High-thermal-stability bionic collagen, cosmetic, medicine, tissue engineering material or medical device and preparation method and application of high-thermal-stability bionic collagen, cosmetic, medicine, tissue engineering material or medical device

Abstract

The invention belongs to the technical field of medical and cosmetic raw materials, and particularly relates to bionic collagen with high thermal stability, a cosmetic, a medicine, a tissue engineering material or a medical device as well as a preparation method and application thereof. The structural formula of the bionic collagen disclosed by the invention is H2N-(P-O-G) n-COOH, wherein P-O-G is a peptide chain composed of proline, hydroxyproline, and glycine residues. According to the invention, through multi-stage temperature control and improvement of the material feeding ratio, the prepared bionic collagen still has a stable triple-helix structure after being subjected to moist heat sterilization; no salting-out phenomenon exists. The bionic collagen has good biological safety, promotes wound healing and promotes cell collagen expression. The bionic collagen keeps a triple helix structure and a fiber network structure in a relatively large temperature range, is good in thermal stability, can be better applied to the fields of cosmetics, medicines, medical instruments, biological materials and the like, and is good in application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical and cosmetic raw materials, and specifically relates to a bionic collagen with high thermal stability, cosmetics, medicine, tissue engineering material or medical device, and a preparation method and use thereof. Background Art

[0002] Biomimetic collagen protein is a synthetic material that simulates the structure and function of natural collagen. It replicates or simulates the properties of collagen in nature through scientific methods, usually has good biocompatibility and biodegradability, can interact well with human tissue, and can be effectively used by the human body. The efficacy of biomimetic collagen products is supported by clinical research. By simulating the structure and function of natural collagen, biomimetic collagen provides new solutions in the fields of medical, beauty and biomaterials.

[0003] Bionic collagen has sufficient plasticity when used; it can stimulate collagen fibroblasts in the body to produce collagen; it can effectively promote the activity of mitochondria, accelerate the cells to eliminate aging molecules and organelles, and achieve anti-aging effects; and its production process is not restricted by the natural environment, which can avoid the safety risks of traditional collagen, which is mainly sourced from deep-sea fish, cattle, and pigs. Therefore, the research and development and preparation of bionic collagen have been enthusiastically sought after by the market and have become a research hotspot for scientific researchers.

[0004] According to existing literature and molecular design principles, the basic repeating unit of natural collagen is the tripeptide "Gly-X-Y", in which the X and Y positions are often replaced by proline and hydroxyproline respectively (i.e., forming "Gly-Pro-Hyp"). This region is the key "code" to maintain the stability of the triple helix folding. Studies have shown that the homology between bovine collagen and the full-length sequence of human type I collagen is about 97.47%, and the homology with type III collagen is about 90.18%. The biomimetic collagen Poly (POG) n The design goal is to simulate the triple helix folding structure of natural collagen by continuously repeating the "POG" (i.e., the core "Gly-Pro-Hyp" in actual sequence expression) unit during chemical synthesis. From the perspective of the core repeating unit, the "Gly-Pro-Hyp" that constitutes the key triple helix region of bionic collagen and natural collagen can be completely consistent, that is, 100% homology is achieved in this part.

[0005] However, there are some problems in the preparation of bionic collagen, such as salt precipitation, which makes it difficult to compound into medical beauty product preparations; poor thermal stability, and precipitation after high temperature or sterilization. These problems seriously reduce the efficacy of bionic collagen and limit its widespread application.

[0006] Therefore, developing a biomimetic collagen with good thermal stability, no salting out and high biological activity is an urgent problem to be solved in this field. Summary of the invention

[0007] In view of the problems of the prior art, the present invention provides a bionic collagen with high thermal stability, a cosmetic, a medicine, a tissue engineering material or a medical device, and a preparation method and use thereof.

[0008] The present invention provides a bionic collagen with high thermal stability, the structural formula of which is shown in Formula I: Formula I: H2N-(POG) n -COOH; Among them, P is a proline residue, O is a hydroxyproline residue, G is a glycine residue, and n ≧ 4; The bionic collagen is prepared according to the following steps: A condensation reaction is carried out using a condensation aid, a tripeptide and a dehydration condensation agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to heat the mixture to 30-50° C. and then cool the mixture to 10-25° C.; The crude product solution is homogenized and separated to obtain; The weight ratio of the condensation aid, the tripeptide and the dehydration condensation agent is 0.7-2.3: 7.2-24.6: 27.4-36.2.

[0009] Preferably, the weight ratio of the condensation aid, the tripeptide and the dehydration condensation agent is 1-2:10-20:30-34.

[0010] Preferably, the weight ratio of the condensation aid, the tripeptide and the dehydration condensation agent is 2:15:30.

[0011] Preferably, the heating temperature is raised to 30°C.

[0012] Preferably, the further cooling temperature is further cooled to 10°C.

[0013] Preferably, the homogenization treatment is carried out in a homogenizer, and the rotation speed of the homogenizer is greater than or equal to 2000 and less than 5000.

[0014] Preferably, the rotation speed of the homogenizer is 2000-4500.

[0015] Preferably, after cooling to 10-25° C., the temperature is kept warm for 34-72 hours.

[0016] Preferably, the dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide, 1,3-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0017] Preferably, the dehydration condensation agent is N,N'-diisopropylcarbodiimide.

[0018] Preferably, the steps include the following: Step 1, preparing a condensation auxiliary agent solution; Step 2, adding a tripeptide having a structural formula of H2N-POG-COOH to the condensation auxiliary agent solution to obtain a tripeptide solution; Step 3, preparing a dehydration condensation agent solution; Step 4, the tripeptide solution is mixed with the dehydration condensation agent solution to carry out a condensation reaction to obtain a crude product solution; Step 5, the crude product solution is homogenized; Step 6, separation and purification, to obtain.

[0019] Preferably, the mixing temperature in step 4 is 0-10°C.

[0020] Preferably, in step 1, after the condensation auxiliary agent solution is prepared, it is stirred at 0-10° C. for 1-4 hours; And / or, in step 2, the tripeptide solution is stirred at 15-30° C. for 1-4 h, then cooled to 0-10° C. and maintained for 1-4 h; And / or, the dehydration condensation agent solution mixed in step 4 is added dropwise to the tripeptide solution within 0.2-1.6 hours; and / or, in step 4, maintaining at 30-50° C. for 1.5-6 h; And / or, the specific process of the homogenization treatment in step 5 is to homogenize at ≤10°C for 0.25-1.2h, and stop for 0.25-1.2h; and repeat this for 2-3 cycles; And / or, the specific steps of separation and purification in step 6 are: adding water 2.5-6 times the volume of the reaction solution, using a hollow fiber membrane with a molecular weight of 3-5W for membrane filtration; and repeating the membrane filtration for 2-3 times.

