the above-mentioned technical problems, one technical solution adopted in this application is to provide a method for preparing telogen effluvium collagen, comprising: preparing a collagen suspension; enzymatically treating the collagen suspension with a protease to remove non-collagenous impurities to obtain a collagen extract, wherein the protease has a higher enzymatic hydrolysis efficiency for non-collagenous impurities than for the telogen peptides of collagen; and dissolving and homogenizing the collagen extract to obtain the telogen effluvium collagen.

[0007] In some embodiments, the enzymatic treatment of the collagen suspension with proteases to remove non-collagenous proteins and obtain a collagen extract includes: adding neutral protease and / or alkaline protease to the collagen suspension to obtain a first mixture; adjusting the pH of the first mixture to the optimal pH range of the neutral protease and / or alkaline protease; allowing the first mixture to react at a first predetermined temperature for a first predetermined time to obtain a second mixture; and centrifuging, filtering, and washing the second mixture to obtain the collagen extract.

[0008] In some embodiments, when the protease includes the neutral protease, the optimal pH range for the neutral protease to function is 6-8; when the protease includes the alkaline protease, the optimal pH range for the alkaline protease to function is 7-10; and / or, the first predetermined temperature is 4°C-20°C; and / or, the first predetermined duration is 4h-8h.

[0009] In some embodiments, when neutral protease and / or alkaline protease are added to the collagen suspension, the preparation method further includes adding an enzyme stabilizer to the collagen suspension.

[0010] In some embodiments, the enzyme stabilizer includes at least one of propylene glycol, polyethylene glycol, sucrose, and mannitol.

[0011] In some embodiments, the protease includes one or more of Bacillus licheniformis alkaline protease, Aspergillus oryzae alkaline protease, Bacillus subtilis alkaline protease, Streptomyces griseus alkaline protease, and Fusarium alkaline protease.

[0012] In some embodiments, prior to enzymatic treatment of the collagen suspension with a protease, the preparation method further includes adding a collagen end-group protectant to the collagen suspension.

[0013] In some embodiments, the collagen end-group protectant comprises at least one of highly unsaturated fatty acids and amino acids.

[0014] In some embodiments, dissolving and homogenizing the collagen extract to obtain the stubble collagen includes: adding an acidic solution to the collagen extract to obtain an acidic solution; and homogenizing the acidic solution under high pressure to obtain the stubble collagen.

[0015] In some embodiments, the acidic solution includes at least one of acetic acid, hydrochloric acid, sulfuric acid, or citric acid.

[0016] In some embodiments, the step of dissolving and homogenizing the collagen extract to obtain the stubular collagen further includes: freeze-drying the stubular collagen to obtain dry stubular collagen.

[0017] In some embodiments, the preparation of collagen suspension includes: screening animal tissues containing collagen; refining the animal tissues into sub-tissues; sequentially treating the sub-tissues with organic reagents, salt solutions, acid-enzyme mixtures, and alkaline solutions to obtain a pretreated product; and homogenizing and grinding the pretreated product to obtain the collagen suspension.

[0018] In some embodiments, homogenizing and grinding the pretreated product to obtain the collagen suspension includes: transferring the pretreated product to a homogenizing and grinding device, adding an alkaline solution, and homogenizing and grinding the pretreated product until the particle size is 20-400 μm to obtain the collagen suspension.

[0019] Another technical solution adopted in this application is to provide a stubble collagen. The stubble collagen is prepared by the preparation method described above.

[0020] In some embodiments, the telopeptide retention rate of the retained collagen is greater than or equal to 80%.

[0021] The method for preparing terminal collagen provided in this application involves enzymatic hydrolysis of a collagen suspension using a protease. The selected protease exhibits higher hydrolysis efficiency for non-collagenous impurities than for telopeptides of collagen, thus removing non-collagenous impurities while minimizing damage to telopeptides. This achieves the removal of non-collagenous impurities and other impurities while retaining telopeptides. The prepared terminal collagen has an intact triple helix structure, few non-collagenous impurities, and a high telopeptide retention rate, showing broad application prospects. The preparation method of terminal collagen in this application is simple and easy to operate, the enzymatic hydrolysis reaction is controllable, the enzyme source is readily available, and the reaction conditions can be achieved with conventional production equipment. The preparation method of terminal collagen in this application enables the industrial-scale production of terminal collagen, possessing significant economic value. [Attached Image Description]

[0022] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0023] Figure 1 is a flowchart of the steps of an embodiment of the preparation method of telogen effluvium collagen provided in this application;

[0024] Figure 2 is a flowchart of the steps of an embodiment of the preparation method of telogen effluvium collagen provided in this application;

[0025] Figure 3 is a flowchart of the steps of an embodiment of the preparation method of telogen effluvium collagen provided in this application;

[0026] Figure 4 is a flowchart of the steps of an embodiment of the preparation method of telogen effluvium collagen provided in this application;

[0027] Figure 5 is a flowchart of the steps of an embodiment of the preparation method of telogen effluvium collagen provided in this application;

[0028] Figure 6 is an SDS-PAGE electrophoresis image of Embodiments 1, 2 and 3 provided in this application;

[0029] Figure 7 is a circular dichroism chromatogram provided in Embodiment 1 of this application;

[0030] Figure 8 is a circular dichroism chromatogram provided in Embodiment 2 of this application;

[0031] Figure 9 is a differential calorimetry scan diagram provided in Embodiment 3 of this application.

Detailed Implementation Methods

[0032] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0033] In this document, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. The term "and / or" in this document merely describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. Furthermore, the character " / " in this document generally indicates that the preceding and following related objects are in an "or" relationship. Additionally, "many" in this document indicates two or more objects.

[0034] Collagen is a crucial component of the extracellular matrix (ECM) in vertebrates, widely distributed in connective tissues including skin, bone, tendons, cartilage, cornea, and teeth. It is the most abundant protein in mammals, accounting for approximately 20%-30% of total protein content. Collagen possesses advantages such as good biocompatibility, biodegradability, low immunogenicity, and unique biological activities, playing a vital role in cell adhesion, cell migration, angiogenesis, tissue morphogenesis, and tissue repair. As a scaffold material, collagen products exhibit tissue affinity and macromolecular properties similar to the natural extracellular matrix, finding wide application in biomedical fields including sutures, tissue replacement and regeneration, medical aesthetics, wound healing, dental dressings, and skin regeneration templates.

