Thermally stabile triple-helix collagen, and preparation method therefor and use thereof

Abstract

Provided are thermally stabile triple-helix collagen, and a preparation method therefor and a use thereof, relating to the technical field of medical and cosmetic raw materials. The structural formula of the triple-helix collagen is as shown in formula I: formula I: H2N-(G-P-О)n-COOH, wherein G-P-O is a peptide chain composed of three different amino acid residues, G is a glycine residue, P is a proline residue, and O is a hydroxyproline residue. The weight average molecular weight distribution thereof is 5.0×103 to 1.5×107. The triple-helix collagen has a triple helix structure, good thermal stability, better wrinkle-resisting, tightening, skin repairing, freckle-removing, and whitening effects, and can be better applied to the fields of cosmetics, medical instruments, etc.

Description

A triple-helix collagen with thermal stability and its preparation method and use Technical Field

[0001] The present invention belongs to the technical field of medical and cosmetic raw materials, and particularly relates to a collagen with thermal stability, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen is the most abundant functional protein in the human body, comprising 70% of human skin. Collagen has a wide range of applications, including medical devices, beauty and skincare, and functional foods.

[0003] In the beauty industry, collagen is widely used in skincare and cosmetics to improve skin moisture retention, elasticity, and radiance. Many functional skincare products, facial masks, serums, and cleansers contain collagen. Collagen replenishes nutrients needed by the skin, improves the living environment of skin cells, and promotes skin tissue metabolism, thereby delaying aging and nourishing the skin. Most collagen skincare products currently on the market are manufactured from high-content collagen raw materials, which are then reduced to smaller molecules through technological means for easier skin absorption. Collagen also has a wide range of applications in the medical plastic surgery industry and medical materials. For example, collagen is used as a key biofiller in facial augmentation, anti-aging plastic surgery, and skin repair. It can also be used clinically to treat pathological changes such as burns and trauma, improving skin condition. In tissue engineering, collagen, as a biodegradable material, can be used to create various tissue engineering scaffolds to support cell growth and tissue repair. Furthermore, due to its excellent biocompatibility and biodegradability, it can also be used to manufacture medical devices such as medical sutures and skin grafts. In the field of bionics, it can be used as a bone tissue engineering scaffold to treat osteoporosis, bone defects and other diseases, and it also has great application prospects as an artificial blood vessel and flexible material.

[0004] The current mainstream collagen extraction and preparation methods include animal-derived extraction, genetic engineering and synthetic collagen. The extraction of animal-derived collagen is mainly industrialized through acid and enzyme methods, which have relatively lower costs. However, there are problems such as animal-derived disease infection, allogeneic collagen that may cause immune rejection or allergic reactions, and production capacity limitations.

[0005] The production of recombinant collagen using genetic engineering technology is a hot topic in the industry. Recombinant collagen is produced by genetic engineering technology. The human collagen gene is subjected to specific sequence design, enzyme cleavage and splicing, and connected to a vector, and then transferred into engineered cells for expression through fermentation to produce collagen. The product obtained by this method has the advantages of good safety, strong processability, and stable quality, but the technical difficulties lie in how to achieve high-density expression of bacteria and yeast strains, ensure biological activity, and separate and purify the recombinant protein. The biggest difficulty is the inability to form a stable triple helical structure. There has been no major breakthrough in the preparation of collagen-like proteins by chemical synthesis. First of all, although the synthesis of polypeptides that mimic the amino acid sequence of human collagen can be achieved in theory, how to correctly fold these protein molecules so that they form an active triple helical structure is a key technical difficulty. In addition, how to prepare collagen with an excellent thermal stability triple helical structure is also one of the major challenges currently faced.