[0021] Preferably, in step 1, the solvent of the condensation aid solution is a phosphate buffer; in step 3, the solvent of the dehydration condensation agent solution is a phosphate buffer; the phosphate buffer is prepared using the following raw materials in molar parts: Potassium chloride 3.2-4.3mM, Sodium hydrogen phosphate 8.8-13.1mM, Potassium dihydrogen phosphate 1.2-2.5mM, Sodium chloride 124-151mM; The pH of the phosphate buffer is 6.9-7.6; And / or, the condensation auxiliary agent is selected from at least one of 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole or N-hydroxysuccinimide.

[0022] The present invention provides a method for preparing the biomimetic collagen with high thermal stability as described in any one of the above items, comprising the following steps: A condensation reaction is carried out using a condensation aid, a tripeptide and a dehydration condensation agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to heat the mixture to 30-50° C. and then cool the mixture to 10-25° C.; The crude product solution is homogenized and separated to obtain; The weight ratio of the condensation aid, the tripeptide and the dehydration condensation agent is 0.7-2.3: 7.2-24.6: 27.4-36.2.

[0023] The present invention provides use of any of the above-mentioned biomimetic collagens with high thermal stability in the preparation of cosmetics, medicines or tissue engineering materials.

[0024] Preferably, the cosmetics are functional skin care products; the tissue engineering materials include tissue engineering materials and bio-based packaging materials used for drug carriers, cosmetic surgery or medical devices; the medical devices include dressings, filling materials or engineering scaffolds for skin, bones, ligaments, peripheral nerves and blood vessels.

[0025] Preferably, the functional skin care product is a facial mask, an essence or a facial cleanser.

[0026] The present invention provides a cosmetic or medicine, which is prepared by using any of the above-mentioned bionic collagen with high thermal stability as an active ingredient and adding cosmetically or pharmaceutically acceptable auxiliary materials.

[0027] The present invention provides a tissue engineering material, which is prepared by using any of the above-mentioned bionic collagens with high thermal stability as an active ingredient and adding medically acceptable auxiliary materials.

[0028] The present invention provides a medical device, which is prepared by using any of the above-mentioned bionic collagen with high thermal stability as an active ingredient and adding medically acceptable auxiliary materials.

[0029] Preferably, the concentration of the biomimetic collagen with high thermal stability is 0.047 wt -0.52 wt .

[0030] The present invention provides a bionic collagen with high thermal stability. Through multi-stage temperature control and material feeding ratio, the prepared bionic collagen still has a stable triple helix structure after wet heat sterilization (121°C); after being treated with a salt solution, there is no salt precipitation phenomenon. The bionic collagen has good biosafety, promotes wound healing, and promotes cell collagen expression. The bionic collagen maintains a triple helix structure and a fiber network structure within a temperature range of 20°C-95°C, has good thermal stability, and can be better applied to cosmetics, drugs, medical devices, biomaterials and other fields. Its effect is similar to that of human collagen, and has good application prospects.

[0031] Bionic collagen is an artificial collagen material designed and constructed through multidisciplinary bionic technology. Its core feature is to accurately simulate the stable triple helix structure of natural collagen and the collagen network fiber structure formed by self-assembly. The biomaterial of the present invention can be called "collagen" and has the same multi-level structure, function and quality controllability as natural collagen.

[0032] The biomimetic collagen material Poly (POG) of the present invention n It is reasonable to call it "collagen". First, Poly (POG) n The multi-level structure is consistent with natural collagen, which includes primary structure, secondary structure, tertiary structure and quaternary structure. In the primary structure, one of the main characteristics of natural collagen is that its amino acid sequence follows the "Gly-XY" repeating tripeptide unit (X / Y is mostly proline and hydroxyproline), which is also the basis for collagen to form a triple helix structure; Poly (POG) n The molecular chain is constructed by repeatedly repeating the POG sequence. It should be noted that "POG" and "GPO" are equivalent in terms of cyclic arrangement, because the key to the function of collagen tripeptide is that there must be a glycine (Gly) in every three amino acids to ensure the tight stacking and stability of the triple helix. Structurally, Poly(POG) n It indeed simulates the basic tripeptide repeating unit of natural collagen, which is consistent with the primary structure of natural collagen. In the secondary structure, natural collagen is based on the tripeptide repeating unit, forming a stable triple helix structure through hydrogen bonds and hydrophobic forces. Through precise chemical design and strict control of the preparation process, Poly(POG) n Successfully achieved a triple helix secondary structure similar to natural collagen; in the tertiary structure, natural collagen forms a compact three-dimensional folded structure through the interaction of molecules inside and outside to maintain its water solubility and multifunctionality; while Poly(POG) nUnder proper hydration conditions, it will also show the molecular aggregation effect of natural collagen, forming a similar tertiary helical network; in the quaternary structure, Poly(POG) n The material can be regulated through process to prepare a composite structure similar to natural collagen fibers, which is widely used in tissue repair and scaffold materials. The bionic collagen molecules can form a highly synergistic collagen fiber structure through non-covalent bonds (such as hydrogen bonds and van der Waals forces), simulating the supramolecular properties of natural collagen.

[0033] In addition, Poly (POG) n The function of Poly(POG) is consistent with that of natural collagen. Natural collagen is the core component of the extracellular matrix (ECM), providing cell adhesion sites and promoting the proliferation and migration of fibroblasts, keratinocytes, endothelial cells, etc. n It has been verified by experiments that it can stably bind to cell surface receptors (such as integrins) and play the same biological role as natural collagen; after natural collagen degradation, it mainly produces small molecules such as glycine, proline and hydroxyproline. These degradation products can be absorbed and utilized by the body, while Poly(POG) n The above non-toxic products are also released after degradation under various physiological conditions (pH, enzyme environment), which is degradable and harmless. Natural collagen is often used as a filling material and structural scaffold in medical and cosmetic applications. Poly(POG) n By adjusting parameters such as molecular weight and cross-linking density, the mechanical properties can be controlled, and when used in scenarios such as soft tissue repair and skin support, they perform consistently with natural collagen.