[0035] The primary tissue sources for collagen production from animal tissues are skin and Achilles tendon. Type I collagen accounts for over 90% of the collagen in skin and Achilles tendon, with a small amount of Type III collagen also present. Both Type I and Type III collagen consist of three parts: a non-helical N-terminal region (N-terminal peptide), a central triple-helical region, and a non-helical C-terminal region (C-terminal peptide). The triple-helical structure comprises three left-handed polypeptide chains (α-chains), linked together by hydrogen bonds between glycine residues (NH) and adjacent polypeptide carbonyl groups (C=O). The triple-helical structure is the active region where collagen exerts its functional properties; an intact triple-helical structure promotes cell adhesion / migration, regulates / promotes cell differentiation, and facilitates healing. C-terminal and N-terminal telopeptides (terminal peptides) are non-helical and contain the uncommon amino acid hydroxylysine. Due to the action of lysyl oxidase, lysine or hydroxylysine residues of adjacent N- and C-terminal peptides are linked end-to-end along the fiber via a covalent aldol reaction. The role of lysyl oxidase is crucial for the formation and stability of collagen fibers. Lysyl oxidase catalyzes the covalent aldol reaction between lysine or hydroxylysine residues in adjacent N- and C-terminal peptides to generate aldol-imine cross-links, thereby increasing the mechanical strength of collagen tissue. Currently, it is believed that the immunogenicity of collagen mainly originates from telopeptides. Treating collagen with telopeptide proteases (such as pepsin and figase) can cleave and remove telopeptides to prepare telopeptide-free collagen. However, immunochemical reactions in collagen implants are often caused by residual cells, extraneous proteins, fats, endotoxins, extraction reagents, or chemical residues from cross-linking treatments. Furthermore, in clinical applications, collagen retaining telopeptides has not shown a significant immune response. Furthermore, C-terminal and N-terminal peptides play important roles in cross-linking and fiber formation; removal of telopeptides often leads to the amorphous arrangement of collagen molecules and the loss of the reconstructed collagen fiber pattern. In summary, telopeptide regions have significant biological importance for collagen materials.

[0036] In view of this, embodiments of this application describe a method for preparing distal collagen. As shown in Figure 1, the method for preparing distal collagen provided in this application includes the following steps:

[0037] S10: Prepare collagen suspension.

[0038] Collagen suspensions can be prepared from animal tissues containing collagen. In collagen suspensions, collagen is in a granular form and retains both N-terminal and C-terminal peptides.

[0039] Of course, if a finished collagen suspension is available, the preparation of the collagen suspension in this step can also represent the operation of taking out the collagen suspension for later use.

[0040] S20: Enzymatic treatment of collagen suspension with protease to remove non-collagenous proteins and obtain collagen extract.

[0041] The protease used exhibits higher enzymatic hydrolysis efficiency for non-collagenous proteins than for the telopeptides of collagen. Proteases with these characteristics bind more readily to non-collagenous proteins and have relatively fewer binding sites for telopeptides. They exhibit relatively high enzymatic activity in hydrolyzing non-collagenous proteins but relatively weak activity in hydrolyzing telopeptides. Therefore, while removing non-collagenous proteins, the protease can minimize damage to telopeptides, achieving the goal of removing non-collagenous proteins and other impurities while retaining telopeptides.

[0042] S30: Dissolve and homogenize the collagen extract to obtain end-cap collagen.

[0043] The collagen extract is dissolved to obtain soluble terminal collagen. The soluble terminal collagen is further homogenized, which helps to further dissolve and extract the terminal collagen, and further removes impurities such as extraneous proteins from the reaction product of step S20, resulting in terminal collagen with higher purity.

[0044] This application utilizes protease to enzymatically hydrolyze collagen suspensions. The selected protease exhibits higher enzymatic hydrolysis efficiency for non-collagenous impurities than for telopeptides of collagen, removing non-collagenous impurities while minimizing damage to telopeptides, thus achieving the removal of non-collagenous impurities and other impurities while retaining telopeptides. The prepared telopeptide-retained collagen has an intact triple helix structure, few non-collagenous impurities, and a high telopeptide retention rate of over 80%, demonstrating broad application prospects. The preparation method of telopeptide-retained collagen in this application is simple and easy to operate, the enzymatic hydrolysis reaction is controllable, the enzyme source is readily available, and the reaction conditions can be achieved with conventional production equipment. The preparation method of telopeptide-retained collagen in this application enables industrial-scale production of telopeptide-retained collagen, possessing significant economic value.

[0045] As shown in Figure 2, in some embodiments, step S10, preparing the collagen suspension, includes the following sub-steps:

[0046] S101: Screening animal tissues containing collagen.

[0047] Animal tissues mainly refer to dermal tissue or Achilles tendon tissue that has undergone preliminary processing and contains only collagen, with visible non-collagenous impurities such as epidermis, fat, and fascia removed. Animal sources include pigs, cattle, sheep, horses, deer, etc., and the animals are generally less than 24 months old, preferably 12-18 months old.

[0048] The tissues can be inspected according to the requirements of the standard "YY / T 0771.2-2020 Animal-derived Medical Devices Part 2: Control of Source, Collection and Disposal". After confirming that they are qualified, the fresh animal tissues are immersed in disinfectant solution for disinfection and cleaning. The disinfected and cleaned tissues are then frozen and stored at -10℃ to -40℃ to obtain frozen tissues.

[0049] Disinfectants include peracetic acid, sodium hypochlorite, polyhexamethylene biguanide, dodecyl dimethyl benzyl ammonium chloride, etc. The disinfection and cleaning operation generally involves soaking in the above disinfectants for 5-10 minutes and rinsing with purified water 1-2 times.

[0050] S102: Subdivide animal tissues into sub-tissues.

[0051] Substances can take various shapes, such as blocky, sheet-like, spherical, or columnar. The volume of substances ranges from 20 to 1000 mm². 2 For example, 50, 200 and 500mm 2 In subsequent step S104, the daughter tissue undergoes further pretreatment, specifically involving soaking it in solutions such as organic reagents, salts, acids, alkalis, and enzymes. Therefore, the size of the daughter tissue affects the surface area in contact with these solutions. When the volume of the animal tissue is fixed, a larger daughter tissue volume results in a smaller specific surface area, leading to a smaller surface area in contact with these solutions, a lower reaction rate, and less likelihood of damaging the collagen components within the daughter tissue. Conversely, a smaller daughter tissue volume results in a larger specific surface area, leading to a larger surface area in contact with these solutions, a higher reaction rate, and greater likelihood of damaging the collagen components within the daughter tissue. Therefore, limiting the daughter tissue volume to the aforementioned range allows for control of the reaction rate between the daughter tissue and subsequent solutions within a certain range, while protecting the collagen components within the daughter tissue.

[0052] Methods for tissue refinement include physical methods such as crushing, pulverizing, grinding, and cutting. For example, a slicer can be used to cut bovine Achilles tendon tissue into thin slices of 2mm-10mm, a pulverizer can be used to grind bovine dermal tissue into granules and pass them through a 5mm sieve, or a tissue grinder can be used to grind pig dermal tissue into tissue fragments of 2mm-10mm.

[0053] S103: The daughter tissue is sequentially treated with organic reagents, salt solution, acid-enzyme mixture, and alkaline solution to obtain the pretreated product.

[0054] Sequential processing can remove impurities such as proteins, fats, polysaccharides, microorganisms, endotoxins, and viruses from the daughter tissues.

[0055] S104: Homogenize and grind the pretreated product to obtain a collagen suspension.