[0006] In the prior art, collagen-like polypeptide molecules can be synthesized using a polypeptide synthesizer. A polypeptide synthesizer is an automated laboratory device specifically designed for synthesizing polypeptide chains in vitro. It is based on solid phase peptide synthesis (SPPS) technology, and by precisely controlling the reaction conditions and steps, it can efficiently construct polypeptide chains consisting of several amino acids to hundreds of amino acids. However, experimental studies have shown that polypeptide molecules prepared using a polypeptide synthesizer are difficult to form a triple helix structure, and even if a triple helix structure is formed, it has the problem of insufficient thermal stability. When collagen is used to make cosmetics or medical devices, there will be a certain need to withstand ambient temperature or undergo high-temperature sterilization. The unstable triple helix structure is difficult to maintain at higher temperatures, which creates certain difficulties for the application of the above-mentioned polypeptides in cosmetics or medical devices.

[0007] Therefore, developing a collagen substitute with better thermal stability is an urgent problem to be solved in this field.

[0008] Summary of the Invention

[0009] In view of the problems of the prior art, the present invention provides a triple-helix collagen with thermal stability, a preparation method and use thereof.

[0010] A triple-helical collagen with thermal stability, the structural formula of which is shown in Formula I:

[0011] Formula I: H2N-(GP-О) n -COOH;

[0012] GP-О is a peptide chain composed of three different amino acid residues; G is a glycine residue, P is a proline residue, and О is a hydroxyproline residue; the weight average molecular weight distribution of the triple helical collagen is 5.0×10 3 ~1.5×10 7 .

[0013] Preferably, the value of n satisfies 5.26×10 4 ≧n≧17.

[0014] Preferably, the triple helix collagen is obtained by condensing a tripeptide having the structural formula H2N-GP-О-COOH as a raw material through a liquid phase reaction.

[0015] The present invention also provides a method for preparing the aforementioned triple-helical collagen with thermal stability, comprising the following steps:

[0016] Step 1: a tripeptide having a structural formula of H2N-GP-О-COOH and a condensation aid are prepared into a tripeptide solution using a phosphate buffer as a solvent; a dehydration condensation agent is prepared into a dehydration condensation agent solution using a phosphate buffer as a solvent;

[0017] The phosphate buffer solution is prepared using the following raw materials in parts by weight:

[0018] 0.18-0.22 parts of potassium chloride,

[0019] 1.0-1.2 parts of sodium dihydrogen phosphate,

[0020] 0.18-0.22 parts of potassium dihydrogen phosphate,

[0021] 995-1005 parts of water;

[0022] The tripeptide solution is prepared using the following raw materials:

[0023] 495-505 parts by volume of phosphate buffer,

[0024] 49.5-50.5 parts by weight of tripeptide,

[0025] 4.9-5.1 parts by weight of condensation aid;

[0026] The dehydration condensation agent solution is prepared using the following raw materials:

[0027] 495-505 parts by volume of phosphate buffer,

[0028] 156-160 parts by weight of a dehydration condensation agent;

[0029] Step 2: adding the dehydration condensation agent solution to the tripeptide solution to carry out a polycondensation reaction; the weight ratio of the tripeptide, the condensation aid, and the dehydration condensation agent in the polycondensation reaction is 49.5-50.5:4.9-5.1:156-160; the temperature of the polycondensation reaction is less than or equal to 20° C., and the time of the polycondensation reaction is 20-26 hours;

[0030] Step 3, separation and purification to obtain.

[0031] Preferably, in step 1, the condensation aid is selected from at least one of 1-hydroxybenzotriazole and 1-hydroxy-7-azabenzotriazole;

[0032] The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide and 1,3-dicyclohexylcarbodiimide;

[0033] The phosphate buffer solution is prepared using the following raw materials in parts by weight:

[0034] 0.2 parts of potassium chloride,

[0035] 1.15 parts of sodium dihydrogen phosphate,

[0036] 0.2 parts of potassium dihydrogen phosphate,

[0037] 1000 parts water;

[0038] The tripeptide solution is prepared using the following raw materials:

[0039] 500 parts by volume of phosphate buffer,

[0040] 50 parts by weight of tripeptide,

[0041] 5 parts by weight of condensation aid;

[0042] The dehydration condensation agent solution is prepared using the following raw materials:

[0043] 500 parts by volume of phosphate buffer,

[0044] 158 parts by weight of dehydration condensation agent.