[0034] The China National Medical Products Administration (NMPA) has clear regulations on the name and application of collagen products in accordance with relevant laws and regulations on drugs, medical devices and cosmetics. The "Technical Specifications for Safety of Cosmetics" (2021 Edition) defines "collagen" as "protein or oligopeptide containing a collagen triple helix structure". Poly (POG) n It meets this definition and is functionally consistent with natural collagen, with no passive risk. China's "Medical Device Classification Rules and Classification Catalog" (2021 Edition) clearly classifies "collagen-based materials" as high-risk Class III medical devices (such as implant filling materials) and Class II dressings. According to the "Regulations on the Supervision and Administration of Cosmetics" and the "Regulations on the Registration and Filing of New Cosmetic Raw Materials", bionic collagen can be filed as a cosmetic raw material, and the safety of the raw material and its skin repair-promoting effect can be supported by existing functional verification methods. Among Class III medical devices, Poly(POG) n It can be used in soft tissue filling materials, nerve regeneration catheters, bone repair scaffolds, etc. Among the second-class dressings, Poly (POG) n Can be used in burns, wound dressings, cosmetics, Poly (POG) nCan be used in anti-aging skin care products, cosmetic raw materials, Poly (POG) n Can be used as raw materials for advanced functional cosmetics.

[0035] Biomimetic Collagen Poly(POG) n Through molecular design and synthesis technology, collagen and its related materials have achieved a multi-level structure, function and application value that is highly consistent with natural collagen, and are superior to naturally extracted collagen in terms of quality safety and production controllability. These characteristics meet the definition of "collagen" by NMPA and relevant regulations, and are highly adaptable in the declaration of medical devices, cosmetics and raw materials, and can normally enter the fields of Class III medical devices, Class II dressings and cosmetics.

[0036] The bionic collagen of the present invention is highly consistent with human type I and type III collagen in sequence and function, but due to the use of chemical synthesis, it is not essentially humanized collagen in the traditional sense and can be regarded as bionic type I / type III collagen. As a new generation of bionic biomaterials that breaks through the bottlenecks of traditional animal-extracted collagen and recombinant collagen, the bionic collagen of the present invention completely gets rid of the dependence on animal-derived ingredients, and eliminates the risks of pathogen carriage, virus contamination, and poor batch stability of materials from the source. By optimizing molecular design or preparation methods, the performance bottlenecks of animal collagen, such as easy degradation, poor thermal stability, and difficulty in chemical modification, as well as the problem of insufficient stability of the triple helix structure of recombinant collagen, are solved, and it has the unique advantages of biocompatibility, structural stability, industrial production, and sustainable development.

[0037] The bionic collagen of the present invention has the following advantages: (1) No animal-derived risk, no reliance on animal extraction, and avoids the potential risks of animal-derived collagen; (2) Highly intelligent controllable, it can precisely regulate molecular weight, molecular structure and amino acid sequence, degradation rate, cross-linking density and pore structure, functional modification, etc. according to specific application requirements; it can design artificial vascular patches with high tensile toughness and stability to meet the physiological conditions of blood flow impact and stress load, and can obtain sufficient mechanical strength without secondary cross-linking treatment. The flexibility of the material is close to that of natural tissue, which is convenient for doctors to operate and use during surgery; by controlling the density of the triple helical collagen sequence, it can regulate its effect on fibroblasts, cells and tissues. The adhesion of smooth muscle cells and endothelial cells provides an ideal scaffold environment for vascular regeneration; (3) Excellent biocompatibility, can be used for artificial collagen vascular patches, gradually degraded with tissue regeneration, and promote the repair of endothelial cells and surrounding vascular tissues; (4) Efficient mass production, compared with the complexity of the natural extraction process, the bionic collagen of the present invention can achieve highly controllable molecular weight and chemical consistency, ensuring uniformity in mass production; (5) Flexible self-assembly ability, can meet short-term and long-term medical needs, short-term use such as wound dressings and functional skin care repair materials, long-term use such as filling injections, artificial scaffolds and transplant materials, providing long-term support for tissue reconstruction. The bionic collagen of the present invention is widely applicable to a variety of tissue engineering and medical fields, especially in high-end functional skin care, wound healing, soft tissue repair, regenerative medicine, tissue engineering scaffolds and vascular repair, showing significant value and application potential.

[0038] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0039] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The circular dichroism spectrum of the biomimetic collagen prepared in Example 1 after heat treatment at 20-95°C, wherein A is the circular dichroism spectrum of the biomimetic collagen before moist heat sterilization after heat treatment at 20-95°C, wherein B is the circular dichroism spectrum of the biomimetic collagen after moist heat sterilization after heat treatment at 20-95°C; Figure 2 This is a circular dichroism spectrum of the comparative collagen after heat treatment at 20-95°C after moist heat sterilization; Figure 3This is a graph showing the molecular weight of the biomimetic collagen prepared in Example 1; Figure 4 This is the infrared spectrum result of the biomimetic collagen prepared in Example 1; Figure 5 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 2 after heat treatment at 20-95°C; Figure 6 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 3 after heat treatment at 20-95°C; Figure 7 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 4 after heat treatment at 20-95°C; Figure 8 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 5 after heat treatment at 20-95°C; Fig. 9 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 6 after heat treatment at 20-95°C; Fig.10 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 7 after heat treatment at 20-95°C; Fig.11 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 8 after heat treatment at 20-95°C; Fig.12 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 9 after heat treatment at 20-95°C; Fig.13 This is a circular dichroism spectrum of the biomimetic collagen prepared in Example 10 after heat treatment at 20-95°C; Fig.14 Figures 1 and 2 are cell viability results when biomimetic collagen is incubated with cells for 1 day, wherein A is a cell viability result when biomimetic collagen prepared in Example 1 is incubated with cells for 1 day, and B is a cell viability result when biomimetic collagen prepared in Comparative Example 1 is incubated with cells for 1 day; Fig.15 Graphs showing cell viability when biomimetic collagen is incubated with cells for 4 days, wherein A is a graph showing cell viability when biomimetic collagen prepared in Example 1 is incubated with cells for 4 days, and wherein B is a graph showing cell viability when biomimetic collagen prepared in Comparative Example 1 is incubated with cells for 4 days; Fig.16 Graphs showing the cell viability results when biomimetic collagen was incubated with cells for 7 days, wherein A is a graph showing the cell viability results when the biomimetic collagen prepared in Example 1 was incubated with cells for 7 days, and wherein B is a graph showing the cell viability results when the biomimetic collagen prepared in Comparative Example 1 was incubated with cells for 7 days; Fig.17 The results of the scratch test of cells treated with bionic collagen in Experimental Example 3 are shown in the first row, where the results of the scratch test of the control group are shown in the first row, and the results of the scratch test of the experimental group are shown in the second row; Fig.18 The results of the scratch test of the cells treated with collagen in Experimental Example 3 are shown in the first row, where the results of the scratch test of the control group are shown in the first row, and the results of the scratch test of the experimental group are shown in the second row; Fig.19 The experimental result diagrams of collagen gene expression in HaCat cells treated with biomimetic collagen in Experimental Example 3, wherein A is the experimental result diagram of the relative expression of Collagen I gene, and wherein B is the experimental result diagram of the relative expression of Collagen III gene; Fig. 20 The experimental result diagrams of collagen gene expression in HSF cells treated with bionic collagen in Experimental Example 3, wherein A is the experimental result diagram of the relative expression of Collagen I gene, and B is the experimental result diagram of the relative expression of Collagen III gene; Fig.21 The experimental result diagrams of collagen gene expression in HaCat cells treated with comparative collagen in Experimental Example 3, wherein A is the experimental result diagram of the relative expression of Collagen I gene, and B is the experimental result diagram of the relative expression of Collagen III gene; Fig. 22 The experimental result diagrams of collagen gene expression in HSF cells treated with comparative collagen in Experimental Example 3, wherein A is the experimental result diagram of the relative expression of Collagen I gene, and B is the experimental result diagram of the relative expression of Collagen III gene; Fig.23 The diagram is a state diagram of collagen in aqueous solution after moist heat sterilization; the diagram of the solution in the left test tube shows the state diagram of comparative collagen in aqueous solution, and the diagram of the solution in the right test tube shows the state diagram of biomimetic collagen prepared in Example 1 in aqueous solution; Fig.24 The diagram is a state diagram of collagen in aqueous solution after PBS treatment; the diagram of the solution in the left test tube shows the state diagram of comparative collagen in aqueous solution, and the diagram of the solution in the right test tube shows the state diagram of biomimetic collagen prepared in Example 1 in aqueous solution; Fig.25The results of the cell scratch test of the biomimetic collagen prepared in Example 1 after wet heat sterilization or filtration sterilization are shown in the figure, wherein the first row of pictures are the results of the control group, the second row of pictures are the results of the biomimetic collagen after wet heat sterilization, and the third row of pictures are the results of the biomimetic collagen after filtration sterilization; Fig.26 The experimental result diagram of collagen gene expression after the bionic collagen prepared in Example 1 was treated with HaCat cells after moist heat sterilization, wherein A is the experimental result diagram of the relative expression of Collagen I gene, and wherein B is the experimental result diagram of the relative expression of Collagen III gene; Fig. 27 These are experimental results of collagen gene expression after HSF cells were treated with moist heat sterilization of the bionic collagen prepared in Example 1, wherein A is an experimental result of the relative expression of Collagen I gene, and B is an experimental result of the relative expression of Collagen III gene. DETAILED DESCRIPTION