[0056] The pretreated product (i.e., the pretreated sub-tissue) obtained after cleaning and impurity removal in step S103 becomes loose and soft, making it easy to break and grind. This pretreated product can be transferred to a closed circulating homogenizer and milled, where an alkaline solution is added for homogenization and milling to obtain a collagen suspension. After homogenization and milling, the particle size of the suspension can be further reduced, which is beneficial for the full exposure of impurities such as extra-protein proteins. The appropriate exposure of collagen telopeptides is beneficial for improving the enzymatic hydrolysis efficiency of non-collagen extra-protein proteins during subsequent enzymatic hydrolysis and for retaining collagen telopeptides. Furthermore, it facilitates the efficiency of post-hydrolysis centrifugation filtration, water washing to remove extra-protein proteins and enzyme reagents, etc.

[0057] As shown in Figure 3, in some embodiments, S103: the daughter tissue is sequentially treated with organic reagents, salt solution, acid-enzyme mixture, and alkaline solution to obtain a pretreated product, including the following steps:

[0058] S1031: The daughter tissue is sequentially treated with organic reagents, including one or more of ethanol, isopropanol and n-propanol, to obtain the first treatment product.

[0059] Ethanol is both hydrophilic and hydrophobic, isopropanol is more hydrophobic, and n-propanol is more hydrophilic. Organic reagents, using one or more of ethanol, isopropanol, and n-propanol, can remove impurities such as inorganic salts, organic acids, fats, and proteins from the daughter tissue. The first treatment product in this step is the daughter tissue treated with the organic reagent, which retains its atomic structure shape, such as blocky, sheet-like, spherical, or columnar.

[0060] In some embodiments, during the organic reagent treatment in step S1031, the volume concentration of the organic reagent is 60%-90%, the weight ratio of the tissue to the organic reagent is 1:3-1:10, the temperature is 25℃-30℃, the stirring speed is 20rpm-60rpm, and the treatment time is 2-3h.

[0061] The organic reagent can be an alcohol, which refers to one or more of ethanol, isopropanol, and n-propanol. The volume concentration of the organic reagent can be, for example, 60%, 75%, 83%, 90%, etc. Specifically, a mixture of 75% ethanol, 90% n-propanol, 60% isopropanol, or 65% ethanol, 10% isopropanol, and 25% n-propanol can be used.

[0062] The weight ratio of tissue samples to organic reagents can be 1:3, 1:5, 1:7, 1:9, 1:10, etc.; the temperature can be 25℃, 27℃, 29℃, 30℃, etc.; the stirring speed can be 20rpm, 40rpm, 45rpm, 60rpm, etc.; and the treatment time can be 2h, 2.5h, 3h, etc. Too high a concentration of organic reagent, too large a dosage, too high a treatment temperature, or too high a stirring speed can easily lead to collagen denaturation, while the opposite will result in poor impurity removal. Therefore, controlling the above parameters within the specified ranges can achieve better impurity removal results.

[0063] S1032: The first processed product is subjected to salt solution treatment, wherein the salt includes one or more of sodium chloride, ammonium chloride, sodium sulfate and ammonium sulfate, to obtain the second processed product.

[0064] The salt solutions, including sodium chloride, ammonium chloride, sodium sulfate, and ammonium sulfate, can remove impurities such as proteins and nucleic acids from the daughter tissue. The second treatment product in this step, namely the daughter tissue treated with the salt solution, retains its atomic structure shape, such as blocky, sheet-like, spherical, or columnar.

[0065] In some embodiments, during the salt treatment in step S1032, the salt solution is a sodium chloride solution with a mass fraction of 5%-10%; or an ammonium chloride solution with a mass fraction of 5%-10%; or an ammonium sulfate solution with a mass fraction of 8%-12%; or a sodium sulfate solution with a mass fraction of 10%-14%, with a weight ratio of tissue to salt solution of 1:2-1:5; the temperature is 20-30℃; the stirring speed is 10-30 rpm; and the treatment time is 4-8 hours. For example, the mass fraction of the sodium chloride solution can be 5%, 8%, 8.5%, 10%, etc.; the mass fraction of the ammonium chloride solution can be 5%, 7%, 8.5%, 10%, etc.; the mass fraction of the ammonium sulfate solution can be 5%, 6.5%, 9.5%, 10%, etc.; and the mass fraction of the sodium sulfate solution can be 5%, 6.5%, 9%, 10%, etc. In this step, the salt solution can remove impurities such as proteins and nucleic acids; if the salt solution concentration is too high or too low, the impurity removal effect will be poor. Therefore, using salt solutions within the above concentration ranges can achieve better impurity removal results.

[0066] S1033: The second processed product is treated with an acid-enzyme mixture, the acid-enzyme mixture including at least one of a mixture of organic acid and acidic protease, and a mixture of inorganic acid and acidic protease, to obtain a third processed product.

[0067] The third treatment product in this step is the sub-tissue treated with the acid-enzyme mixture, which retains the shape of the atomic structure, such as blocky, sheet-like, spherical, or columnar. For example, the second treatment product is left in a container, soaked in the acid-enzyme mixture, then washed with water to remove small fragments, and the pH of the washing solution is adjusted to 6.0-7.0. The third treatment product is then collected by filtration. Using a mixture of organic acid and acidic protease or a mixture of inorganic acid and acidic protease can remove acid-soluble proteins and further expand and loosen the second treatment product, approximately 3-5 times its original size. A small amount of tissue fragments are generated during this process, which can be removed by washing with water.

[0068] It should be noted that the acid solution can adjust the pH of the reaction system, so that the enzyme is in a weakly acidic environment and only maintains a certain activity. However, it is not the optimal pH for acidic proteases (such as pepsin) to react. Although this step inevitably hydrolyzes a small amount of telopeptides, under limited reaction conditions, its purpose is not to remove telopeptides, but to make the tissue block swell and loosen, and to remove surface impurities. It can swell approximately 3 to 5 times, accompanied by the production of a small amount of tissue fragments, which can be removed by washing with water.

[0069] In some embodiments, during the acid-enzyme mixture treatment in step S1033, the weight ratio of the second treatment product to the acid-enzyme mixture is 1:5 to 1:10, for example, 1:5, 1:6, 1:8, or 1:10; the acid solution is acetic acid, citric acid, or hydrochloric acid; the enzyme is pepsin, and its amount is 1 / 5000 to 1 / 1000 of the weight of the second treatment product. The treatment temperature is 15℃ to 25℃, for example, 15℃, 17℃, 21℃, or 25℃; the treatment time is 4h to 8h, for example, 4h, 6h, or 8h. In this step, the pH value of the reaction environment of the acidic protease is 5.0 to 7.0, that is, the acidic protease is in a weakly acidic environment, its activity value is low, and its hydrolytic power on the telopeptides in the collagen of the second treatment product (i.e., the daughter tissue treated with salt solution) is low, so that most of the telopeptides are retained. Similarly, excessively high acid concentrations can damage the telopeptide structure of collagen, while excessively low concentrations result in poor impurity removal. Conversely, excessively high enzyme concentrations can cause overly strong telopeptide cleavage, while excessively low concentrations result in poor impurity removal. Therefore, controlling these parameters within the specified ranges allows for the preservation of telopeptides while simultaneously causing the tissue block to swell and loosen, and removing surface impurities.