[0045] Preferably, in step 2, the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent in the dehydration condensation agent solution and the tripeptide solution is 50:5:158;

[0046] The polycondensation reaction time is 24 hours.

[0047] Preferably, in step 3, the specific steps of separation and purification are: cyclic grinding with a homogenizer, filtering through a 50,000 molecular weight cut-off membrane to obtain a solution containing the triple helical collagen.

[0048] The present invention also provides use of the triple-helix collagen with thermal stability in the preparation of cosmetics, medicines, cosmetic and plastic surgery materials or medical devices.

[0049] Preferably, the cosmetics are functional skin care products;

[0050] The medical device is a filling material or an engineering scaffold for skin, bones, and ligaments.

[0051] Preferably, the functional skin care product is a facial mask, essence or facial cleanser.

[0052] The present invention also provides a cosmetic, which is prepared by using the triple-helix collagen with thermal stability as an active ingredient and adding auxiliary materials.

[0053] Preferably, the concentration of the thermally stable triple-helical collagen is 0.0005 wt%-0.05 wt%.

[0054] Preferably, the concentration of the thermally stable triple-helical collagen is 0.0005 wt %.

[0055] The "triple helical collagen" provided by the present invention refers to a protein having a tripeptide repeating unit and a triple helical structure. It is a mixture of proteins with different degrees of polymerization with a tripeptide repeating unit as the repeating structure. The "triple helical structure" is the basic structure of natural collagen, which is a triple helical structure formed by three polypeptide chains entangled with each other. The method for detecting whether a protein or polymer has a triple helical structure similar to that of natural collagen is to use circular dichroism spectroscopy to detect whether the polymer has a positive peak at 220-230nm. The ratio of "parts by volume" to "parts by weight" is determined as follows:

[0056] 1 part by volume: 1 part by weight = 1 ml: 1 g.

[0057] The present invention provides a new collagen polymer with thermal stability, which is a liquid-phase synthesized polypeptide chain obtained by condensation polymerization with three amino acids as a unit. Existing collagen products will gel at temperatures exceeding 40°C and cannot maintain the triple helix structure. They cannot be used as raw materials in cosmetics (except for cosmetics used in cold storage). In addition, if used as medical devices, they cannot be heated and sterilized (above 60°C) or disinfected (autoclave). The collagen polymer (polypeptide chain) provided by the present invention can maintain the triple helix structure under normal temperature and high temperature (40-60°C) conditions, and its thermal stability is better than that of existing similar synthetic collagen products, making it better for use in cosmetics or medical devices. In addition, the collagen polymer provided by the present invention has the ability to promote the expression of the Elastin gene in fibroblasts, has anti-wrinkle and firming effects, and has the best effect at a concentration of 0.005wt%. In addition, the collagen polymer of the present invention also has the effects of repairing skin and removing spots and whitening.

[0058] Therefore, the collagen polymer of the present invention has good application prospects.

[0059] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0060] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG1 is a circular dichroism spectrum of triple helical collagen after heat treatment at 20-60° C. in Experimental Example 1;

[0062] FIG2 is a circular dichroism spectrum of the commercial polypeptide in Experimental Example 1 after heat treatment at 20-60° C.;

[0063] FIG3 is a graph showing the molecular weight test results of triple helical collagen in Experimental Example 1;

[0064] FIG4 is a graph showing the experimental results of the relative expression level of the Elastin gene in Experimental Example 2. DETAILED DESCRIPTION

[0065] The reagents and raw materials used in the following examples and experimental examples are all commercially available unless otherwise specified.

[0066] Example 1: Thermally stable triple-helical collagen

[0067] This embodiment provides a triple-helical collagen with thermal stability, which is a protein with a triple-helical structure and has the following structural formula:

[0068] H2N-(GP-О) n -COOH

[0069] In the formula, G, P and O are composed of the following amino acid residues:

[0070] P: proline,

[0071] O: Hydroxyproline,

[0072] G: glycine;

[0073] n≧4. Since the heat-stable triple-helical collagen of 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.

