Triple-helical collagen, preparation method therefor and use thereof

Abstract

Provided are a triple-helical collagen, a preparation method therefor, and a use thereof. The structural formula of the triple-helical collagen is as shown in Formula I: H2N-(O-G-P)n-COOH, O-G-P being a peptide chain composed of three different amino acid residues, G being a glycine residue, P being a proline residue, and O being a hydroxyproline residue. The weight-average molecular weight distribution of the triple-helical collagen is 3.0×103-8.0×104. The triple-helical collagen has a triple helix structure and good thermal stability, and exhibits better moisturizing, antioxidant, damaged hair repair and hair growth effects, making it more suitable for applications in cosmetics, medical devices and other fields, with promising application prospects.

Description

Triple helix collagen 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 triple-helix collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen, the most abundant functional protein in the human body, plays a crucial role in physiological functions, thanks to its unique triple-helix structure, which weaves into a robust network. This protein is widely distributed throughout various tissues, including skin, bones, and ligaments, and is crucial for maintaining the structural stability and proper function of various tissues and organs. With the rapid advancement of technology, synthetic collagen has been widely incorporated into cosmetic formulations, medical plastic surgery, and the development and application of medical materials, greatly expanding its application in various health and beauty fields.

[0003] In the beauty and skincare industry, collagen, owing to its crucial role in skin health, is widely incorporated into various skincare and makeup products, aiming to enhance core skin properties such as hydration, elasticity, and radiance. Many high-quality skincare products, including creams, masks, serums, and cleansers, incorporate collagen into their formulas to supply the skin with essential nutrients, optimize cellular survival, and stimulate skin metabolism, thereby combating aging and deeply nourishing the skin. Currently, numerous collagen skincare brands are launching collagen ingredients that are highly concentrated and processed into small molecules to ensure more efficient skin penetration. Furthermore, collagen has also shown broad application potential in the medical plastic surgery and medical materials industries. As a leading biofiller, it plays an irreplaceable role in facial contouring, anti-aging treatments, and skin repair and regeneration. Furthermore, collagen can also be effectively used in clinical treatments for burns, scalds, and other injuries, improving the condition of damaged skin. In tissue engineering technology, collagen is used to make various tissue engineering scaffolds due to its biodegradability. These scaffolds provide an ideal support environment for cell proliferation and tissue regeneration. In addition, collagen is also used in the research and development and production of medical sutures, skin transplants and other related medical devices due to its excellent biocompatibility and biodegradability. Furthermore, collagen has also made remarkable achievements in bionics and regenerative medicine. For example, in the field of bone tissue engineering, it can be used as a scaffold material to assist in the treatment of diseases such as osteoporosis and bone defects, and has even been extended to the development of artificial blood vessels and flexible implant materials, indicating that collagen has unlimited potential and broad application prospects in future biomedical materials and tissue regeneration science.

[0004] Currently, synthetic collagen comes from two main sources: animal-derived collagen, obtained through specific extraction processes from animal tissues such as cattle, pigs, chickens, and fish; and recombinant collagen, produced using modern biotechnology methods such as fermentation, purification, and genetic engineering. However, the preparation and application of both types of artificial collagen face technical challenges. First, extracting collagen from animal tissue inevitably carries potential immunological risks, such as the potential for transmission of zoonotic infectious diseases like mad cow disease, posing a threat to human health. Second, amino acid sequence differences between animal-derived collagen and human collagen may result in unsatisfactory bioactivity, stability, and safety, preventing them from perfectly mimicking the functional properties of collagen in the human body. Furthermore, while recombinant collagen theoretically ensures an amino acid sequence closer to that of human collagen, its production process is complex and costly, and in practice, it may not guarantee the formation of a complete triple helical structure in every batch, thus affecting its bioactivity. Therefore, the bioactivity of recombinant collagen requires further verification and improvement.

[0005] However, there have been no significant breakthroughs in the chemical synthesis of collagen-like proteins. First, while the synthesis of peptides that mimic the amino acid sequence of human collagen is theoretically feasible, correctly folding these protein molecules into an active triple-helical structure remains a key technical challenge. Furthermore, the production of collagen with a triple-helical structure that exhibits excellent thermal stability is also a major challenge.