[0070] In some embodiments, during the acid-enzyme mixture treatment in step S1033, the inorganic acid includes hydrochloric acid, the organic acid includes at least one of acetic acid and citric acid, and the acidic protease includes pepsin, etc. Hydrochloric acid, acetic acid, or a mixture of hydrochloric acid and acetic acid can be used. The acid solution can adjust the pH of the reaction system, placing the enzyme in a weakly acidic environment and inhibiting its activity.

[0071] In some embodiments, during the enzyme-enzyme mixture treatment in step S1033, the weight ratio of the enzyme solution to the second treatment product is 1:5000-1:1000. For example, the weight ratio of the enzyme solution to the second treatment product can be 1:5000, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, etc. As mentioned above, if the concentration of the enzyme solution is too high, the cleavage force on the telopeptides will be too strong; if it is too low, the impurity removal effect will be poor. Therefore, controlling the weight ratio of the enzyme solution to the second treatment product within the above range can retain the telopeptides while removing impurities.

[0072] S1034: The third treatment product is subjected to alkaline solution treatment, wherein the alkaline includes at least one of sodium hydroxide and potassium hydroxide, to obtain a pretreated product.

[0073] Since there is acid residue in the third treatment product in step S1033, the third treatment product will shrink after the addition of alkaline solution in this step. However, as the acid is neutralized by the alkali, the third treatment product will swell by about 2-3 times and produce tissue fragments, which can be removed by washing with water. After washing with water to remove small fragments and washing until the pH of the washing solution is 7.0-8.5, the pretreated product can be obtained by filtration and collection.

[0074] In addition, this step can also remove some alkali-soluble proteins.

[0075] In some embodiments, during the alkaline solution treatment of the third processed product in step S1034, the mass fraction of the alkaline solution is 0.1M-0.5M, the weight ratio of the third processed product to the alkaline solution is 1:5-1:8, the treatment temperature is 4℃-18℃, and the treatment time is 8h-12h. For example, the mass fraction of the alkaline solution can be 0.1M, 0.3M, 0.4M, 0.5M, etc.; the weight ratio of the third processed product to the alkaline solution can be 1:5, 1:65, 1:8, etc.; the treatment temperature can be 4℃, 8℃, 15℃, 18℃, etc.; and the treatment time can be 8h, 10h, 12h, etc. If the concentration of the alkaline solution is too high, it can easily damage the collagen structure; if it is too low, the impurity removal effect will be poor. If the weight ratio of the third processed product to the alkaline solution is too high, the alkaline solution content will be low, resulting in poor impurity removal. If the treatment temperature is too high, it can easily damage the collagen structure; if the treatment time is too short, it will be difficult to achieve sufficient impurity removal. Therefore, controlling the above parameters within the above range can protect the structure of collagen while effectively removing impurities.

[0076] In some embodiments, S104 involves homogenizing and grinding the pretreated product to obtain a collagen suspension, comprising the following steps:

[0077] S1041: Transfer the pretreated product to a homogenizing and grinding device, add an alkaline solution, and homogenize and grind the pretreated product until the particle size is 20-400μm to obtain a collagen suspension.

[0078] Adding an alkaline solution can make the tissue fluffy. Homogenizing the pretreated product to a particle size of 20-400 μm provides a suitable particle size range, which is beneficial for improving the enzymatic hydrolysis efficiency of non-collagenous proteins and the retention rate of collagen telopeptides. Furthermore, it facilitates efficient extraction processes such as centrifugation, filtration, and washing to remove impurities and enzyme reagents after enzymatic hydrolysis.

[0079] The added alkaline solution has a pH value of 8.0-10.0, such as 8.0, 8.5, 9.5, 10.0, etc.

[0080] The processing temperature is 15-25℃; the particle size of collagen in the collagen suspension is 20-400μm. The processing temperature can be 15℃, 18℃, 22℃, 25℃, etc.; the particle size of collagen in the collagen suspension can be 20μm, 80μm, 100μm, 160μm, 200μm, 250μm, 320μm, 400μm, etc.

[0081] Since an acid-enzyme mixture was used in the previous step S103, the pH of the reaction system is acidic. In this step, sodium hydroxide or potassium hydroxide solution can be used to adjust the pH of the pretreated product to between 8.0 and 10.0, thus removing some alkali-soluble impurities. Excessively high processing temperatures can easily denature collagen, while excessively low temperatures result in poor impurity removal. If the collagen particle size is too large, its specific surface area is too small, hindering its ability to fully contact other solutions in the next step; if the collagen particle size is too small, its specific surface area is too large, easily damaging the collagen structure. Therefore, controlling the above parameters within the specified range can protect the collagen structure while effectively removing impurities.

[0082] As shown in Figure 4, in some embodiments, step S20 involves enzymatically treating the collagen suspension with a protease to remove non-collagenous impurities, thereby obtaining the collagen extract, and includes the following steps:

[0083] S201: Neutral protease and / or alkaline protease are added to collagen suspension to obtain a first mixture, and the pH value of the first mixture is adjusted to the optimal pH range of neutral protease and / or alkaline protease.

[0084] On the one hand, adding neutral and / or alkaline proteases to the collagen suspension, and adjusting the pH of the first mixture to the optimal pH range for these proteases, results in a slightly neutral or alkaline pH. This prevents swelling under acidic conditions and avoids exposing more telopeptide cleavage sites, thus stabilizing the collagen suspension and collagen molecular structure. This helps reduce telopeptide hydrolysis while preserving the telopeptides in the collagen. On the other hand, neutral and / or alkaline proteases have different enzymatic efficiencies for different substrates, making them more likely to bind to non-collagenous proteins. They have relatively fewer telopeptide binding sites, resulting in higher enzymatic activity for non-collagenous proteins and relatively lower activity for telopeptide hydrolysis. Therefore, this method effectively removes non-collagenous proteins while minimizing telopeptide damage, achieving the removal of non-collagenous proteins and other impurities while retaining telopeptides. This leads to a high telopeptide retention rate in the obtained collagen.

[0085] It should be noted that neutral proteases do not refer to a class of proteases whose optimal pH value for function is necessarily 7.0, but rather to proteases that can have good activity in the range of neutral, slightly acidic, or slightly alkaline conditions.

[0086] Specifically, in one embodiment of this application, the optimal pH range for the neutral protease is 6-8, such as 6.0, 6.5, 6.7, 7.1, 7.3, 7.6 or 8.0.

[0087] Specifically, in one embodiment of this application, the optimal pH range for the alkaline protease is 7-10.

[0088] The collagen suspension can be treated with neutral protease alone, alkaline protease alone, or a mixture of both.

[0089] It is readily apparent that the optimal pH range for the protease selected in this embodiment is slightly acidic, neutral, or slightly alkaline. At this pH, the enzyme activity is moderate, effectively removing non-collagenous proteins while minimizing damage to telopeptides. Furthermore, within the aforementioned pH range, the collagen suspension and its molecular structure remain stable. The collagen suspension does not swell under acidic conditions, preventing the exposure of more telopeptide cleavage sites. This stability helps reduce telopeptide hydrolysis while preserving the telopeptides of the collagen.