[0074] The preparation method of the above-mentioned triple-helix collagen with thermal stability is as follows:

[0075] 1. Preparation of phosphate buffer

[0076] To 1000 ml of high-purity water, add 0.2 g of potassium chloride (KCl), 1.15 g of sodium dihydrogen phosphate (Na2HPO4), and 0.2 g of potassium dihydrogen phosphate (KH2PO4), and stir to prepare phosphate buffer solution (Buffer-PB, hereinafter referred to as PB solution).

[0077] 2. Preparation of tripeptide solution

[0078] 50 g of tripeptide GP-O (manufactured by Uniqs Inc.) and 5 g of 1-hydroxybenzotriazole (HOBt) as a condensation aid were added to 500 ml of the PB solution, and the mixture was stirred to prepare a tripeptide solution (hereinafter referred to as GPO solution).

[0079] 3. Preparation of dehydration condensation agent solution

[0080] 158 g of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide (EDC) was added to 500 ml of the PB solution, and the mixture was stirred to prepare a dehydration condensation agent solution (hereinafter referred to as EDC solution).

[0081] 4. Polycondensation reaction

[0082] The prepared GPO solution was poured into a 2L separatory funnel and cooled to 2°C in a thermostatic bath. EDC solution, which had also been cooled to 2°C, was then gradually added (the volume ratio of GPO solution to EDC solution was 1:1). The reaction temperature was maintained below 20°C for 24 hours of polycondensation. The reaction solution was transferred to a 10L container, circulated and ground using a homogenizer, and filtered through a 50,000 molecular weight cutoff membrane (Asahi Kasei AHP-1013D) using a rotary pump to obtain an aqueous solution containing the target product, a heat-stable triple-helical collagen.

[0083] Example 2 Moisturizing lotion for anti-wrinkle and firming

[0084] This embodiment provides a moisturizing lotion, the composition of which includes:

[0085] 0.0005 wt% of triple helical collagen prepared in Example 1,

[0086] Glycerol 10wt%,

[0087] Polyethylene glycol 15wt%,

[0088] Hyaluronic acid 5wt%,

[0089] The rest is water.

[0090] The technical solution of the present invention is further illustrated by experiments below.

[0091] Characterization of Experimental Example 1 Products

[0092] 1. Experimental Methods

[0093] This experimental example characterizes the triple helical collagen prepared in Example 1, including:

[0094] 1. Circular dichroism spectroscopy characterization

[0095] A commercial peptide was selected as a reference substance, and its molecular formula is H2N-(POG) 10 -COOH (purchased from Anhui Guoping Pharmaceutical Co., Ltd.) The amino acid sequence of this reference substance is similar to that of the sample prepared in Example 1. The main difference between this reference substance and the sample prepared in Example 1 is that this reference substance is prepared using a peptide synthesizer.

[0096] The triple-helical collagen from Example 1 or a commercially available peptide reference substance was prepared as a 0.25 mg / ml aqueous solution and heat-treated at 20°C, 40°C, and 60°C for 1 hour. 5 ml of each solution was sampled and analyzed on a circular dichroism spectrometer to confirm the presence of a positive peak unique to triple-helical collagen.

[0097] 2. Molecular weight characterization

[0098] The measurement was performed by light scattering using a Wyatt multi-angle light scattering detector (DAWN HELEOS II) and a Wyatt differential refractive index detector (Optilab T-rEX) and a Shodex SB-806M column.

[0099] 2. Experimental Results

[0100] 1. Circular dichroism spectroscopy characterization

[0101] The characterization results of triple-helical collagen are shown in Figure 1. As can be seen from the figure, after treatment at 20°C, 40°C, and 60°C, positive peaks in the 220-230nm range were detected in all three groups of samples, and the peak heights remained almost unchanged, indicating that triple-helical collagen can remain stable within the temperature range of 20°C-60°C. This shows that the triple-helical collagen provided by the present invention can form a stable triple helical structure within the temperature range of 20°C-60°C.