[0041] The reagents and raw materials used in the following examples and experimental examples are all commercially available unless otherwise specified.

[0042] Example 1 A biomimetic collagen with good thermal stability and no salting out and high biological activity This embodiment provides a biomimetic collagen with good thermal stability and high biological activity without salting out. It is a protein with a triple helix structure, and its structural formula is as follows: H2N-(POG) n -COOH P, O and G in the formula are composed of the following amino acid residues: P: Proline, O: Hydroxyproline, G: glycine; n≧4. Since the triple-helical biomimetic collagen in this embodiment is a mixture of molecules with different degrees of polymerization, there are many possible values ​​of n, and its range can be estimated based on the characterization of the molecular weight.

[0043] The preparation method of the triple helix collagen is as follows: 1. Condensation auxiliary solution (1) Dilute the commercially available PBS solution (100 mM Na2HPO4·12H2O, 18 mM KH2PO4, 27 mM KCl, 1370 mM NaCl, pH = 7.4) ten-fold to obtain phosphate buffered saline, hereinafter referred to as PB solution.

[0044] (2) Weigh 2 g of condensation aid 1-hydroxybenzotriazole (HOBt), add 500 mL of PB solution, and stir at 4 °C for 2 h to form a uniform suspension.

[0045] 2. Preparation of tripeptide solution Add 15 g of tripeptide POG (manufactured by Uniqs Inc.) to the suspension, raise the temperature to 25°C, and stir for 2 hours; then lower the temperature to 4°C and keep warm for 1 hour to obtain the product.

[0046] 3. Preparation of dehydration condensation agent solution Weigh 30 g of dehydration condensation agent 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide (EDC) and add 500 mL of PB solution to obtain the product.

[0047] 4. Polycondensation reaction At 4°C, add the prepared dehydration condensation agent solution to the prepared tripeptide solution, and all the solution will drip in 1 hour. Raise the temperature to 40°C and maintain for 3 hours; cool down to 25 degrees and maintain for 36 hours. Add 1000mL of deionized water to the reaction solution, cool down to 4 degrees and maintain for 1 hour. Load the reaction solution into a homogenizer, homogenize at 2000 speed for 1 hour, and stop for 1 hour. Repeat this 3 cycles. During this period, the temperature control device is used to ensure that the system does not exceed 10°C.

[0048] 5. Separation and purification 8L of deionized water was added to the reaction solution, and membrane filtration was performed using a hollow fiber membrane with a molecular weight of 5W, and the membrane filtration cycle was repeated 3 times. During the membrane filtration process, the volume of the solution gradually decreased, and deionized water was added to 10L each time it decreased to 2L. Finally, an aqueous solution containing the target product biomimetic collagen was obtained.

[0049] Example 2 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 was followed, except that the amount of the condensation aid 1-hydroxybenzotriazole (HOBt) was 2 g, the amount of the tripeptide POG was 10 g, and the amount of the dehydration condensation agent 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide (EDC) was 34 g. The biomimetic collagen prepared in this example had similar stability to that of Example 1.

[0050] Example 3 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 was followed, except that the amount of the condensation aid 1-hydroxybenzotriazole (HOBt) was 1 g, the amount of the tripeptide POG was 20 g, and the amount of the dehydration condensation agent 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide (EDC) was 30 g. The biomimetic collagen prepared in this example had similar stability to that of Example 1.

[0051] Example 4 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 is followed, except that in step 4, "raising the temperature to 40° C." is changed to "raising the temperature to 50° C.". The biomimetic collagen prepared in this example has similar stability to that of Example 1.

[0052] Example 5 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 is followed, except that in step 4, "raising the temperature to 40° C." is changed to "raising the temperature to 30° C.". The biomimetic collagen prepared in this example has similar stability to that of Example 1.

[0053] Example 6 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 is followed, except that in step 4, "cooling to 25 degrees and maintaining for 36 hours" is changed to "cooling to 25 degrees and maintaining for 70 hours". The biomimetic collagen prepared in this example has similar stability to that of Example 1.

[0054] Example 7 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 is followed, except that in step 4, "cooling to 25 degrees and maintaining for 36 hours" is changed to "cooling to 10 degrees and maintaining for 36 hours". The biomimetic collagen prepared in this example has similar stability to that of Example 1.

[0055] Example 8 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 is used, except that in step 4, the temperature when the prepared dehydration condensation agent solution is added dropwise to the prepared tripeptide solution is changed to 10° C. The biomimetic collagen prepared in this example has similar stability to that of Example 1.