[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 response to the problems of the prior art, the present invention provides a triple-helix collagen and a preparation method and use thereof.

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

[0011] Formula I: H2N-(O-GP) n -COOH;

[0012] Among them, О-GP 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 3.0×10 3 ~8.0×10 4 .

[0013] Preferably, the triple helix collagen is obtained by condensing a tripeptide having the structural formula H2N-О-GP-COOH as a raw material through a liquid phase reaction.

[0014] The present invention also provides a method for preparing the triple-helical collagen, comprising the following steps:

[0015] Step 1: a tripeptide having a structural formula of H2N-О-GP-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;

[0016] The phosphate buffer solution is prepared using the following raw materials in parts by weight:

[0017] 0.18-0.22 parts of potassium chloride,

[0018] 1.0-1.2 parts of sodium dihydrogen phosphate,

[0019] 0.18-0.22 parts of potassium dihydrogen phosphate,

[0020] 995-1005 parts of water;

[0021] The tripeptide solution is prepared using the following raw materials:

[0022] 495-505 parts by volume of phosphate buffer,

[0023] 49.5-50.5 parts by weight of tripeptide,

[0024] 4.9-5.1 parts by weight of condensation aid;

[0025] The dehydration condensation agent solution is prepared using the following raw materials:

[0026] 495-505 parts by volume of phosphate buffer,

[0027] 156-160 parts by weight of a dehydration condensation agent;

[0028] 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;

[0029] Step 3, separation and purification to obtain.

[0030] Preferably, in step 1, the condensation aid is selected from at least one of 1-hydroxybenzotriazole and 1-hydroxy-7-azabenzotriazole;

[0031] The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide and 1,3-dicyclohexylcarbodiimide;

[0032] The phosphate buffer solution is prepared using the following raw materials in parts by weight:

[0033] 0.2 parts of potassium chloride,

[0034] 1.15 parts of sodium dihydrogen phosphate,

[0035] 0.2 parts of potassium dihydrogen phosphate,

[0036] 1000 parts water;

[0037] The tripeptide solution is prepared using the following raw materials:

[0038] 500 parts by volume of phosphate buffer,

[0039] 50 parts by weight of tripeptide,

[0040] 5 parts by weight of condensation aid;

[0041] The dehydration condensation agent solution is prepared using the following raw materials:

[0042] 500 parts by volume of phosphate buffer,

[0043] 158 parts by weight of dehydration condensation agent.

[0044] 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;

[0045] The polycondensation reaction time is 24 hours.

[0046] 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.

[0047] The present invention also provides use of the triple-helix collagen in preparing cosmetics, medicines, cosmetic and plastic surgery materials or medical devices.

[0048] Preferably, the cosmetics are functional skin care products;

[0049] The medical device is a filling material or an engineering scaffold for skin, bones, and ligaments.

[0050] Preferably, the functional skin care product is a facial mask, essence or facial cleanser.

[0051] The present invention also provides a cosmetic, which is prepared by using the triple helix collagen as an active ingredient and adding auxiliary materials.

[0052] Preferably, the concentration of the triple helical collagen is 0.0005 wt%-0.1 wt%.

[0053] 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:

[0054] 1 part by volume: 1 part by weight = 1 ml: 1 g.

[0055] 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 a medical device, 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 function of promoting the expression of AQP3 in fibroblasts and has a moisturizing effect. In addition, the collagen polymer of the present invention also has the effects of anti-oxidation, repairing damaged hair, and promoting hair growth.

[0056] Therefore, the collagen polymer of the present invention has good application prospects.

[0057] 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.