[0102] The characterization results of a commercial peptide control are shown in Figure 2. As can be seen from the figure, after treatment at 20°C, 40°C, and 60°C, certain positive peaks can be detected in the 220-230nm range, indicating that the commercial peptide can also form a triple helix structure. However, as the temperature increases, the height of the positive peak in the 220-230nm range decreases, indicating that the triple helix structure formed by this commercial peptide molecule easily disappears under heat treatment conditions, indicating that its triple helix structure has poor thermal stability.

[0103] The experimental results show that for a polypeptide, whether it can form a stable triple helix structure is related to its preparation method and amino acid sequence, and the triple helical collagen prepared by the liquid phase synthesis method provided by the present invention can meet the requirements for forming a stable triple helix structure.

[0104] 2. Molecular weight characterization

[0105] As shown in FIG3 , the results show that the weight average molecular weight Mw of the triple helical collagen prepared in Example 1 is 5.0×10 3 ~1.5×10 7 (g / mol).

[0106] Experimental Example 2: Detection of the Effect of Promoting the Expression of Fibroblast-Anti-Wrinkle and Firming Related Genes

[0107] 1. Test Purpose

[0108] Elastin is the primary component of elastic fibers. Elastic fibers are primarily found in ligaments and blood vessel walls. Elastic fibers coexist with collagen fibers, giving tissues their elasticity and tensile strength. Although elastin only accounts for 2% of the total protein in the dermis, it plays a vital role in skin elasticity. Elastin is the primary cause of sagging, drooping, and fine wrinkles in aging skin. As the skin ages, elastic fibers break down more and more significantly. The elastin gene (often abbreviated as ELN) is the genetic material that encodes tropoelastin, the precursor molecule of elastin. Therefore, characterizing the expression of elastin, such as the elastin gene, can be used to determine whether a product has anti-wrinkle and firming properties.

[0109] 2. Experimental Methods

[0110] 1. Cell sample processing

[0111] Adherent cells: Rinse the cells twice with PBS, then carefully scrape them off with a cell scraper. Centrifuge the culture medium at 3000 rpm for 10 minutes. Discard the supernatant and retain the cell pellet, then transport on dry ice.

[0112] For suspension cells: Centrifuge the culture medium at 3000 rpm for 10 minutes. Discard the supernatant and retain the cell pellet, then transport on dry ice.

[0113] Cell supernatant: Centrifuge the sample at 3000 rpm for 15 minutes at 2-8°C to obtain the supernatant. Use the supernatant immediately for experiments or aliquot and store at -20°C or -80°C. Avoid repeated freezing and thawing.

[0114] 2. The specific settings of the test group are as follows:

[0115] A blank control (BC), a negative control (NC), a positive control (PC) and three sample groups (0.0005%, 0.005%, 0.05%) were set up. Among them, the blank control was not treated with drug and radiation; the negative control was only treated with 9J / cm 2 UVA irradiation; positive control group was irradiated with 9J / cm 2 UVA irradiation, the drug was 100 μg / mL vitamin C and 7 μg / mL vitamin E; three sample groups (0.0005%, 0.005%, 0.05%) were irradiated with 9 J / cm 2 UVA irradiation was performed, and the drugs were triple-helical collagen (prepared according to the method of Example 1) at concentrations of 0.0005 wt%, 0.005 wt%, and 0.05 wt%.

[0116] 6. ELISA experimental steps

[0117] Follow the instructions of the kit. The operation of different indicators is different. Please refer to the instructions of the ordered kit (see attachment) for details. The basic steps are as follows:

[0118] 1) Coating: Dilute the antibody to a protein content of 1-10 μg / ml in carbonate coating buffer. Add 100 μl to each well of a polystyrene ELISA plate and incubate at 4°C overnight. The next day, discard the solution and wash the wells three times with wash buffer, each for 3 minutes. (Commercial kits often come pre-coated with the antibody; this step can be omitted.)