[0056] Example 9 A biomimetic collagen with good thermal stability and no salt precipitation and high biological activity The method of Example 1 is followed, except that in step 4, the speed of the homogenizer is changed to 4500. The biomimetic collagen prepared in this example has similar stability to that of Example 1.

[0057] Example 10 A biomimetic collagen with good thermal stability and no salting out and high biological activity The method of Example 1 is followed, except that the dehydration condensation agent is N,N'-diisopropylcarbodiimide. The biomimetic collagen prepared in this example has similar stability to that of Example 1.

[0058] Example 11 Moisturizing lotion for anti-wrinkle and firming This embodiment provides a moisturizing lotion, the composition of which includes: Triple helical biomimetic collagen prepared in Example 1 0.05 wt , Glycerol 10wt%, Polyethylene glycol 15wt%, Hyaluronic acid 5wt%, The rest is water.

[0059] Example 12 Moisturizing lotion for anti-wrinkle and firming This embodiment provides a moisturizing lotion, the composition of which includes: Triple helical biomimetic collagen 0.5 wt% prepared in Example 1 , Glycerol 10wt%, Polyethylene glycol 15wt%, Hyaluronic acid 5wt%, The rest is water.

[0060] Example 13 Moisturizing lotion for anti-wrinkle and firming This embodiment provides a moisturizing lotion, the composition of which includes: Triple helical biomimetic collagen 5 wt% prepared in Example 1 , Glycerol 10wt%, Polyethylene glycol 15wt%, Hyaluronic acid 5wt%, The rest is water.

[0061] Comparative Example 1 This comparative example provides the control sample used in the experiment.

[0062] The control sample is collagen prepared according to the scheme of Example 1 described in the patent with publication number CN 117903293 A. The control sample has the same sequence and similar structure as the biomimetic collagen prepared in Example 1. The difference lies in that no multi-stage temperature control is performed during the preparation process, the material feed ratio is different, and the membrane passing conditions after the reaction are different.

[0063] The technical solution of the present invention is further illustrated by experiments below.

[0064] Experimental Example 1 Characterization of Products 1. Experimental Methods This experimental example characterizes the biomimetic collagen prepared in Example 1, including: 1. Circular dichroism spectroscopy characterization The collagen of the comparative example and the biomimetic collagen of Example 1 before and after moist heat sterilization (121°C, 15 min) were prepared into aqueous solutions with a concentration of 0.2 mg / ml, and heat-treated at 20°C, 40°C, 60°C, 80°C, and 95°C for 1 hour. 5 ml of each solution was taken and analyzed on a circular dichroism spectrometer to confirm whether each sample had a positive peak unique to triple helical collagen.

[0065] 2. Molecular weight characterization The SEC-MALS technique was used for the determination, using a Wyatt multi-angle light scattering detector DAWN HELEOS II and a Shodex SB-806M column.

[0066] 3. Infrared characterization The infrared spectrometer Thermo Fisher Scientific Nicolet iS20 was used to perform infrared spectroscopy detection on the biomimetic collagen of Example 1.

[0067] 2. Experimental Results 1. Circular dichroism spectroscopy characterization The circular dichroism spectroscopy characterization results of the biomimetic collagen prepared in Example 1 before and after moist heat sterilization are as follows: Figure 1 As shown. As can be seen from the figure, before moist heat sterilization, the biomimetic collagen sample can detect the positive peak in the range of 220-230nm, indicating that the biomimetic collagen prepared in Example 1 has a triple helix structure. After treatment at 20°C, 40°C, 60°C, and 80°C, the peak height of the positive peak in the range of 220-230nm has almost no change, and after heat treatment at 95°C, the peak height has slightly decreased, indicating that the biomimetic collagen sample has good thermal stability and its triple helix structure is not destroyed by high temperature. After moist heat sterilization, the positive peak in the range of 220-230nm still exists in the biomimetic collagen sample, and after treatment at 20°C, 40°C, and 60°C, the peak height of the positive peak in the range of 220-230nm has almost no change, and after heat treatment at 80°C, the peak height has slightly decreased, indicating that the biomimetic collagen sample still has good thermal stability after moist heat sterilization, and its triple helix structure is not destroyed by high temperature.

[0068] The characterization results of the collagen in the comparative example after moist heat sterilization are as follows Figure 2As shown in the figure, it can be seen that for the comparative collagen sample, positive peaks can also be detected in the range of 220-230nm, but the peak height is greatly reduced. As the heat treatment temperature increases, the peak height of the positive peak in the range of 220-230nm is further reduced, especially when the heat treatment exceeds 40°C, which has a great impact on the peak height. This shows that the triple helix structure of the comparative collagen sample is destroyed after wet heat sterilization, and when it is heat treated above 40°C, its triple helix structure is further destroyed, and the thermal stability is poor.

[0069] The experimental results show that the biomimetic collagen prepared in the embodiment of the present invention has a triple helix structure, and the triple helix structure has good thermal stability. Even after moist heat sterilization and heat treatment at 95°C, its triple helix structure can still be stably maintained. In addition, for biomimetic collagen, whether it can form a stable triple helix structure is related to its preparation method. The present invention uses multi-stage temperature control, adjustment of material feed ratio, sequence and interval, and membrane conditions after reaction, etc., to prepare a triple helix biomimetic collagen that can meet the requirements of forming a highly thermally stable triple helix structure.

[0070] 2. Molecular weight characterization like Figure 3 As shown in the results, the weight average molecular weight Mw of the biomimetic collagen prepared in Example 1 is 6.646×10 6 (g / mol).

[0071] 3. Infrared characterization results The results are as follows Figure 4 and as shown in Table 1. The results show that 3327.16 cm -1 is the stretching vibration absorption peak of -OH and -NH-; 2949.38 cm -1 is the stretching vibration absorption peak of saturated -CH; 1626.35 cm -1 is the stretching vibration absorption peak of C=O on amide; 1551.99 cm -1 The bending vibration absorption peak of NH on amide; 1446.35 cm -1 is the bending vibration absorption peak of saturated -CH2; 1402.73 cm -1 is the bending vibration absorption peak of -CH; 1334.43 cm -1 is the stretching vibration absorption peak of CN; 1233.78 cm -1 is the stretching vibration absorption peak of CO. The results show that the present invention successfully synthesizes the bionic collagen product.

[0072] Table 1 Infrared spectrum data analysis results

[0073] Experimental Example 2 Effect of Preparation Method on Thermal Stability 1. Experimental Methods In this experimental example, the biomimetic collagen prepared in Examples 1-10 and Comparative Example 1 was sterilized by moist heat (121°C, 15 min) and then prepared into an aqueous solution with a concentration of 0.2 mg / ml. The solution was then heat treated at 20°C, 40°C, 60°C, 80°C and 95°C for 1 hour, and 5 ml of each solution was analyzed on a circular dichroism spectrometer to evaluate the thermal stability of the biomimetic collagen of the present invention.