[0090] Furthermore, the enzyme used in one embodiment of this application is derived from microorganisms, such as bacteria, actinomycetes, and molds. It belongs to the serine proteolytic enzyme class of endopeptidases and has the ability to catalyze the hydrolysis of amino acid amide bonds and ester bonds in proteins. It can hydrolyze certain proteins into peptides or amino acids. The key catalytic active sites are serine, histidine, aspartic acid, and alanine.

[0091] Specifically, the proteases include one or more of the following: Bacillus subtilis protease, Bacillus licheniformis protease, Aspergillus oryzae protease, Streptomyces griseus protease, and Fusarium protease.

[0092] The amount of protease used can be related to the weight of animal tissue used to prepare the collagen suspension. If at least one of Bacillus licheniformis alkaline protease or Aspergillus oryzae alkaline protease is used, the weight ratio of the protease to the animal tissue used to prepare the collagen suspension is 1:500-1:50, for example, 1:500, 1:450, 1:350, 1:300, 1:250, 1:200, 1:100, 1:50, 1:20, etc. If at least one of Bacillus subtilis alkaline protease, Streptomyces griseus alkaline protease or Fusarium alkaline protease is used, the weight ratio of the protease to the animal tissue used to prepare the collagen suspension is 1:100-1:20, for example, 1:100, 1:75, 1:50, 1:20, etc.

[0093] In some embodiments, in step S201, when adding neutral protease and / or alkaline protease to the collagen suspension, an enzyme stabilizer may also be added to the collagen suspension to obtain a first mixture. The enzyme stabilizer can stabilize and protect the enzyme, enabling it to stably carry out the enzymatic hydrolysis reaction.

[0094] Specifically, enzyme stabilizers include at least one of propylene glycol, polyethylene glycol, sucrose, and mannitol.

[0095] To protect the telopeptides, in some embodiments, the preparation method further includes the following steps prior to enzymatic treatment of the collagen suspension with a protease:

[0096] S200: Collagen end-group protectant added to collagen.

[0097] Adding a collagen telomerase protectant before the enzymatic digestion reaction protects the telomeres from hydrolysis during the digestion reaction, which can further protect the telomeres of collagen and improve the retention rate of telomeres in the extracted collagen.

[0098] Specifically, collagen terminator protectants include at least one of highly unsaturated fatty acids and amino acids. Highly unsaturated fatty acids include at least one of arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, linoleic acid, and linolenic acid. The principle is that highly unsaturated fatty acids produce dilute aldehydes and partial peroxides under oxidative conditions. These peroxides can readily bind to the carboxyl or amino groups of the terminal peptides of collagen fibers, resisting enzymatic hydrolysis and preserving the terminal peptides. Amino acids include at least one of glycine / polyglycine, lysine, polylysine, glutamic acid, and polyglutamic acid. The principle is that they bind to the exposed carboxyl or amino groups of the terminal peptides to form amide compounds, protecting the terminal peptides from enzymatic hydrolysis.

[0099] S202: The first mixture is reacted at a first predetermined temperature for a first predetermined time to obtain a second mixture.

[0100] The first mixture reacts at a first predetermined temperature for a first predetermined time, allowing the protease to fully exert its enzymatic hydrolysis of impurities such as non-collagenous proteins.

[0101] Specifically, the first predetermined temperature is 4℃-20℃, such as 4℃, 7℃, 10℃, 16℃, or 20℃. By controlling the enzymatic hydrolysis temperature of the protease at the first predetermined temperature, the state of the collagen suspension and the collagen molecular structure can be stabilized, the triple helix structure of collagen can be protected, and the activity of the protease can be better controlled, reducing its ability to cleave telomeres.

[0102] Specifically, the first predetermined reaction time is 4-8 hours, such as 4 hours, 5.5 hours, 7.2 hours, or 8 hours. During the reaction of the first mixture, the mixture can also be stirred. The purpose of stirring is to ensure that the protease and the collagen in the collagen suspension come into full contact and react, thereby effectively removing impurities such as non-collagenous proteins.

[0103] The stirring speed is 10 rpm to 20 rpm, for example, 10 rpm, 15 rpm, or 20 rpm. Specifically, in step S201, the collagen suspension can be transferred to a stirred reaction vessel in a clean environment, followed by the addition of protease. Therefore, the stirred reaction vessel can stir the first mixture.

[0104] S203: The second mixture is centrifuged, filtered, and washed with water to obtain a collagen extract.

[0105] In step S202, the impurities formed after the enzymatic hydrolysis reaction and the enzyme protein used must be separated from the collagen.

[0106] Centrifugal filtration and washing refers to a method of continuously introducing water to achieve centrifugation and washing simultaneously. This method utilizes the water solubility of small molecule impurities such as proteases and hydrolyzed proteins, as well as the centrifugal force generated during the centrifugation process, to efficiently, quickly, and thoroughly remove water-soluble impurities, thereby retaining larger collagen suspension particles.

[0107] Specifically, separation can be achieved by repeated water washing and filtration, and repeated centrifugal filtration. In some embodiments, step S203 involves centrifuging and washing the second mixture to obtain collagen extract, wherein the centrifugation speed is 2000 rpm-4000 rpm; and the water washing volume is 3 to 5 times the volume of the second mixture. For example, the centrifugation speed can be 2000 rpm, 2500 rpm, 2800 rpm, 3500 rpm, 3800 rpm, 4000 rpm, etc.; and the water washing volume can be 3 times, 3.5 times, 4 times, 4.5 times, 5 times, etc., the volume of the second mixture. Controlling the centrifugation speed and water washing volume within the above range allows impurities to precipitate sufficiently, effectively removing some salts and organic reagents to obtain collagen with high purity.

[0108] In some embodiments, step S203, which involves centrifuging, filtering, and washing the second mixture to obtain the collagen extract, includes the following sub-steps:

[0109] S2031: The second mixture is placed in a device capable of simultaneously centrifuging, filtering and washing, the device being equipped with a filter bag with a pore size of 800-1000 mesh.

[0110] In step S202, the first mixture is stirred, and the resulting second mixture contains collagen, hydrolyzed proteins from weak alkaline protease and / or alkaline protease, and other impurities. The target collagen is precipitated by centrifugation, and the supernatant containing impurities is removed. Filtration can further remove other impurities with molecular weights inconsistent with collagen; the pore size of the filter bag can be set to 800 mesh, 850 mesh, 900 mesh, 950 mesh, 1000 mesh, etc. Washing with water can remove water-soluble proteins, organic reagents, and other impurities.

[0111] Steps S201 to S204 of this application are crucial for the retention of telopeptides. Only under the specific conditions of enzymatic hydrolysis temperature, enzymatic hydrolysis time, and enzyme dosage disclosed in this patent can impurities such as non-collagenous proteins be removed while retaining telopeptides. The retention rate of telopeptides is high, and the collagen extract obtained after enzymatic treatment is a delicate, flexible, and smooth white solid.

[0112] As shown in Figure 5, in some embodiments, step S30, which involves dissolving and homogenizing the collagen extract to obtain telogen effluvium collagen, includes the following steps:

[0113] S301: Add an acidic solution to the collagen extract to obtain an acidic solution.