[0119] 2) Blocking: Add 200 μl of blocking solution to each well and incubate at 37°C for 1-2 hours.

[0120] 3) Washing: Carefully remove the sealing film, place the plate in a plate washer, and wash 3-5 times. Alternatively, wash the plate manually by discarding the liquid, adding 300 μl of wash solution to each well, soaking for 1-2 minutes, patting dry on absorbent paper, and repeating 3-5 times. (Commercial kits generally come pre-coated with antibodies, so the first three steps can be omitted.)

[0121] 4) Sample Addition: Add 100 μl of the appropriately diluted sample to the coated wells. (Also, prepare blank wells, wells containing the standard sample at a fractional dilution, and, if possible, negative and positive control wells as quality control points.)

[0122] 5) Incubation: Seal the plate with sealing film and incubate at 37°C for 1-2 hours.

[0123] 6) Washing: Same as step 3.

[0124] 7) Add antibody: Add 100 μl of diluted biotinylated antibody working solution to each well.

[0125] 8) Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 hour.

[0126] 9) Washing: Same as step 3.

[0127] 10) Add enzyme conjugate: Add 100 μl of the diluted enzyme conjugate working solution to each well.

[0128] 11) Incubation: Seal the plate with a sealing film and incubate at 37°C in the dark for 30 min.

[0129] 12) Washing: Same as step 3.

[0130] 13) Add colorimetric substrate: Add 100 μl of TMB substrate solution to each well and incubate at 37°C in the dark for 10-30 min until a clear color gradient appears in the wells containing the serially diluted standard.

[0131] 14) Stop the reaction: Add 100 μl of 2 M sulfuric acid to each reaction well. The color will change from blue to yellow.

[0132] 15) Result determination: Within 10 minutes, measure the OD value of each well on a microplate reader at 450 nm, adjusting the blank control well to zero.

[0133] 3. Experimental Results

[0134] The results of Elastin gene content detection are shown in Figure 4:

[0135] The results showed that compared with the BC group, the NC group fibroblasts received a total dose of 9J / cm 2 After UVA radiation, the expression of Elastin gene decreased significantly (p<0.01), indicating that the radiation stimulation was effective.

[0136] Compared with the NC group, vitamin C and vitamin E in the PC group could significantly increase the expression of Elastin gene (p<0.01), indicating that the positive control test was effective.

[0137] Compared with the NC group, the expression of Elastin gene in fibroblasts was significantly increased at concentrations of 0.0005wt%, 0.005wt%, and 0.05wt% (p<0.05). In particular, the 0.0005wt% concentration showed the best effect in increasing Elastin gene expression in fibroblasts, significantly surpassing that of the PC group.

[0138] In the t-test statistical analysis, significance is indicated by # when comparing the NC group with the BC group, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. Significance is indicated by * when comparing the sample group or PC group with the NC group, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0139] The above experimental results show that the triple-helical collagen of the present invention (especially when administered at a concentration of 0.0005 wt%) can promote the expression of the Elastin gene in fibroblasts and has anti-wrinkle and firming effects.

[0140] Through the above embodiments and experimental examples, it can be seen that the present invention provides a triple-helix collagen with thermal stability, which has the same triple-helix structure as natural collagen, can promote the expression of the Elastin gene in fibroblasts, has anti-wrinkle and firming effects, and can maintain the triple-helix structure in the temperature range of 20°C-60°C. It has good thermal stability and can be better applied in the fields of cosmetics, medical devices, etc., and has good application prospects.

Claims

1. A triple-helical collagen with thermal stability, characterized in that Its structural formula is shown in Formula I: Formula I: H2N-(G-P-О) n -COOH; GP-О is a peptide chain composed of three different amino acid residues; G is a glycine residue, P is a proline residue, and О is a hydroxyproline residue; the weight average molecular weight distribution of the triple helical collagen is 5.0×10 3 ~1.5×10 7 .

2. The heat-stable triple-helical collagen according to claim 1, characterized in that: The value of n satisfies 5.26×10 4 ≧n≧17.