[0074] 2. Experimental Results The circular dichroism spectroscopy characterization results of the biomimetic collagen prepared in Example 1 and Comparative Example 1 after wet heat sterilization are as follows: Figure 1 B. As shown in 2, the circular dichroism spectroscopy characterization results of the biomimetic collagen prepared in Example 2-10 after moist heat sterilization are as follows Figure 5-13 As shown: after moist heat sterilization, the positive peak in the range of 220-230nm still exists, and the peak height has almost no change. The peak height of the bionic collagen prepared in Examples 4-8 and 10 in the range of 220-230nm is higher than that of the bionic collagen prepared in Example 1, indicating that the bionic collagen prepared by the present invention still has a stable triple helix structure after high temperature moist heat sterilization.

[0075] After the biomimetic collagen prepared in Examples 2 and 9 was further heat-treated at 20°C, 40°C, 60°C, 80°C, and 95°C, the peak height of the positive peak in the range of 220-230nm was almost unchanged ( Figure 5 , 12 After the biomimetic collagen prepared in Examples 1, 5, and 7 was further heat-treated at 20°C, 40°C, 60°C, and 80°C, the peak height of the positive peak in the range of 220-230nm remained almost unchanged ( Figure 1 B, 8, 10). After the biomimetic collagen prepared in Examples 3, 4, 6, 8, and 10 was further heat-treated at 20°C, 40°C, and 60°C, the peak height of the positive peak in the range of 220-230nm remained almost unchanged ( Figure 7 , 9 , 11, 13), indicating that the thermal stability of the bionic collagen prepared by the present invention is better than that of the bionic collagen of the comparative example; the composition and ratio of raw materials and temperature control have a certain influence on the thermal stability of the bionic collagen. Compared with the collagen of the comparative example, the triple helical structure of the collagen prepared in Examples 2-10 is not destroyed after wet heat sterilization, and it still has good thermal stability after further heat treatment. Specific comparative data are shown in Table 2.

[0076] Table 2 Circular dichroism spectra of collagen after moist heat sterilization

[0077] Therefore, after wet heat sterilization, the triple helix structure of the biomimetic collagen prepared in the embodiment of the present invention is not destroyed and still has good thermal stability. In particular, the raw material composition and ratio, multi-stage temperature control method, homogenizer speed, etc. in the preparation method have an impact on the thermal stability of the product.

[0078] Experimental Example 3 Effects of biomimetic collagen on cell proliferation, migration and collagen expression This experimental example uses human cells to study the effect of the biomimetic collagen prepared in Example 1 on cell proliferation, migration and collagen expression, so as to evaluate whether the sample to be tested has biological safety, promotes wound healing and has anti-wrinkle and firming effects.

[0079] 1. Experimental Methods 1. Cytotoxicity assay The Cell Counting Kit-8 (CCK8) detection method was used to determine the effects of biomimetic collagen or comparative collagen in the concentration range of 0.1%-10% on the proliferation of human skin fibroblasts (HSF) cells.

[0080] 2. Cell scratch test The scratch test is used to study the cell migration ability. Human immortalized keratinocytes (HaCaT) cells were used. The experimental group cells were treated with 0.5 mg / mL biomimetic collagen or 0.5 mg / mL comparative collagen for 0, 12 h, and 24 h; the control group cells were treated with the same volume of PBS. Then the scratch test was performed separately. The specific steps include the following: (1) Cell culture: First, culture HACAT cells in a culture dish or 96-well plate until they form a monolayer.

[0081] (2) Create a scratch: Use a dedicated scratching tool to draw a straight line on the cell monolayer to form a "wound".

[0082] (3) Washing: Gently wash the cells to remove any cells that may have fallen off, ensuring that there are no cells in the scratched area.

[0083] (4) Culture and observation: Place the cells back into the incubator for continued culture, and observe and record the cell migration and coverage of the scratch area at different time points (0 h, 12 h, and 24 h).

[0084] 3. Determination of collagen expression (1) Main reagents Low-glucose DMEM culture medium (Solebol), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), cell lysis buffer (Novozyme).

[0085] (2) Main equipment CO2 incubator (Thermo, 160i), biological safety cabinet (Sujing Antai, BSC-1604ⅡA2), inverted microscope (Leica, DMi8), QPCR instrument (Roche), floor-standing half-body ultraviolet therapy device (Sigma High, SS-03AB).

[0086] (3) Test method The specific settings of the test group are as follows: A blank control group (BC), a negative control group (NC), a positive control group (PC), and a collagen group (0.05 , 0.5 , 5 The blank control group was not given any medication or radiation treatment; the negative control group was only given 9 J / cm 2 UVA irradiation; positive control group was irradiated with 9 J / cm 2 UVA irradiation, the drug is 25 μg / mL vitamin C and 7 μg / mL vitamin E; the collagen components are the comparative collagen group and the biomimetic collagen group prepared in Example 1. After administration, the collagen group was subjected to 9 J / cm 2 UVA irradiation, the drug in the comparative collagen group was 0.05 , 0.5 , 5 The concentration of the comparative collagen; the drug of the biomimetic collagen group was 0.05 , 0.5 , 5 concentration of biomimetic collagen (prepared according to the method of Example 1).

[0087] The specific steps are as follows: 1) Cell seeding: HaCaT cells or HSF cells were plated at 8×10 4 Cells were inoculated into 24-well plates at 100 μl / well and incubated overnight in an incubator (37°C, 5% CO2) using DMEM as the culture medium.

[0088] 2) Administration: Administration was performed when the cell plating rate in the 24-well plate reached 40%-60%. 1 mL of cell culture medium (DMEM medium) was added to each well of the blank control group and the negative control group; 1 mL of culture medium (DMEM medium) containing 25 μg / mL vitamin C and 7 μg / mL vitamin E was added to each well of the positive control group; 1 mL of culture medium (DMEM medium) containing the corresponding concentration of sample was added to each well of the comparative collagen group and the collagen group.

[0089] 3) Radiation: 24 hours after the administration, the total dose received by the negative control group, positive control group, comparative collagen group and biomimetic collagen group was 9 J / cm 2 At the same time, the blank control group was placed in the same environment (UVA radiation dose of 0 J / cm 2 ).

[0090] 4) Collect cells: After incubation for 24 hours, add 0.5 mL of lysis buffer to each well and leave at room temperature for 5 minutes to allow for complete lysis. Transfer the cells to a 1.5 mL RNase-free Eppendorf tube and extract RNA according to the operating procedures.