[0114] Adding acidic solvents to collagen extracts can improve collagen dissolution efficiency, allowing the enzymatically hydrolyzed collagen suspension to dissolve further and obtain soluble collagen.

[0115] The acidic solution includes at least one of acetic acid, hydrochloric acid, sulfuric acid, or citric acid.

[0116] Specifically, acetic acid can be used as the acidic solution, as it has good dissolving and leaching effects. The collagen extract can be placed in a sealed container, with a mass ratio of collagen extract to acidic solution of 1:5 to 1:10. The acidic solution should be a 5mM to 10mM acetic acid solution. The stirring temperature should be 20℃ to 25℃, and the stirring speed 10rpm to 15rpm. After stirring, most of the solid collagen extract will disperse and dissolve into a gel-like state in the acidic solution system.

[0117] Of course, in other embodiments, an acidic solution may not be added; instead, purified aqueous solution or PBS buffer solution (pH 6.0-7.5) may be used for extraction. In this embodiment, using an acidic solution to dissolve and extract collagen is more efficient.

[0118] S302: Homogenize the acid solution under high pressure to obtain telogen collagen.

[0119] High-pressure homogenization helps to further dissolve and extract telopeptide-retaining collagen. Specifically, a high-pressure fluid homogenizer can be used, with homogenization conditions of first treating at 200-400 bar pressure and then at 400-600 bar pressure, and homogenization temperature of 10℃-18℃, to obtain gel-state collagen with telopeptides retained.

[0120] In some embodiments, other forms of telogen effluvium collagen can also be obtained. Step S30, which involves dissolving and homogenizing the collagen extract to obtain telogen effluvium collagen, further includes:

[0121] S303: Freeze-dry gel-state collagen to obtain dry-state telogen collagen.

[0122] Freeze-drying can be performed using low-temperature drying methods well known to those skilled in the art. Its purpose is to remove organic acids and moisture from collagen gel to obtain dry collagen, making it easier to preserve.

[0123] This application also provides a terminal collagen. The terminal collagen is prepared using the method described above. The terminal collagen provided in this application uses a mild combination of physical, chemical, and enzymatic methods to remove non-collagenous impurities, microorganisms, endotoxins, potential viruses, extraneous proteins, inorganic salts, organic solvents, and other impurities while preparing medical collagen that retains terminal peptides. Unlike existing technologies, the terminal collagen provided in this application addresses the material defects of existing de-terminated collagen, such as fewer cross-linking sites, weak fibroblasting properties, poor thermal stability, poor mechanical properties, and poor in vitro self-assembly / gel performance. It provides more intermolecular cross-linking sites, offering wider applications for tissue engineering products with higher requirements for structural integrity, strength, and stiffness. Furthermore, the terminal collagen provided in this application has a complete triple helix structure, thorough impurity removal, and high purity. The preparation method is simple, especially the purification step, which does not employ cumbersome and complex methods such as salting out, dialysis, ultrafiltration, and ion exchange chromatography. The extraction cycle is short, the cost is low, and it can be produced on an industrial scale.

[0124] The beneficial effects of this application are further illustrated below with reference to the embodiments.

[0125] To make the technical problems, technical solutions, and beneficial effects solved by the embodiments of this application clearer, the following will provide a more detailed description in conjunction with the embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit this application or its applications. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0126] I. Implementation Examples

[0127] Example 1

[0128] 1. Animal tissue screening: Examine fresh 18-month-old bovine dermal tissue and confirm its appearance, color and odor. Immerse qualified bovine dermal tissue in 0.15% peracetic acid solution for 8 minutes, wash twice with purified water, and freeze in a -18℃ freezer for later use.

[0129] 2. Tissue Refinement: Take out the frozen dermal tissue, crush it in a frozen state using a universal pulverizer, and pass it through a 3cm stainless steel sieve to prepare tissue fragments with a particle size of less than 3cm.

[0130] 3. Cleaning and removing impurities:

[0131] 3.1 Organic reagent treatment: In a cleanroom environment, the tissue fragments were put into a stirred reaction vessel, and 75% ethanol solution with 3 times the weight of the tissue fragments was added. The temperature was set at 28℃ and the stirring speed was 40 rpm. After treatment for 3 hours, the solution was removed, and the tissue fragments were retained in the stirred reaction vessel by the filter screen. The vessel was then washed twice with an equal amount of purified water.

[0132] 3.2 Salt solution treatment: In a cleanroom environment, add 5 times the weight of the tissue fragments in a 7.5% sodium chloride solution to the stirred reaction vessel, set the temperature to 25℃, the stirring speed to 30 rpm, and treat for 8 hours. After treatment, remove the solution. The tissue fragments are trapped in the stirred reaction vessel by the filter screen. Add an equal amount of purified water and wash twice.

[0133] 3.3 Treatment of acid-enzyme mixture: In a cleanroom environment, add 5 times the weight of the tissue fragments in 0.5M acetic acid solution and 1 / 1000 the weight of the tissue fragments in pepsin to the stirred reaction vessel. Set the temperature to 20℃, let it stand for 8 hours, and then remove the solution. The tissue fragments are trapped in the stirred reaction vessel by the filter screen. Wash with purified water until the pH of the washing solution is 6.0-7.0.

[0134] 3.4 Alkaline solution treatment: In a cleanroom environment, add 8 times the weight of the tissue fragments in 0.5M sodium hydroxide solution to the stirred reaction vessel, set the temperature to 4℃, let it stand for 12 hours, and then remove the solution. The tissue fragments are trapped in the stirred reaction vessel by the filter screen. Wash with purified water until the pH of the washing solution is 7.0-8.5.

[0135] 4. Homogenization and Grinding: In a cleanroom environment, the cleaned and purified tissue fragments are transferred to a circulating homogenizing and grinding device. A sodium hydroxide solution with a pH of 9.0 is added at 10 times the weight of the tissue fragments. The temperature is set at 15℃, and the tissue fragments are ground into a collagen suspension.

[0136] 5. Extraction and purification: In a cleanroom environment, the collagen suspension was transferred to a stirred reaction vessel, an equal volume of purified water was added, and then 1 / 100 weight of Bacillus subtilis alkaline protease from tissue fragments was added. The temperature was set at 15℃ and the stirring speed at 20 rpm. After stirring for 6 hours, the extract was passed into a centrifuge and filtered while being washed with water. The filter bag had a pore size of 1000 mesh and the centrifugation speed was 4000 rpm.

[0137] 6. Dissolution and homogenization: In a cleanroom environment, the collagen extract obtained by centrifugation is placed in a stirred reaction vessel, and 10 mM acetic acid solution with 10 times the weight of the collagen extract is added. The temperature is set at 25℃, the stirring speed is 15 rpm, and the mixture is stirred and dissolved for 12 hours. The acid solution is then treated once each at 200 bar and 400 bar using a high-pressure fluid homogenizer, with a homogenization temperature of 18℃. The resulting gel-like collagen is then collected.

[0138] 7. Freeze-drying: Gel-state collagen materials are freeze-dried to obtain dry collagen.