3. The heat-stable triple-helical collagen according to claim 1, characterized in that: The triple helix collagen is obtained by condensing a tripeptide having a structural formula of H2N-GP-О-COOH as a raw material through a liquid phase reaction.

4. The method for preparing the heat-stable triple-helical collagen according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: a tripeptide having a structural formula of H2N-GP-О-COOH and a condensation aid are prepared into a tripeptide solution using a phosphate buffer as a solvent; a dehydration condensation agent is prepared into a dehydration condensation agent solution using a phosphate buffer as a solvent; The phosphate buffer solution is prepared using the following raw materials in parts by weight: 0.18-0.22 parts of potassium chloride, 1.0-1.2 parts of sodium dihydrogen phosphate, 0.18-0.22 parts of potassium dihydrogen phosphate, 995-1005 parts of water; The tripeptide solution is prepared using the following raw materials: 495-505 parts by volume of phosphate buffer, 49.5-50.5 parts by weight of tripeptide, 4.9-5.1 parts by weight of condensation aid; The dehydration condensation agent solution is prepared using the following raw materials: 495-505 parts by volume of phosphate buffer, 156-160 parts by weight of a dehydration condensation agent; Step 2: adding the dehydration condensation agent solution to the tripeptide solution to carry out a polycondensation reaction; the weight ratio of the tripeptide, the condensation aid, and the dehydration condensation agent in the polycondensation reaction is 49.5-50.5:4.9-5.1:156-160; the temperature of the polycondensation reaction is less than or equal to 20° C., and the time of the polycondensation reaction is 20-26 hours; Step 3, separation and purification to obtain.

5. The method for preparing triple-helical collagen with thermal stability according to claim 4, characterized in that: In step 1, the condensation auxiliary agent is selected from at least one of 1-hydroxybenzotriazole and 1-hydroxy-7-azabenzotriazole; The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide and 1,3-dicyclohexylcarbodiimide; The phosphate buffer solution is prepared using the following raw materials in parts by weight: 0.2 parts of potassium chloride, 1.15 parts of sodium dihydrogen phosphate, 0.2 parts of potassium dihydrogen phosphate, 1000 parts water; The tripeptide solution is prepared using the following raw materials: 500 parts by volume of phosphate buffer, 50 parts by weight of tripeptide, 5 parts by weight of condensation aid; The dehydration condensation agent solution is prepared using the following raw materials: 500 parts by volume of phosphate buffer, 158 parts by weight of dehydration condensation agent.

6. The method for preparing triple-helical collagen with thermal stability according to claim 4, characterized in that: In step 2, the weight ratio of the tripeptide, condensation aid and dehydration condensation agent in the dehydration condensation agent solution and the tripeptide solution is 50:5:158; The polycondensation reaction time is 24 hours.

7. The method for preparing triple-helical collagen with thermal stability according to claim 4, characterized in that: In step 3, the specific steps of separation and purification are: cyclic grinding with a homogenizer, filtering through a 50,000 molecular weight cut-off membrane to obtain a solution containing the triple helical collagen.

8. Use of the thermally stable triple-helical collagen according to any one of claims 1 to 3 in the preparation of cosmetics, medicines, cosmetic materials or medical devices.

9. The use according to claim 8, characterized in that: The cosmetics are functional skin care products; The medical device is a filling material or an engineering scaffold for skin, bones, and ligaments.

10. The use according to claim 9, characterized in that: The functional skin care product is a facial mask, essence or facial cleanser.

11. A cosmetic, characterized in that: The invention is prepared by using the triple-helix collagen with heat stability as claimed in any one of claims 1 to 3 as an active ingredient and adding auxiliary materials.

12. The cosmetic according to claim 11, characterized in that: The concentration of the triple-helical collagen with thermal stability is 0.0005 wt%-0.05 wt%.

13. The cosmetic according to claim 12, characterized in that: The concentration of the triple-helical collagen with thermal stability is 0.0005 wt %.

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