[0091] 5) Reverse transcription: Synthesize cDNA according to the instructions of the RNA reverse transcription kit.

[0092] 6) Perform qRT-PCR detection on CollagenⅠ and CollagenⅢ.

[0093] 3. Experimental Results 1. Toxicity of biomimetic collagen to cells The results are as follows Figure 14-16 The results showed that biomimetic collagen or comparative collagen had no effect on the proliferation behavior of HSF cells, indicating that raw collagen or comparative collagen had no toxicity to HSF cells, and both products had good biosafety.

[0094] 2. Effect of biomimetic collagen on cell migration The scratch test results are as follows Fig.17 , 18 As shown in the figure: Compared with the control group, the proliferation of cells treated with bionic collagen at the scratch site increased significantly at 12 hours, and the scratch was almost healed at 24 hours, indicating that bionic collagen promoted cell migration and was beneficial to wound healing. However, the proliferation behavior of cells treated with comparative collagen at the scratch site was less than that of the control group, and the width of the scratch was greater than that of the control group at 24 hours, proving that comparative collagen inhibited cell migration and was not conducive to wound healing.

[0095] 3. Effect of biomimetic collagen on collagen expression The qPCR results of HaCaT cells are shown in Fig.19 As shown: For the CollagenⅠ gene, the content of CollagenⅠ gene in cells treated with biomimetic collagen increased significantly, especially when the drug concentration was 0.05 and 0.5 When the expression of CollagenⅠ gene in cells was increased, the effect of increasing the expression of CollagenⅠ gene in cells was the best, and the increasing effect was significantly better than that in the blank control group. For CollagenⅢ gene, the content of CollagenⅢ gene in cells treated with bionic collagen was equivalent to or slightly lower than that in the negative control group. The results show that the bionic collagen prepared in Example 1 of the present invention promotes the expression of CollagenⅠ gene in HaCaT cells.

[0096] The qPCR results of HSF cells are shown in Fig. 20 As shown: for the Collagen III gene, the content of the Collagen III gene in the cells treated with biomimetic collagen was significantly increased compared with the negative control group, and increased with the increase of the biomimetic collagen concentration. The results show that the biomimetic collagen prepared in Example 1 of the present invention has a good promoting effect on the expression of the Collagen III gene in HSF cells.

[0097] The results of the detection of the content of CollagenⅠ and CollagenⅢ genes in the cells treated with collagen in the comparative example are as follows Fig.21 , 22 As shown: compared with the negative control group, the expression of CollagenⅠ in HaCaT cells treated with comparative collagen increased slightly, but was significantly lower than that in the bionic collagen group; the expression of CollagenⅢ in HSF cells treated with comparative collagen was also significantly lower than that in the bionic collagen group. The results show that the promoting effect of comparative collagen on CollagenⅠ and CollagenⅢ genes in cells is significantly weaker than that of the collagen prepared in Example 1 of the present invention.

[0098] The above experimental results show that the biomimetic collagen of the present invention has good biological safety and good ability to promote wound healing. In terms of collagen expression, the biomimetic collagen of the present invention promotes the expression of Collagen I gene in HaCaT cells, especially at a dosage of 0.05 and 0.5 When the amount of collagen III gene in HSF cells was increased, the expression of collagen III gene in HSF cells was promoted. This indicates that bionic collagen has the effect of promoting the expression of collagen in cells. Therefore, the collagen of the present invention has the effect of anti-wrinkle and firming. Moreover, judging from the different effects of the comparative collagen and the bionic collagen prepared in Example 1 of the present invention on cell migration and promoting the expression of collagen, the preparation method has a significant effect on the effect of bionic collagen.

[0099] Experimental Example 4 Effects of sterilization and salt concentration on the properties of biomimetic collagen The comparative example collagen and the biomimetic collagen prepared in Example 1 of the present invention were sterilized with moist heat (121° C., 15 min) or treated with PBS, and the properties of the products were observed.

[0100] The specific steps of PBS treatment are: taking comparative example collagen and biomimetic collagen prepared in Example 1 of the present invention, dissolving them in water to prepare 0.5% aqueous solution; and then adding one tenth volume of 10× PBS solution thereto.

[0101] The properties of the product after moist heat sterilization are as follows Fig.23 As shown: the biomimetic collagen prepared in Example 1 is clear after sterilization and has no visual changes; while the comparative example collagen becomes a suspension after wet heat sterilization, with a large amount of white flocs appearing, and cannot be restored at room temperature.

[0102] The properties of the products before and after PBS treatment are as follows Fig.24 As shown: the biomimetic collagen prepared in Example 1 was clear after being treated with PBS, and no change was observed visually; while the comparative example collagen had white flocculent precipitation after adding PBS, and it could not be restored.

[0103] This experimental example also prepares biomimetic collagen according to the method of comparative example 1, the difference is that a series of buffer solutions with different proportions are used, and the products all have salting-out problems; this experimental example also prepares biomimetic collagen according to the method of embodiment 1, the difference is that the reaction temperature is 20 degrees throughout the process, and precipitation will occur after wet heat sterilization of the product.

[0104] The results show that the bionic collagen prepared in the embodiment of the present invention has no salting out, good thermal stability, and will not be denatured by wet heat sterilization operation.

[0105] The above results show that the bionic collagen prepared in Example 1 has more stable properties and still maintains stable and uniform properties after being treated with salt solution and high temperature.

[0106] Experimental Example 5 Biological activity of collagen after sterilization This experimental example carried out the cell scratch test and collagen expression test according to the method of Experimental Example 3, except that the collagen used was sterilized by moist heat (121°C) or filtration.

[0107] The results of cell scratch assay are as follows Fig.25 As shown in Figure 2, collagen still has good cell migration activity after wet heat sterilization or filtration sterilization and can be used to promote wound healing.

[0108] The qPCR results of cells are as follows Fig.26 , 27As shown, the bionic collagen prepared in Example 1 still has a significant effect of promoting the expression of Collagen I gene after wet heat sterilization.

[0109] The above results show that the biomimetic collagen prepared in Example 1 of the present invention still has a good effect of promoting cell proliferation, migration and collagen expression after wet heat sterilization, and has good biological activity.