[0058] 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

[0059] FIG1 is a circular dichroism spectrum of triple helical collagen after heat treatment at 20-60° C. in Experimental Example 1;

[0060] FIG2 is a circular dichroism spectrum of the commercial polypeptide in Experimental Example 1 after heat treatment at 20-60° C.;

[0061] FIG3 is a graph showing the molecular weight test results of triple helical collagen in Experimental Example 1;

[0062] FIG4 is a graph showing the experimental results of the relative expression level of AQP3 in Experimental Example 2. DETAILED DESCRIPTION

[0063] The reagents and raw materials used in the following examples and experimental examples are all commercially available unless otherwise specified.

[0064] Example 1 Triple Helical Collagen

[0065] This embodiment provides triple helical collagen, which is a polypeptide with a triple helical structure, and its structural formula is as follows:

[0066] H2N-(О-GP) n -COOH

[0067] In the formula, G, P and O are composed of the following amino acid residues:

[0068] P: proline,

[0069] O: Hydroxyproline,

[0070] G: glycine;

[0071] n≧4. Since the 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 molecular weight.

[0072] The preparation method of the triple helix collagen is as follows:

[0073] 1. Preparation of phosphate buffer

[0074] 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).

[0075] 2. Preparation of tripeptide solution

[0076] 50 g of tripeptide O-GP (manufactured by Uniqs Inc.) and 5 g of condensation aid 1-hydroxybenzotriazole (HOBt) were added to 500 ml of PB solution, and the mixture was stirred to prepare a tripeptide solution (hereinafter referred to as OGP solution).

[0077] 3. Preparation of dehydration condensation agent solution

[0078] 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).

[0079] 4. Polycondensation reaction

[0080] The prepared OGP solution was poured into a 2L separatory funnel and cooled to 2°C in a thermostatic bath. EDC solution, which had been cooled to 2°C, was then gradually added (the volume ratio of OGP 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, triple-helical collagen.

[0081] Example 2 Moisturizing lotion for anti-wrinkle and firming

[0082] This embodiment provides a moisturizing lotion, the composition of which includes:

[0083] 0.1 wt% of triple helical collagen prepared in Example 1,

[0084] Glycerol 10wt%,

[0085] Polyethylene glycol 15wt%,

[0086] Hyaluronic acid 5wt%,

[0087] The rest is water.

[0088] The technical solution of the present invention is further illustrated by experiments below.

[0089] Experimental Example 1 Characterization of Products

[0090] 1. Experimental Methods

[0091] This experimental example characterizes the triple helical collagen prepared in Example 1, including:

[0092] 1. Circular dichroism spectroscopy characterization

[0093] 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.

[0094] 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.

[0095] 2. Molecular weight characterization

[0096] 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.

[0097] 2. Experimental Results

[0098] 1. Circular dichroism spectroscopy characterization

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 2. Molecular weight characterization

[0103] As shown in FIG3 , the results show that the weight average molecular weight Mw of the triple helical collagen prepared in Example 1 is 3.0×10 3 ~8.0×10 4 (g / mol).

[0104] Experimental Example 2: Detection of the Effect of Promoting the Expression of Fibroblast-Anti-Wrinkle and Firming Related Genes

[0105] 1. Test Purpose

[0106] Aquaporin 3 (AQP3) is a cell membrane substance located in the basal layer of the epidermis. It regulates skin biological functions, such as water retention and transepidermal water loss. Responsible for the transport of substances such as water, glycerol, and urea, it is a transporter protein factor primarily expressed in keratinocytes and skin fibroblasts. AQP3 not only participates in skin hydration and barrier function, but also plays a vital role in skin damage, repair, and healing. It is crucial for maintaining skin moisture, normal morphology, and function.

[0107] 2. Experimental Methods

[0108] 1. Main reagents

[0109] Low-glucose DMEM culture medium (Solebro), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), and cell lysis buffer (Novozyme).

[0110] 2. Main equipment

[0111] 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, SS-03AB).

[0112] 3. Test methods

[0113] The specific settings of the test group are as follows:

[0114] Hacat cells were plated in serum-supplemented DMEM medium (10^5 cells / well) for 24 hours. After adherence, the cells were treated with 200 μmol / L hydrogen peroxide for 2 hours to establish a model. The cells were then cultured in clean DMEM supplemented with calf serum (0.1 wt% collagen prepared in Example 1 was added to the sample group). After 2 days, the cells were harvested and protein extracted for ELISA analysis. A blank control was prepared without hydrogen peroxide or collagen, with all other treatments remaining the same. A negative control was prepared without collagen, with all other treatments remaining the same.