[0139] Example 2

[0140] 1. Animal tissue screening: Examine fresh 12-month-old pig dermal tissue and confirm its appearance, color and odor. Immerse qualified bovine dermal tissue in 0.08% sodium hypochlorite solution for 5 minutes, wash twice with purified water, and freeze in a -40℃ freezer for later use.

[0141] 2. Tissue refinement: Take out the frozen dermal tissue and grind it into tissue fragments with a particle size of 3cm-4cm using a tissue homogenizer.

[0142] 3. Cleaning and removing impurities:

[0143] 3.1 Organic reagent treatment: In a cleanroom environment, the tissue fragments were put into a stirred reaction vessel, and 10 times the weight of the tissue fragments in 60% isopropanol solution was added. The temperature was set at 30℃ and the stirring speed at 60 rpm. After treatment for 2 hours, the solution was removed. The tissue fragments were retained in the stirred reaction vessel by the filter screen and washed once with an equal amount of purified water.

[0144] 3.2 Salt treatment: In a cleanroom environment, add 10% sodium sulfate solution with 4 times the weight of tissue fragments to the stirred reaction vessel, set the temperature to 30℃, the stirring speed to 30 rpm, and treat for 4 hours. After treatment, remove the solution. The tissue fragments are trapped in the stirred reaction vessel by the filter screen. Add an equal amount of purified water to wash 3 times.

[0145] 3.3 Acid-Enzyme Mixture Treatment: In a cleanroom environment, add 10 times the weight of the tissue fragments in 0.01M hydrochloric acid solution and 1 / 5000 weight of the tissue fragments in pepsin to the stirred reaction vessel. Set the temperature to 15℃, let it stand for 4 hours, then remove the solution. The tissue fragments are retained in the stirred reaction vessel by the filter screen. Wash with purified water until the pH of the washing solution is 6.0-7.0.

[0146] 3.4 Alkaline solution treatment: In a cleanroom environment, add 8 times the weight of the tissue fragments in 0.5M sodium hydroxide solution to the stirred reaction vessel, set the temperature to 4℃, let it stand for 8 hours, and then remove the solution. The tissue fragments are trapped in the stirred reaction vessel by the filter screen. Wash with purified water until the pH of the washing solution is 7.0-8.5.

[0147] 4. Homogenization and Grinding: In a cleanroom environment, the cleaned and purified tissue fragments are transferred to a circulating homogenizing and grinding device. A sodium hydroxide solution with a pH of 10.0 is added at 10 times the weight of the tissue fragments. The temperature is set to 25°C, and the tissue fragments are ground into a protein suspension.

[0148] 5. Extraction and purification: In a cleanroom environment, the collagen suspension was transferred to a stirred reaction vessel, 1.5 times the volume of purified water was added, and 1 / 500 weight of Bacillus licheniformis alkaline protease was added. The temperature was set at 18℃ and the stirring speed at 20 rpm. After stirring for 8 hours, the extract was passed into a centrifuge and filtered while being washed with water. The filter bag had a pore size of 800 mesh and the centrifugation speed was 3000 rpm.

[0149] 6. Dissolution and homogenization: In a cleanroom environment, the collagen extract obtained by centrifugation is placed in a stirred reaction vessel, and 5mM acetic acid solution with 5 times the weight of the collagen extract is added. The temperature is set at 20℃, the stirring speed is 10rpm, and the mixture is stirred and dissolved for 24h. The acid solution is then treated once each at 400bar and 600bar using a high-pressure fluid homogenizer at a homogenization temperature of 10℃. The gel-state collagen material is then collected.

[0150] 7. Freeze-drying: Gel-state collagen materials are freeze-dried to obtain dry collagen.

[0151] Example 3

[0152] 1. Animal tissue screening: Examine fresh 18-month-old bovine Achilles tendon tissue and confirm its appearance, color and odor. Immerse qualified bovine Achilles tendon tissue in 0.16% polyhexamethylene biguanide (PHMB) solution for 5 minutes, wash twice with purified water, and freeze in a -40℃ freezer for later use.

[0153] 2. Tissue refinement: Take out the frozen bovine Achilles tendon tissue and cut it into 2-4cm thin slices using a slicing machine, and collect them for later use.

[0154] 3. Cleaning and removing impurities:

[0155] 3.1 Organic reagent treatment: In a cleanroom environment, tissue slices were placed into a stirred reaction vessel and a mixed alcohol solution (65% ethanol: 10% isopropanol: 15% n-propanol mixture: 10% purified water) was added. The temperature was set at 25℃ and the stirring speed at 20 rpm. After treatment for 3 hours, the solution was removed. The tissue slices were retained in the stirred reaction vessel by the filter screen and washed twice with an equal amount of purified water.

[0156] 3.2 Salt solution treatment: In a cleanroom environment, add 5 times the weight of the tissue slices in a 12% sodium sulfate solution to the stirred reaction vessel, set the temperature to 20℃ and the stirring speed to 30 rpm, and remove the solution after 6 hours. The tissue slices are retained in the stirred reaction vessel by the filter screen and washed twice with an equal amount of purified water.

[0157] 3.3 Acid-Enzyme Mixture Treatment: In a cleanroom environment, add 5 times the weight of the tissue slices in 0.1M acetic acid solution and 1 / 2000 the weight of the tissue slices in pepsin to the stirred reaction vessel. Set the temperature to 20℃, allow it to stand for 4 hours, then remove the solution. The tissue slices are retained in the stirred reaction vessel by a filter screen. Wash with purified water until the pH of the washing solution is 6.0-7.0.

[0158] 3.4 Alkaline solution treatment: In a cleanroom environment, add 5 times the weight of the tissue slices in 0.2M sodium hydroxide solution to the stirred reaction vessel, set the temperature to 18℃, let it stand for 12 hours, and then remove the solution. The tissue slices are trapped in the stirred reaction vessel by the filter screen. Wash with purified water until the pH of the washing solution is 7.0-8.5.

[0159] 4. Homogenization and Grinding: In a cleanroom environment, the cleaned and purified tissue slices are transferred to a circulating homogenizer and grinding equipment. A sodium hydroxide solution with a pH of 8.0 and 15 times the weight of the tissue is added. The temperature is set to 15°C, and the tissue slices are ground into a protein suspension.

[0160] 5. Extraction and purification: In a cleanroom environment, the collagen suspension was transferred to a stirred reaction vessel, an equal volume of purified water was added, and then 1 / 60 weight of Fusarium alkaline protease from tissue slices was added. The temperature was set at 10℃ and the stirring speed at 15 rpm. After stirring for 6 hours, the extract was passed into a centrifuge and filtered while being washed with water. The filter bag had a pore size of 800 mesh and the centrifugation speed was 4000 rpm.

[0161] 6. Dissolution and homogenization: In a cleanroom environment, the collagen extract obtained by centrifugation is placed in a stirred reaction vessel, and 7.5mM acetic acid solution with 8 times the weight of the collagen extract is added. The temperature is set at 25℃, the stirring speed is 15rpm, and the mixture is stirred and dissolved for 18h. The acid solution is then treated twice by a high-pressure fluid homogenizer at 400bar pressure and the homogenization temperature is 15℃. The gel-state collagen is then collected.