[0110] It can be seen from the above embodiments and experimental examples that the present invention provides a bionic collagen with high thermal stability, and the bionic collagen structural formula is H2N-(POG) n -COOH; wherein POG is a peptide chain composed of proline, hydroxyproline and glycine residues. The present invention uses multi-stage temperature control and improved material ratios to ensure that the prepared biomimetic collagen still has a stable triple helix structure after wet heat sterilization (121°C); after being treated with a salt solution, there is no salting out phenomenon. The biomimetic collagen has good biosafety, promotes wound healing, and promotes cellular collagen expression. The biomimetic collagen maintains a triple helix structure within a temperature range of 20°C-95°C, has good thermal stability, and can be better applied in the fields of cosmetics, pharmaceuticals, medical devices, biomaterials, etc., with good application prospects. Especially in the fields of tissue engineering and regenerative medicine such as artificial skin, vascular stents, cartilage repair matrices and wound dressings, surgical sutures and implants such as targeted drug delivery carriers, surgical suture materials or surface coatings of implanted devices, cosmetics and high-end skin care products such as anti-aging and skin barrier repair, bio-ink materials, organ model printing materials, antibacterial, anti-fouling and other engineered functional materials, functional foods such as anti-aging beverages, plant-based foods, functional food additives, high-end textiles and consumer products such as medical textiles, skin care clothing, degradable plastics and packaging materials, and experimental tools for scientific research such as collagen substrates, etc., there are broad prospects.

Claims

1. A bionic collagen with high thermal stability, characterized in that: Its structural formula is shown in Formula I: Formula I: H2N-(P-O-G) n -COOH; Among them, P is a proline residue, O is a hydroxyproline residue, G is a glycine residue, and n ≧ 4; The bionic collagen is prepared according to the following steps: A condensation reaction is carried out using a condensation aid, a tripeptide and a dehydration condensation agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to heat the mixture to 30-50° C. and then cool the mixture to 10-25° C.; The crude product solution is homogenized and separated to obtain; The weight ratio of the condensation aid, the tripeptide and the dehydration condensation agent is 0.7-2.3: 7.2-24.6: 27.4-36.

2.

2. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: The weight ratio of the condensation auxiliary agent, the tripeptide and the dehydration condensation agent is 1-2:10-20:30-34.

3. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: The heating temperature is raised to 30°C; And / or, the further cooling temperature is further cooled to 10°C.

4. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: The homogenization process is carried out in a homogenizer, and the rotation speed of the homogenizer is greater than or equal to 2000 and less than 5000.

5. The biomimetic collagen with high thermal stability according to claim 4, characterized in that: The speed of the homogenizer is 2000-4500.

6. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: After cooling to 10-25° C., the mixture is kept warm for 34-72 hours.

7. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide, 1,3-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

8. The biomimetic collagen with high thermal stability according to claim 1, characterized in that: The steps include the following: Step 1, preparing a condensation auxiliary agent solution; Step 2, adding a tripeptide having a structural formula of H2N-POG-COOH to the condensation auxiliary agent solution to obtain a tripeptide solution; Step 3, preparing a dehydration condensation agent solution; Step 4, the tripeptide solution is mixed with the dehydration condensation agent solution to carry out a condensation reaction to obtain a crude product solution; Step 5, the crude product solution is homogenized; Step 6, separation and purification, to obtain.

9. The biomimetic collagen with high thermal stability according to claim 8, characterized in that: In step 1, after the condensation auxiliary agent solution is prepared, it is stirred at 0-10° C. for 1-4 hours; And / or, in step 2, the tripeptide solution is stirred at 15-30° C. for 1-4 h, then cooled to 0-10° C. and maintained for 1-4 h; And / or, the dehydration condensation agent solution mixed in step 4 is added dropwise to the tripeptide solution within 0.2-1.6 hours; and / or, in step 4, maintaining at 30-50° C. for 1.5-6 h; And / or, the mixing temperature in step 4 is 0-10°C; And / or, the specific process of the homogenization treatment in step 5 is to homogenize at ≤10°C for 0.25-1.2h, and stop for 0.25-1.2h; and repeat this for 2-3 cycles; And / or, the specific steps of separation and purification in step 6 are: adding water 2.5-6 times the volume of the reaction solution, using a hollow fiber membrane with a molecular weight of 3-5W for membrane filtration; and repeating the membrane filtration for 2-3 times.

10. The biomimetic collagen with high thermal stability according to claim 8, characterized in that: In step 1, the solvent of the condensation auxiliary agent solution is phosphate buffer; in step 3, the solvent of the dehydration condensation agent solution is phosphate buffer; the phosphate buffer is prepared using the following raw materials in molar parts: Potassium chloride 3.2-4.3mM, Sodium hydrogen phosphate 8.8-13.1mM, Potassium dihydrogen phosphate 1.2-2.5mM, Sodium chloride 124-151mM; The pH of the phosphate buffer is 6.9-7.6; And / or, the condensation auxiliary agent is selected from at least one of 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole or N-hydroxysuccinimide.

11. The method for preparing the biomimetic collagen with high thermal stability according to any one of claims 1 to 10, characterized in that: The steps include: A condensation reaction is carried out using a condensation aid, a tripeptide and a dehydration condensation agent to obtain a crude product solution; the reaction procedure of the condensation reaction is to heat the mixture to 30-50° C. and then cool the mixture to 10-25° C.; The crude product solution is homogenized and separated to obtain; The weight ratio of the condensation aid, the tripeptide and the dehydration condensation agent is 0.7-2.3: 7.2-24.6: 27.4-36.

2.

12. Use of the biomimetic collagen with high thermal stability according to any one of claims 1 to 10 in the preparation of cosmetics, medicines or tissue engineering materials.

13. Use of the biomimetic collagen with high thermal stability according to claim 12 in the preparation of cosmetics, medicines or tissue engineering materials, characterized in that: The cosmetics are functional skin care products; the tissue engineering materials include tissue engineering materials and bio-based packaging materials used for drug carriers, cosmetic surgery or medical devices; the medical devices include dressings, filling materials or engineering scaffolds for skin, bones, ligaments, peripheral nerves and blood vessels.

14. Use of the biomimetic collagen with high thermal stability according to claim 13 in the preparation of cosmetics, medicines or tissue engineering materials, characterized in that: The functional skin care product is a facial mask, an essence or a facial cleanser.

15. A cosmetic or medicine, characterized in that: The invention is prepared by using the bionic collagen with high thermal stability as claimed in any one of claims 1 to 10 as an active ingredient and adding cosmetically or pharmaceutically acceptable auxiliary materials.

16. A tissue engineering material, characterized in that: The invention is prepared by using the bionic collagen with high thermal stability as claimed in any one of claims 1 to 10 as an active ingredient and adding medically acceptable auxiliary materials.

17. A medical device, characterized in that: The invention is prepared by using the bionic collagen with high thermal stability as claimed in any one of claims 1 to 10 as an active ingredient and adding medically acceptable auxiliary materials.

18. The cosmetic or medicine according to claim 15, the tissue engineering material according to claim 16, or the medical device according to claim 17, characterized in that: The concentration of the biomimetic collagen with high thermal stability is 0.047 wt -0.52 wt .

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