[0115] ELISA experimental steps:

[0116] 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:

[0117] 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.)

[0118] 2) Blocking: Add 200 μl of blocking solution to each well and incubate at 37°C for 1-2 hours.

[0119] 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.)

[0120] 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.)

[0121] 5) Incubation: Seal the plate with sealing film and incubate at 37°C for 1-2 hours.

[0122] 6) Washing: Same as step 3.

[0123] 7) Add antibody: Add 100 μl of diluted biotinylated antibody working solution to each well.

[0124] 8) Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 hour.

[0125] 9) Washing: Same as step 3.

[0126] 10) Add enzyme conjugate: Add 100 μl of the diluted enzyme conjugate working solution to each well.

[0127] 11) Incubation: Seal the plate with a sealing film and incubate at 37°C in the dark for 30 min.

[0128] 12) Washing: Same as step 3.

[0129] 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 minutes until a clear color gradient appears in the wells containing the serially diluted standard.

[0130] 14) Stop the reaction: Add 100 μl of 2 M sulfuric acid to each reaction well. The color will change from blue to yellow.

[0131] 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.

[0132] 3. Experimental Results

[0133] The AQP3 content test results are shown in Figure 4:

[0134] The results showed that compared with the blank control group, the expression level of AQP3 in the negative control group Hacat cells was significantly increased after being treated with hydrogen peroxide (p < 0.01), indicating that under the initial damage stimulation of hydrogen peroxide, AQP3 would be highly expressed to repair the damage, and the modeling was effective.

[0135] Compared with the negative control group, the AQP3 expression level of Hacat cells in the sample group at a concentration of 0.1wt% was further significantly increased (p<0.05). This indicates that triple-helical collagen can promote AQP3 expression, thereby improving the effect of repairing damage.

[0136] When the t-test method is used for statistical analysis, the significance of the negative control group compared with the blank control group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **. When the sample group is compared with the negative control group, the significance of the negative control group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0137] The above experimental results show that after Hacat cells are stimulated by hydrogen peroxide, the expression of the hydrated protein AQP3 increases to repair damage. The use of the triple-helical collagen of the present invention can promote the expression of AQP3 in Hacat cells and enhance the effect of repairing damage. This shows that the triple-helical collagen provided by the present invention has an antioxidant effect.

[0138] From the above embodiments and experimental examples, it can be seen that the present invention provides a triple helix collagen, which has the same triple helix structure as natural collagen, has antioxidant effect, 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 cosmetics, medical devices and other fields, and has good application prospects.

Claims

1. A triple helical collagen, characterized in that Its structural formula is shown in Formula I: Formula I: H2N-(O-G-P) n -COOH; Among them, О-GP 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 3.0×10 3 ~8.0×10 4 .

2. The 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-O-GP-COOH as a raw material through a liquid phase reaction.

3. The method for preparing triple helical collagen according to claim 1 or 2, characterized in that: The steps include: Step 1: a tripeptide having a structural formula of H2N-О-GP-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.

4. The method for preparing triple helical collagen according to claim 3, 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.

5. The method for preparing triple helical collagen according to claim 3, 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.

6. The method for preparing triple helical collagen according to claim 3, 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.

7. Use of the triple helical collagen according to claim 1 or 2 in the preparation of cosmetics, medicines, cosmetic and plastic surgery materials or medical devices.

8. The use according to claim 7, 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.

9. The use according to claim 8, characterized in that: The functional skin care product is a facial mask, essence or facial cleanser.

10. A cosmetic, characterized in that: The invention is prepared by using the triple helix collagen as claimed in claim 1 or 2 as an active ingredient and adding auxiliary materials.

11. The cosmetic according to claim 10, characterized in that: The concentration of the triple helical collagen is 0.0005 wt%-0.1 wt%.

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