[0162] 7. Freeze-drying: Dry collagen can be obtained by freeze-drying gel-state collagen materials.

[0163] II. Performance Testing

[0164] According to the medical standard "YY / T 1453-2016 Characterization Method of Type I Collagen in Tissue-Engineed Medical Device Products", the content of miscellaneous proteins, collagen, hydroxyproline, total sugar, fat, microbial limits, and endotoxins were tested, and the content of N-terminal peptides and C-terminal peptides was detected by ELISA.

[0165] Table 1 Performance Testing Methods

[0166] Referring to Table 2, in order to compare with the embodiments of the present invention, the applicant purchased commercial bovine Achilles tendon type I collagen material for simultaneous testing of the corresponding items. The bovine Achilles tendon type I collagen material is a determinate collagen extracted by a combination of acid and protease hydrolysis to remove telopeptides.

[0167] Table 2. Test results of Examples 1, 2, 3 and commercially available products.

[0168] As can be seen from the results in Table 2, compared with commercially available products, the collagen prepared by the method provided in the embodiments of this application has a retention of more than 80% of N-terminal peptides and C-terminal peptides, and a higher purity. The collagen content is more than 98%, and the content of impurities, total sugar and fat is very low, all below 0.5%. The levels of microorganisms and endotoxins are also low, with endotoxins less than 0.02 EU / ml, which meets the requirements of medical standards.

[0169] III. SDS-PAGE Electrophoresis Analysis

[0170] Please refer to Figure 6. Figure 6 shows four bands of electrophoresis. The first band is the marker electrophoresis pattern, and the second, third, and fourth bands are the collagen electrophoresis patterns corresponding to Examples 1, 2, and 3, respectively. As can be seen from Figure 6, the triple helix structure of the collagen from Examples 1, 2, and 3 was opened after electrophoresis. The molecular weight of the α1 and α2 chains is approximately 100 kDa, proving that the triple helix of collagen was preserved. The various enzymes used in the preparation method did not have a significant hydrolytic effect on the triple helix structure. The molecular chains are intact and not damaged.

[0171] IV. Circular Dichroism (CD) Chromatography Analysis

[0172] CD spectroscopy is used to characterize the triple helix structure of collagen. The CD spectrum of physiological collagen shows a negative peak at approximately 195 nm and a positive peak at approximately 220 nm. The positive absorption peak is a typical characteristic of circular dichroism spectroscopy for the L-polyproline configuration. Combined with the position of the negative peak, this indicates a complete triple helix structure in collagen. If no positive peak is detected at 220 nm, it indicates the absence of a triple helix structure. The higher the ratio of positive to negative peaks, the more complete the triple helix structure and the higher its content.

[0173] Referring to Figures 7 and 8, which show the circular dichroism (CD) chromatograms of the collagen corresponding to Examples 1 and 2, respectively, both collagen from Examples 1 and 2 exhibit a characteristic positive peak at approximately 220 nm and a characteristic negative peak at approximately 195 nm. Therefore, the collagen from Examples 1 and 2 possesses a triple helix structure.

[0174] V. Differential Calorimetry (DSC) Analysis

[0175] Please refer to Figure 9, which shows the differential calorimetry (DCT) spectrum of the collagen corresponding to Example 3. A melting peak appears at 118.49℃, indicating that the collagen structure is intact and has a high melting temperature.

[0176] The above description is merely an embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A method for preparing a tail collagen, characterized by, include: Preparation of collagen suspension; The collagen suspension was treated with a protease to remove non-collagenous impurities, resulting in a collagen extract. The protease was more efficient at hydrolyzing non-collagenous impurities than at hydrolyzing telopeptides of collagen. The collagen extract was dissolved and homogenized to obtain the terminal collagen.

2. The production method according to claim 1, wherein The process of enzymatically treating the collagen suspension with protease to remove non-collagenous proteins and obtain a collagen extract includes: Neutral protease and / or alkaline protease are added to the collagen suspension to obtain a first mixture, and the pH value of the first mixture is adjusted to the optimal pH range of the neutral protease and / or alkaline protease. After the first mixture reacts at a first predetermined temperature for a first predetermined time, a second mixture is obtained; The second mixture was centrifuged, filtered, and washed with water to obtain the collagen extract.

3. The production method according to claim 2, wherein When the protease includes the neutral protease, the optimal pH range for the neutral protease to function is 6-8; when the protease includes the alkaline protease, the optimal pH range for the alkaline protease to function is 7-10; and / or, the first predetermined temperature is 4°C-20°C; and / or, the first predetermined duration is 4h-8h.

4. The production method according to claim 2, wherein When neutral protease and / or alkaline protease are added to the collagen suspension, the preparation method further includes: An enzyme stabilizer is added to the collagen suspension.

5. The preparation method according to claim 4, characterized in that, The enzyme stabilizer includes at least one of propylene glycol, polyethylene glycol, sucrose, and mannitol.

6. The preparation method according to any one of claims 1-5, characterized in that, The protease includes one or more of the following: Bacillus licheniformis alkaline protease, Aspergillus oryzae alkaline protease, Bacillus subtilis alkaline protease, Streptomyces griseus alkaline protease, and Fusarium alkaline protease.

7. The preparation method according to any one of claims 1-5, characterized in that, Prior to the enzymatic treatment of the collagen suspension with protease, the preparation method further includes: A collagen end-group protectant is added to the collagen suspension.

8. The preparation method according to claim 7, characterized in that, The collagen end-group protectant includes at least one of highly unsaturated fatty acids and amino acids.

9. The preparation method according to any one of claims 1-5, characterized in that, The process of dissolving and homogenizing the collagen extract to obtain the terminal collagen includes: An acidic solution was added to the collagen extract to obtain an acidic solution. The acid solution was homogenized under high pressure to obtain the telogen effluvium collagen.

10. The preparation method according to claim 9, characterized in that, The acidic solution includes at least one of acetic acid, hydrochloric acid, sulfuric acid, or citric acid.

11. The preparation method according to claim 9, characterized in that, The process of dissolving and homogenizing the collagen extract to obtain the telogen effluvium collagen further includes: Dry-state collagen was obtained by freeze-drying the collagen at the ends of the head.

12. The preparation method according to any one of claims 1-5, characterized in that, The preparation of the collagen suspension includes: Screening animal tissues containing collagen; The animal tissue is further subdivided into sub-tissues; The sub-tissue was sequentially treated with organic reagents, salt solution, acid-enzyme mixture, and alkaline solution to obtain a pretreated product. The pretreated product is homogenized and ground to obtain the collagen suspension.

13. The preparation method according to claim 12, characterized in that, The pretreated product is homogenized and ground to obtain the collagen suspension, comprising: The pretreated product is transferred to a homogenizing and grinding device, an alkaline solution is added, and the pretreated product is homogenized and ground until the particle size is 20-400 μm to obtain a collagen suspension.

14. A type of telogen effluvium collagen, characterized in that, The telogen effluvium collagen is prepared by any one of claims 1 to 13.

15. The telogen effluvium collagen according to claim 14, characterized in that, The telopeptide retention rate of the retained collagen is greater than or equal to 80%.

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